BIOCENTER OULU

Transcription

BIOCENTER OULU

BIOCENTER OULU
2015
ANNUAL REPORT
BIOCENTER OULU
ANNUAL REPORT 2015
TABLE OF CONTENTS
Governing Board Administrative Staff Biocenter Office Research Project Leaders FiDiPro Professor Junior Investigators Coordinators Comments of the Scientific Director Coordinator's Corner
Doctoral Programme Corner Visiting Writers Core Facilities Funding of Biocenter Oulu in 2015 ADMINISTRATIVE
STAFF
In the front row:
Seppo Vainio, Johanna Myllyharju and Pirkko Huhtala
In the back:
Teija Luoto, Anne Vainionpää, Antti Viklund,
Ritva Saastamoinen and Irmeli Nykyri
Missing from the photo:
Eeva-Riitta Savolainen
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RESEARCH PROJECTS
Kalervo Hiltunen 30
Thomas Kietzmann 34
Petri Kursula 37
Johanna Myllyharju 41
Taina Pihlajaniemi 45
Lloyd Ruddock 49
Markku Savolainen, Karl-Heinz Herzig and Marjo-Riitta Järvelin 52
Outi Savolainen and Mikko Sillanpää 60
Seppo Vainio 63
Robert Winqvist 67
JUNIOR INVESTIGATORS
Lari Lehtiö Gonghong Wei 72
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COORDINATORS´ PROJECTS
AND SELECTED CORE FACILITIES
EDITORS
Pirkko Huhtala
Teija Luoto
Biocenter Oulu
PHOTOS
Biocenter Oulu Groups
Group Photos: Greystone Oy / Juho Alatalo
Kulmakuvaamo (page 116–117)
Cover Figure: Petri Kursula
LAYOUT
Greystone Oy
BIOCENTER OULU 4 Annual Report 2015
Sinikka Eskelinen André H. Juffer
Aki Manninen Minna Männikkö Raija Soininen Raija Sormunen Rik K. Wierenga BIOCENTER OULU PROJECTS 2016–2019
EVENTS
Photo Gallery
Doctoral Programme Events 2015 Seminar Series 2015 Doctoral Programme Events 2016 Doctoral Programme – Curriculum Outline 2017–2018 BIOCENTER OULU 5 Annual Report 2015
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BIOCENTER CONTACTS
BIOCENTER CONTACTS
GOVERNING BOARD 2015
ADMINISTRATIVE STAFF
BIOCENTER OFFICE
Professor Petri Kursula, Ph.D.
Professor Robert Winqvist, Ph.D.
André Juffer, Ph.D.
Faculty of Biochemistry and Molecular Medicine
Laboratory of Cancer Genetics and Tumour
Biocomputing Coordinator
Professor Jyrki Heino
Professor Johanna Myllyharju, Ph.D.
Teija Luoto
& Biocenter Oulu, University of Oulu
Biology, Cancer and Translational Medicine
University of Turku
Scientific Director of Biocenter Oulu
Tel. Department of Biomedicine,
Research Unit
University of Oulu, P.O. Box 5000,
e-mail [email protected]
University of Bergen
Faculty of Medicine
FI-90014 University of Oulu, Finland
Tel. [email protected]
+ 358 (0)294 48 6103
Professor Riitta Keiski
University of Oulu
Irmeli Nykyri
Tel.
Tel.
Tel.
Professor Johanna Myllyharju
+ 358 (0)294 48 5740
[email protected]
+ 358 (0)294 48 6104
+ 358 8 315 3228
[email protected]
Professor Johanna Myllyharju, Ph.D.
University of Oulu
Professor Seppo Vainio, Ph.D.
Anne Vainionpää
Professor Tomi Mäkelä
Director of Biocenter Oulu Doctoral Programme
Tel.
University of Helsinki
+ 358 (0)294 48 6105
Professor Kyösti Oikarinen
Biocenter Oulu
University of Oulu
Tel. University of Oulu, P.O. Box 5000,
+ 358 (0)294 48 6084
+ 358 (0)294 48 5740
[email protected]
Docent Aki Manninen, Ph.D.
Coordinator in Cell Biology
FiDiPro PROFESSOR
Tel.
+ 358 (0)294 48 1161
Biocenter Oulu
Professor Susan Quaggin, M.D., Ph.D.
FiDiPro Distinguished Professor
Tel. of the Academy of Finland
[email protected]
Professor Taina Pihlajaniemi, M.D., Ph.D.
Faculty of Biochemistry and Molecular
+ 358 (0)294 48 6081
Medicine, University of Oulu
Docent Katri Pylkäs, Ph.D.
Docent Eeva-Riitta Savolainen, M.D., Ph.D.
Street address:
and Northwestern University Feinberg
Laboratory of Cancer Genetics and Tumour
Administrative Coordinator
Aapistie 5A, FI-90220 Oulu, Finland
School of Medicine
Biology, Cancer and Translational Medicine
Business Development Manager
Hematology Laboratory
www.oulu.fi/biocenter/
Tel.
Division of Nephrology,
Research Unit
Jouko Uusitalo
Oulu University Hospital, P.O. Box 500,
303 East Superior Street
Faculty of Medicine
Technopolis Plc, Oulu
FI-90029 Oulu, Finland
Chicago, IL 60611, USA
MSc Laura Pietikäinen
University of Oulu
[email protected]
[email protected]
RESEARCH PROJECT LEADERS
Professor Matti Weckström
University of Oulu
+ 358 (0)294 48 5800
Professor Lloyd Ruddock, Ph.D.
Tel.
[email protected]
Pirkko Huhtala, Ph.D.
Professor Karl-Heinz Herzig, M.D., Ph.D.
Coordinator
Research Unit of Metabolism
Academy Professor Seppo Ylä-Herttuala
Biocenter Oulu
Nutrition and Environment
Tel. University of Eastern Finland (Chairperson)
Institute of Biomedicine, Faculty of Medicine
Tel.
Tel.
+ 358 (0)294 48 6101
[email protected]
+ 358 (0)294 48 5280
Docent Ritva Saastamoinen, Ph.D.
Lari Lehtiö, Ph.D.
Coordinator in Transgenic Facility
Biocenter Oulu
Professor Markku Savolainen, M.D., Ph.D.
Research Unit of Internal Medicine
Faculty of Medicine
Tel. Tel.
[email protected]
Gonghong Wei, Ph.D.
Docent Raija Sormunen, Ph.D.
Coordinator of Electron Microscopy
Biocenter Oulu
Professor Outi Savolainen, Ph.D.
Department of Biology
Tel. FI-90014 University of Oulu, Finland
Faculty of Science
Professor Kalervo Hiltunen, M.D., Ph.D.
Biocenter Oulu
Tel.
[email protected]
Tel.
Tel.
[email protected]
+ 358 (0)294 48 1150
[email protected]
Docent Raija Soininen, Ph.D.
+ 358 (0)294 48 1683
Doctoral Programme Coordinator
+ 358 (0)294 48 6102
+ 358 (0)294 48 5101
Professor Marjo-Riitta Järvelin, M.D., Ph.D.
Centre For Life-Course Epidemiology
Oulu University Hospital
Tel.
Faculty of Medicine
[email protected]
+ 358 (0)294 48 1782
+ 358 (0)294 48 1169
+ 358 (0)294 48 6111
Tel.
+ 358 (0)294 48 6135
+ 358 (0)294 48 6112
[email protected]
COORDINATORS
Professor Rik K. Wierenga, Ph.D.
Coordinator in Structural Biology
Docent Lauri Eklund, Ph.D.
Professor Mikko Sillanpää, Ph.D.
Light Microscopy Coordinator
[email protected]
Research Unit of Statistical Models and Data
Biocenter Oulu
Analysis, Department of Mathematical Sciences
Tel. Professor Thomas Kietzmann, M.D., Ph.D.
Faculty of Science
[email protected]
Tel. University of Oulu, P.O. Box 5000,
Tel. Tel. e-mail [email protected]
+ 358 (0)294 48 7713
+ 358 (0)294 48 6073
+ 358 (0)294 48 1500
Docent Sinikka Eskelinen, Ph.D.
Light Microscopy Coordinator
Professor Seppo Vainio, Ph.D.
Biocenter Oulu
Tel.
Tel.
[email protected]
+ 358 (0)294 48 6084
+ 358 (0)294 48 4106
[email protected]
+ 358 8 315 3650
+ 358 (0)294 48 1199
COMMENTS OF THE SCIENTIFIC DIRECTOR
UNIVERSITY OF OULU AND BIOCENTER OULU
– STEPPING FROM 2015 TO 2016 UNDER NEW UNIVERSITY OF OULU ORGANIZATIONAL AND STRATEGIC
GUIDELINES AND WITH A NEW COMPOSITION OF
BIOCENTER OULU RESEARCH PROJECTS
The new University of Oulu strategy for 2016–2020 was
launched in February 2016. It is based on five thematic, internationally significant research focus areas: Creating sustainability through materials and systems, Molecular and environmental basis of lifelong health, Digital solutions in sensing and
interactions, Earth and near-space system and environmental
change, and Understanding humans in change. In these areas
of strength, the goal of the University of Oulu is to “participate in solving global challenges and produce new knowledge
for building a more sustainable, healthier, more intelligent and
more humane world”.
COMMENTS
OF THE SCIENTIFIC
DIRECTOR
Scientific Director of Biocenter Oulu
Johanna Myllyharju
The organizational structure of the University of Oulu was renewed during 2015 and has been in effect since January 2016.
Although the mission and tasks of Biocenter Oulu remain the
same – internationally high-level science, PhD training and
infrastructure services – the renewal meant quite substantial
changes to the administration of Biocenter Oulu. First of all,
Biocenter Oulu and the other focus area institutes of the University of Oulu no longer have their own bylaws and Governing Boards. Biocenter Oulu has had a Governing Board since
its establishment in 1986 and the Boards during this almost
30-year period have had a huge impact in guiding, sparring
and supporting Biocenter Oulu in reaching its goals of excellence. All Biocenter Oulu Boards and especially the last one
that started its term in January 2015, i.e. Academy Professor
Seppo Ylä-Herttuala (Chair since 2010, Member of the Board
since 2003), Professors Jyrki Heino (Vice Chair since 2011),
Tomi Mäkelä, Riitta Keiski, Kyösti Oikarinen, the late Matti
Weckström, Business Development Manager Jouko Uusitalo
and Laura Pietikäinen, MSc, are warmly thanked and appreciated for their extremely valuable efforts and input for the
benefit and success of Biocenter Oulu and the University of
Oulu. In the future, a Scientific Advisory Board (SAB) will
be nominated for Biocenter Oulu, but unlike the previous
Boards, the SAB will not be a decision-making body.
Secondly, although Biocenter Oulu research projects will continue to be selected for 4-year terms based on international
evaluation, changes have occurred in their funding principles.
The University of Oulu continues to allocate strategic funding to the projects selected to Biocenter Oulu in the form of
post-doc and PhD student positions, but starting from 2016,
funding is provided to and job contracts are made in the host
Faculty of the project instead of Biocenter Oulu. Productivity
funding is no longer allocated to Biocenter Oulu projects from
the Biocenter Oulu budget, but instead all research groups/
units in the University of Oulu “earn” their basic funding
from the Faculty budgets according to Ministry of Education funding principles. Biocenter Oulu research projects for
2016–2019 were selected by the Biocenter Oulu Board in
2015 based on evaluation carried out by a panel of esteemed
international professors: Kathryn Cheah – University of Hong
Kong (Chair), Janos Hajdu – Uppsala University, Rita Horvath – Newcastle University and Lars Klareskog – Karolinska
Institute. Thirteen projects were selected for Biocenter Oulu
for 2016–2019 (see p. 104), five of them being new (start of
one new project pending) and three continuing ones being
also renewed in the form of inclusion of new PIs.
Thirdly, Biocenter Oulu infrastructure core facilities are now
part of a new University of Oulu infrastructure unit where
strategic infrastructures serving a wide customer base are included. In addition to Biocenter Oulu infrastructures, this new
unit includes the Center for Microscopy and Nanotechnology, the Laboratory Animal Center, the Learning & Interaction
Observation Forum Laboratory, the Biodiversity Unit (Zoological Museum, Botanical Museum, Botanical Gardens and
Bothnian Bay Research Station) and the Oulanka Research
Station. Biocenter Oulu infrastructures as well as the other
infrastructures within the University level unit form their own
entities with their own Directors and separate budgets. The
purpose of the University of Oulu infrastructure unit is to
provide strategic support for research, combine information
concerning the infrastructures into a “single entry point” and
to share and implement good practices.
KEY FIGURES OF BIOCENTER OULU
Biocenter Oulu is a multidisciplinary umbrella organization
within the molecular and environmental basis of lifelong health
research focus area of the University of Oulu. In 2015, in Biocenter Oulu there were 10 research projects (some containing
subgroups), two junior investigator projects and six coordinator projects (some of the core facilities are coordinated by Biocenter Oulu research project leaders). These projects are from
the Kontinkangas Campus (Faculty of Medicine and Faculty
of Biochemistry and Molecular Medicine) and the Linnanmaa Campus (Faculty of Science), reflecting our truly multidisciplinary nature. The numbers of Biocenter Oulu personnel
in different categories are indicated in Figure 1. The numbers
of international research personnel in Biocenter Oulu have
been increasing steadily and currently seven of the Biocenter
Oulu project leaders are of foreign origin, and, altogether,
46% of the scientific personnel are of international origin. Biocenter Oulu scientists published 150 articles in international
peer-reviewed publication series in 2015 (Figure 2). Biocenter
Oulu projects are successful in attracting outside competitive
funding, including, for example, 3,8 M€ Academy of Finland
funding, 2,2 M€ from the EU and 1 M€ from foundations.
A formal Biocenter Oulu Doctoral Programme (DP) with a
structured curriculum has been in operation since 1995. The
20th year of Biocenter Oulu DP was celebrated in the event
“Career Seminar & 20 Years of Doctoral Training in BCODP” where all previous Biocenter Oulu DP coordinators,
Drs Erja Heikkinen, Sinikka Eskelinen, Pekka Kilpeläinen
and Anthony Heape, told how it all started and developed
onwards, and invited Biocenter Oulu DP alumni shared their
career stories in science and beyond. The Biocenter Oulu DP
currently has 25 of the 140 University of Oulu-funded doctoral training positions and a total of 110 PhD students. By
the end of 2015 the Biocenter Oulu DP had produced 305
PhD (64,3 %) and MD-PhD (35,7 %) graduates, 12 doctoral
theses being completed in 2015.
The 19th Discovery of the Year made by Biocenter Oulu young
scientists was selected by the board of the Biocenter Oulu DP
with the help of an outside expert, and was presented orally
along with four other selected studies at the Discovery of the
Year Symposium in December 2015. The 2015 Discovery of
the Year award was given to PhD students Marjut Nätynki
and Jaakko Kangas from Lauri Eklund’s group for their shared
authorship in an excellent paper on deciphering common and
specific cellular and molecular features of Tie-2 mutations that
are known to cause venous malformations. We warmly congratulate the whole Eklund group for this great team effort
and look forward to Marjut and Jaakko’s dissertations on April
8th 2016. Also shortlisted and presented were studies by Antti
Salo on the manifestation of extracellular matrix abnormalities and chondrodysplasia in mice with compromized collagen
prolyl 4-hydroxylase activity, by Kateryna Kubaichuk on the
discovery that lack of manganese superoxide dismutase causes
mitochondrial dysfunction and promotes hepatocarcinogenesis, by Sandhanakrishnan Cattavarayane on the finding that
long-term self-renewal of murine embryonic stem cells in the
absence of leukemia inhibitory factor is dependent on α6β1
and αV integrins, and by Justiina Ronkainen showing that the
fat mass- and obesity-associated gene Fto affects the dietary
response in mouse white adipose tissue.
The Best Poster award went to Heli Härönen, showing that
both transmembrane and shed collagen XIII are indispensable
for maintenance and plasticity of neuromuscular junctions. In
addition, the Biocenter Oulu DP board decides on an annual
special prize to a person who has made a significant or otherwise memorable contribution to Biocenter Oulu activities.
This year the special grant recipient was Dr. Raija Sormunen
for her unique contribution to the Biocenter Oulu EM core
facility since 1998. Raija has made an immense contribution
to the research work carried out by young scientists of Biocenter Oulu, as she is a collaborator in about 100 PhD theses
from the Biocenter Oulu DP. Raija will retire in March 2016
and we are going to miss her dearly. In the same event, special
thanks were given to Minna Männikkö, Sini Skarp and Mari
Taipale for their contribution to the Biocenter Oulu DNA
analysis core facility and Sinikka Eskelinen for her contribution as the Biocenter Oulu light microscopy coordinator.
From 2016 onwards these core facilities will operate under
new leadership of Drs. Katri Pylkäs and Lauri Eklund, respectively.
BIOCENTER OULU INFRASTRUCTURES, BIOCENTER
FINLAND AND INFRASTRUCTURE ROADMAPS
Biocenter Finland (and Biocenter Oulu infrastructure facilities, BCO being a Biocenter Finland member) is in Finland’s
roadmap for research infrastructures 2014–2020. A midterm
evaluation of this national roadmap will take place in 2017.
Inclusion in the national roadmap is an important factor with
respect to the Academy of Finland funding instrument for research infrastructures (FIRI), from which Biocenter Finland
has now obtained close to 3.5 M€ in the first three FIRI calls.
The panel statement of the FIRI2015 application of Biocenter
Finland stated that “the Biocenter Finland is an outstanding
collaboration in the life science area” and that “Biocenter Finland is an integral part of the seven biocenters located at the
six host universities where a majority of Finnish biomedical
research is conducted. Nationally there is a very clear collaboration with strong governance and engagement structures
in place”. All Biocenter Finland technology platforms were
evaluated at the beginning of 2016 by the Biocenter Finland
Scientific Advisory Board, Professors Carl-Henrik Heldin –
Uppsala University, Ole-Petter Ottersen – University of Oslo,
Marja Jäättelä – University of Copenhagen, Matthias Wilmanns – The European Molecular Biology Laboratory and
Gunnar von Heijne – Stockholm University. According to
their statement of Biocenter Finland overall, it has been and
is very successful.
of the European Research Area”. The Health and Food ESFRI
landmarks are the biobanking and biomolecular resources research infrastructure (BBMRI), the European advanced translational research infrastructure in medicine (EATRIS), the European clinical research infrastructure network (ECRIN), the
distributed infrastructure for life-science information (ELIXIR), INFRAFRONTIER and INSTRUCT.
Several European Strategy Forum on Research Infrastructure
(ESFRI) roadmap projects are also in Finland’s roadmap for
research infrastructures 2014–2020. The ESFRI projects in
which Biocenter Oulu is directly involved are the European
infrastructure for phenotyping and archiving of model mammalian genomes (Infrafrontier), Euro-BioImaging, and integrated structural biology (Instruct) projects. Biocenter Oulu
coordinates Finnish participation in the Infrafrontier ESFRI
project. Biocenter Oulu also coordinates the Instruct Finland
National Affiliated Center, the partners being the Universities
of Helsinki, Eastern Finland, Tampere and Turku and Åbo
Akademi. In addition, Biocenter Oulu is a partner in the Finnish Euro-BioImaging Node with Åbo Akademi (Coordinator)
and the Universities of Turku and Helsinki. All these Finnish
ESFRI participations were granted funding from the latest
FIRI2015 call. Special thanks go to Dr. Raija Soininen, Prof.
Rik Wierenga and Dr. Lauri Eklund for being instrumental in
the Infrafrontier, Instruct and Euro-Bioimaging activities of
Biocenter Oulu. Biocenter Oulu was also granted European
Regional Development funding for 2016–2017 for a tissue
imaging centre project that will be carried out in collaboration with Infotech Oulu. The aim of this project is to further
develop expertise and the capacity for advanced imaging and
automated image analysis.
Several changes effective from 2016 onwards have now taken
place: renewal of the entire University of Oulu organization,
renewal of Faculty organizations into research groups/units instead of departments, divisions etc., establishment of the new
University of Oulu research infrastructure unit, new project
composition of Biocenter Oulu for 2016–2019 and the new
strategy of the University of Oulu for 2016–2020. And we
do anticipate more to come… However, our main duty has
not changed, it is to fulfil the aim of excellence in the three
cornerstones of our operations: internationally high-level re-
The third update of the ESFRI roadmap after its first publication in 2006 was published on March 10th, 2016. ESFRI
Roadmap 2016 includes 21 ESFRI projects consisting of nine
from the 2008 Roadmap, six from the 2010 Roadmap and five
new projects and one reoriented project selected from 20 eligible proposals that were submitted in March 2015. One new
ESFRI project in the Health and Food area was selected for
the 2016 Roadmap, this being the European infrastructure for
multi-scale plant phenomics and simulation for food security
in a changing climate (EMPHASIS). The continuing Health
and Food ESFRI projects from the 2008 and 2010 roadmaps
are the infrastructure for analysis and experimentation on
Ecosystems (AnaEE), the European marine biological resource
centre (EMBRC), the European research infrastructure on
highly pathogenic agents (ERINHA), the European Infrastructure of Open Screening Platforms for Chemical Biology
(EU-OPENSCREEN), the European research infrastructure
for imaging technologies in biological and biomedical sciences
(Euro-BioImaging), the infrastructure for systems biology Europe (ISBE) and the microbial resource research infrastructure
(MIRRI). In addition, the 2016 Roadmap includes 29 ESFRI
landmarks which have reached the implementation phase and
are “pan-European hubs of scientific excellence, generating
new ideas and pushing the boundaries of science and technology”. The ESFRI Landmarks are “the RIs that were implemented or started implementation under the ESFRI Roadmap
and are now established as major elements of competitiveness
BIOCENTER OULU 2016 AND ONWARDS
search, PhD training in a high-quality and effective DP, and
development and operation of state-of-the-art infrastructures
that serve a wide user base ranging from local to international customers. According to the evaluators for the selection of
the new Biocenter Oulu projects 2016–2019 “Biocenter Oulu
supports high-level research in a multidisciplinary environment. Biocenter Oulu is a powerful constellation, which has
produced frontier science in biomedicine, biotechnology and
other areas of biosciences. The panel sees the strength of the
Biocenter in promoting interactions between different institutes and disciplines, permitting new investigators to start a
career within a good infrastructure, and building on previous
strength while giving room for new initiatives”. These aims
we keep clear in our minds and carry out following the new
motto of the University of Oulu “Science with arctic attitude”.
Oulu, March 2016
Johanna Myllyharju
109 Ph.D. and
M.D., Ph.D. students
Figure 1.
PERSONNEL
68 Post-doctoral fellows
2 Junior investigator project leaders
7 Coordinator project leaders
13 Project leaders
18 Senior scientists
56,5 Laboratory technicians and others
Figure 2.
PUBLICATIONS:
PUBLICATION FORUM CATEGORY RANKING
(JUFO3 highest)
JUFO3
19%
51%
29%
JUFO2
JUFO1
COORDINATOR'S CORNER
1. BCO Day and the first PhD Satellite Symposium
BCO Day is our traditional scientific meeting planned and
organized by our PhD students. The organizing committee
decides the topic, selects and invites the speakers and contacts
sponsors, and makes all practical arrangements. This is a superior learning experience for the students. The topic of the
25th BCO Day was “Personalized Medicine: Hype or Hope?”
Can it be any more timely? This year there was an additional
PhD Satellite Symposium aimed at all BCO-DP students to
present their own work in posters or in oral presentations. This
is a good opportunity to socialize, discuss and learn from one
another.
2. Oulu Bioimaging (OBI) Day with Image contest
The OBI network brings together scientists from different
disciplines to apply and promote development of imaging
technologies and image analysis within the University of Oulu
and Oulu University Hospital. This year, besides presentation
of the latest achievements in imaging, there was an OBI Day
Science Image Contest for the University of Oulu and Oulu
University Hospital community. In addition to being shown
at the event, and on our www-pages, the beautiful top three
images were on display for the public at Tietomaa, the Science
Theatre of Oulu.
Coordinator of Biocenter Oulu
Pirkko Huhtala
MY TOP TEN FAVORITE EVENTS
AT BIOCENTER OULU IN 2015
I have listed in chronological order my top ten favorites of our special
activities at BCO in 2015. Enthusiastic people and their ideas have generated
these happenings, and the active participants have made them a success.
3. Visits of secondary-school and high-school students
We encourage local school classes interested in biosciences
and medicine to visit our facilities, meet our researchers and
students and learn about our work. We have also organized
hands-on workshops during the University of Oulu Science
Days. It is very rewarding to see a genuine interest in the eyes
of these teenagers, planning their careers.
4. The first Nobel Prize Campus event
A new Kontinkangas Medical Campus event was started in
a collaborative effort by Biocenter Oulu, Medical Research
Center Oulu, the Faculty of Medicine and the Faculty of
Biochemistry and Molecular Medicine. On October 6, we
gathered together to follow a webcast from the Karolinska Institute concerning the exciting announcement of the Nobel
Prize 2015 in Physiology or Medicine. In addition, we learned
about the previous prize. This event has a good possibility to
develop into a great get-together event with great science!
5. Visit to Biocenter Kuopio
We at BCO have a tradition to have a yearly mini-symposium
with Biocenter Kuopio (BCK). We meet either in Kuopio or in
Oulu. This year it was time to take a busload of our researchers
to Kuopio, to enjoy the hospitality of BCK. The program was
excellent and I heard that all the eager Kuopio speakers could
not even fit into it. Next year we have to try to make it even
better! By the way, it will be the 20th joint meeting!
6. Joint BCO-DP – Ulm Graduate School meeting
BCO-DP has a Joint PhD Program with the International
Graduate School in Molecular Medicine, Ulm, Germany. This
in brief means that a student has two supervisors, one in each
country, and he/she spends part of his/her study period in the
partner laboratory, and receives a degree from both universities. The fall meeting was organized in Oulu with many group
leaders and students from both countries already participating
or planning to join this very important new collaboration forum, beneficial for all participants.
7. “Our Genetic Future” seminar
Genes and Society is a two-year (2015–2017) Argumenta
project funded by the Finnish Cultural Foundation and coordinated from BCO. This funding is intended to stimulate
dialogue between researchers in various fields of science, concerning significant subjects of current research. Genes and
Society project brings together people to discuss the current
state and future prospects of genetic research and its applications. A Seminar Day “Our Genetic Future” (Geneettinen
tulevaisuutemme) with national top-class speakers was held in
Finnish and it was designed for the general public interested
in the topic. A high number of participants (over 300) and
a lively discussion demonstrated the importance and need of
this type of outreach activity.
8. Oulu Bioscience Networking Event
Networking is important for everybody, but it is particularly
important for our soon-to-be PhDs, and PhDs looking for
their next working possibilities. Oulu Bioscience Networking
Event provides a great platform to hear company presentations and to learn to know people in the field. Currently, BCO
does not have any externally funded development projects that
are focused on the development of working life, business and
entrepreneurial skills for PhDs. However, by supporting and
participating in this event, organized mainly by young members of the local Biochemistry society, we encourage this type
of important activity.
9. 20 years of doctoral training and career symposium
BCO is the oldest bio-institute in Finland (established in
1986) and it has already had structural doctoral training for
two decades. It was fun to hear about the development of PhD
training into today´s structured doctoral program, described
to us by former school coordinators. The key elements have
always been there: quality, internationality and multidisciplinarity. The Career symposium continued with BCO graduates
showing examples of how a PhD in biosciences can lead to a
career anywhere your interests are.
10. Discovery of the Year
The traditional Discovery of the Year is a favorite event for
many of us. The articles published during the year are ranked
by an outside evaluator and the top-ranked articles are then
presented by junior members. We try to keep the list secret
until the speakers, one by one, are asked to the podium. The
best are rewarded and special rewards are given for important
inputs. This exciting event completes the year of BCO joint
activities and (almost) starts the refreshing holiday season.
Looking back to my list I notice that it covers quite well the
tasks we have. In addition to high quality research and doctoral training we support local, national and international
networking in science, we are connected to the surrounding
society and entrepreneurs, and we give opportunities for general public to get a glimpse of the world of science.
P.S. I want to add here one more special event (not organized
by BCO): The solar eclipse on the 20th of March, when the
moon covered about 90% of the sun at noon. The eagerness of
us all to see it was inspiring! Let’s keep that attitude!
Oulu, January 2016
Pirkko Huhtala
DOCTORAL PROGRAMME CORNER
The Biocenter Oulu Doctoral Programme (BCO-DP) had its
20-year anniversary in 2015. This landmark was celebrated
at the end of a busy year by our common career seminar and
birthday cake occasion in December. All our former coordinators (Erja Heikkinen, Sinikka Eskelinen, Pekka Kilpeläinen
and Anthony Heape) were there to share their thoughts with
us about the highlights and developments of their period. We
also heard interesting stories from our alumni on what they
had done with their PhDs, and perspectives presented by one
of our current doctoral students. It was a pleasure to experience this overview.
Doctoral training truly entered a new time when the first official Finnish graduate schools were established 20 years ago.
Biocenter Oulu was one of the pioneers in establishing its own
graduate school, with its first 4-year PhD positions covering
1995–98. The most recent set of such 4-year positions, 11 of
them for 2016-19, was subject to an open international call
for applicants in 2015. We have introduced many good and
previously unseen practices, trying to create an ideal doctoral
training environment. The list of items covering what we have
done that no one in Oulu had done before is something we
can be proud of, and it includes:
A NEW ERA OF ANOTHER 20 YEARS
FOR TRAINING GREAT DOCTORS
IN BCO HAS BEGUN!
Coordinator of Biocenter Oulu Doctoral Programme
Ritva Saastamoinen
Secretary of Biocenter Oulu Doctoral Programme
Anne Vainionpää
Director of Biocenter Oulu Doctoral Programme
Seppo Vainio
• The first graduate school in Oulu: Biocenter Oulu
Graduate School (of Biomedical Sciences); since 2010 the
Biocenter Oulu Doctoral Programme (BCO-DP)
• A structured training curriculum to support the full doctoral training programme, 1995–
• 4-year Graduate School/doctoral training positions, 1995–
• Strong promotion of international mobility, 1995–
• Annual thesis follow-up system, 1996–
• Promotion of business and working life skills, 1996– :
“BioPD Programme”, “Doctors to working life”, “Working
life and Business skills for PhDs”
• International Joint Doctoral Degree Programme (Oulu–
Ulm), 2014–
• Active promotion of local interdisciplinary cooperation
(e.g. “Multidisciplinary Imaging Workshop” 2014, interdisciplinary “BRIDGE” PhD position, 2014–2017)
• Public outreach (e.g. Finnish Cultural Foundation Argumenta project “Genes and Society” 2015–2017)
Our central mission has always been to train skilled multitalented doctors in an excellent international research community by offering a training curriculum that supports both career
development of scientific expertise and other skills required in
job markets. As the world changes all the time, our practices
have also been adapted and changed over time.
From its start BCO has offered doctoral students business-minded education, which complements their science-oriented fundamental understanding of living organisms. Health
and wellness monitoring, bioeconomy, green energy, clean
tech and other biotech-related subjects have become megatrends, so today such interest is very timely and should be
actively utilized. Although the unemployment rate of the doctors who have graduated from BCO has been lower than that
in biosciences in general, the employment prospects of bioscience doctors have recently become more challenging than
ever before. Competition for academic positions after a PhD
is hard and resources are limited. It is very important for all
doctoral students to understand the fact that most graduated doctors in all fields will be employed outside of academia.
That’s why we need to provide a doctoral training environment enabling also professional development towards industry and enterprises. We need to build bridges within academia
and between academia and industry in all “Triple-I” dimensions (Interdisciplinary, Intersectorial, International).
How does society appreciate scientists and do we have too
many doctors? The answer depends on what skills and expertise we expect from them. What a modern doctorate
should produce is a creative, critical, autonomous intellectual
risk-taker who is capable of adapting to different and perhaps
unforeseen tasks in society. The responsibility of the supervisors is to offer exciting but always feasible projects to doctoral
students and to support them enough to help them learn
timely skills that will make them employable. The doctoral
students’ own expectations and attitudes also matter a lot: how
flexible one’s mindset is towards new challenges. Too many
tend to underestimate the compatibility of their own skills and
personal strengths with non-academic jobs. It is not just scientific expertise that is acquired during doctoral training; it is
the skills common to all fields of science that make someone
good in a wider perspective. That’s why everyone should remember to focus on developing their whole professional profile to gain core competence in communication, negotiation
and management, as well as the ability to apply creative thinking and the capacity to adapt to different contexts and deal
with complex and multidisciplinary work. Training in these
transferable skills is today given in our collaborative doctoral
education programme under the University of Oulu Graduate
School (UniOGS) umbrella. Professor David Bogle, chair of
the League of European Universities (LERU) Doctoral Studies Community, has cited Anna Upton, who has expressed all
this in a nice way: “Subject knowledge, connections gained and
the ability to read critically all continue to be extremely important. My ability to manage projects, multi-task, solve problems
and think in an analytical way were developed during my doctorate. I can manage my time effectively, motivate myself, and deal
with difficult people. Perhaps these things are the most important
transferable benefits gained from my doctorate.” (LERU 2010:
Doctoral degrees beyond 2010: Training talented researchers
for society).
We have active talented people in the research community.
They are very brave; working with short contracts, persevering with hardships and still able and self-driven to make discoveries and gain success. Our doctoral training and research
environment creates plenty of opportunities to become friends
with colleagues from other scientific fields. We all depend
on interactions and sharing. One important element of the
BCO-DP is to offer all of us platforms to interact, to meet
physically in seminars and other common events, to develop
our social networks. We are living in the time of the internet,
virtual networks and virtual seminars and meetings, but these
can never replace the power of scientists coming together in
seminars and congresses to share ideas. These moments can be
like quantum leaps! So let’s all enjoy our beloved work in all
imaginable ways to develop our scientific and social excellence!
Seppo & Ritva
VISITING WRITER
My name is Mari Aikio. I defended my doctoral thesis in December 2013 after working in Professor Taina Pihlajaniemi’s
group. I became greatly interested in energy metabolism
and the regulation of adipogenesis during my PhD studies
when working on mice deficient in a ubiquitous basement
membrane protein, collagen type XVIII. The mutant mice
for this collagen show previously unidentified alterations in
their fat deposition and adipogenesis. Thus, after completing
my PhD, I started to search for a suitable laboratory where
I could broaden my knowledge and extend my scientific activities in energy homeostasis and fat cell development, and
transcriptional control of these processes. It was quite clear to
me from the beginning that I would like do my first postdoc
studies abroad. While preparing my thesis I became aware of
Professor Bruce Spiegelman’s impressive achievements in adipogenesis and metabolism, and decided to write to him to ask
about the possibility of obtaining a postdoctoral position in
his laboratory at Harvard Medical School and Dana-Farber
Cancer Institute. I was invited for interview and eventually
was offered a position as a fellow in Prof. Spiegelman’s group.
After meeting Bruce and seeing his enthusiasm and talent it
was very easy for me to nail down my decision. Six months later I received a Sigrid Jusélius Fellowship and in January 2015
my husband and I eventually moved to Boston.
POST-DOC’ING IN BEANTOWN
– THE HOLLYWOOD OF SCIENCE
They say being a scientist in Boston is the same as being an actor
in Hollywood. I guess it is not a totally wrong assumption, considering
that several world class universities, such as Harvard and MIT, are
located in the city.
Mari Aikio
Post doctoral fellow
Harvard Medical School
Dana-Farber Cancer Institute
Boston, USA
My project is related to activation of brown adipose tissue and
browning of white adipose tissue. In particular, I am exploring the existence of alternative pathways of thermogenesis and
the transcriptional and translational regulation of Prdm16.
Ultimately, these approaches have great potential to reveal
exciting new pathways important in energy metabolism. Dayto-day work at the lab is not that different from that in Finland. On the one hand, being at Harvard means that you have
huge resources at your disposal, easy access to a wide variety
of specialized methods and courses, and are surrounded by
highly skillful fellow researchers. That, in turn, could mean
that sometimes the environment is competitive. On the other
hand, by Finnish standards the lab is very messy – writing
desks are located next to the lab benches, so you can sometimes find someone dissecting a mouse next to you while you
are putting together a PowerPoint presentation for your next
lab meeting.
We live in a comfortable two-room apartment at Coolidge
Corner, which is one of the liveliest and coziest neighborhoods
in Boston. From the beginning we have felt at home in Boston, partly because of its strong European influences. Boston
is one of the oldest cities in the US and its prestigious universities and red-brick buildings, narrow and compact streets
combined with modern skyscrapers make the city’s culture
and architecture unique. Boston has a climate of extremes;
during our first year, we have seen record-breaking snowfall
and one of the sunniest summers, with temperatures staying
above 30 °C for several weeks. Boston’s location on the Atlantic coast means freezing winds in the winter, a wide variety of
marine activities in the summer, such as whale watching and
island hopping, and last but not least, excellent seafood. Boston is arguably one of the greatest sports cities in the world,
featuring NHL, MLB, NBA and NFL teams. The city is also
known for its annual marathon, and for a recreational runner,
such as myself, the city also offers great and picturesque running paths where one can sweat away the work stress.
The first year has gone very fast. Moving abroad, especially
to the US, requires an unimaginable amount of paperwork,
challenges and planning (and nerves). In our case, we did
everything ourselves while still in Finland, and, for example,
finding an apartment was not an easy task. But it has definitely
all been worth it! We have really enjoyed our time and had
great experiences professionally and personally, and met many
interesting people, such as President Tarja Halonen, who was a
visiting lecturer at Harvard last spring. I recommend working
oversees to anyone; it is the adventure of a lifetime!
Mari Aikio
Our lab is fairly big. At the moment we are PI, 13 postdocs,
one PhD student and four technicians, representing nine nationalities in all. Every fellow is in charge of their own projects but there is also great support around from the other lab
members and Bruce. On top of frequent ad hoc discussions,
Bruce has one-to-one meetings with every fellow bi-weekly.
These meetings provide a chance to have a private conversation about data and project milestones, and they also often
include a discussion about career plans and aspirations. Our
weekly lab meetings are a forum to present work to other lab
members 3–4 times a year. It is a great opportunity to receive
critique, questions and advice on experimental work. An additional training method is an annual lab retreat where the
whole lab goes off-site for a group brain-storming session.
Everyone, including Bruce, is required to give a presentation
with one key rule: you are not allowed to talk about your current work. Instead, you have to come up with an idea about a
potential future project. It is a beneficial and fun way to make
everybody think “out of the box”.
VISITING WRITER
I came to Oulu to complete my PhD right after obtaining a
Master’s Degree in Medical Biology at the University of Vilnius, Lithuania.
I still vividly recall the first weeks at the Department of Biochemistry. I was asking postdocs and PhD students how long
it took/would take for them to get their degrees. Their answers
puzzled me, as they were showing that on average it took about
eight years to complete a PhD at that time. That sounded like
such a long period of time and I just could not understand
why it took so long. I intended to graduate in four years. That
was the time when the number of publications was a criterion
for granting the degree, and not the number of years of study.
In the end it took twice the time I planned, if not longer,
which followed the general trend in the faculty. With the time
came understanding that science on a large scale develops rapidly, but when it comes to bench work, generating results and
publishing is a long and slow process.
While working on a PhD I’ve learned that the scientific world
can be quite competitive. How on earth can somebody come
up with identical ideas and work on similar projects on the
other side of the world? This just happens, and then years of
your work and that of your team-mates becomes worthless
and requires much more input to be suitable for publishing. I
liked how my PhD supervisor Prof. Seppo Vainio commented
on such a situation: ‘it is good that others are doing science
as well.’
Renata Prunskaitė-Hyyryläinen
Post doctoral fellow
TRANSITION FROM
PhD STUDENT TO POSTDOC
Baylor College of Medicine
Houston, USA
Along with the main projects of PhD work I was continuously
exploring new fields and taking sidetracks in order to expand
and broaden my understanding. I was also aiming to find new
points of view and bring a new perspective to my own work
and thinking. I attended various seminars, conferences, and
courses including those that were outside of my main PhD
topic.
One such sidetrack was my work with the optical projection
tomography (OPT) core facility. The aim of answering our
own scientific questions encouraged us to develop this technique together with Dr. Ilkka Pietilä, Antti Viklund and Hannele Härkman. This technique turned out to be interesting
and useful for many scientists in Finland and abroad. I also
found that collaboration was an interesting way of learning
new things and a way to apply my own knowledge in a novel
and creative way. While collaborating with Prof. Ulf Ahlgren
(Sweden) on developing and testing new image fusion OPT
software, we were able to obtain more accurate data on otherwise difficult-to-image specimens. Our knowledge of immunohistochemistry has led to stimulating collaboration with
Zeiss, which enabled us to demonstrate the suitability of lightsheet microscopy for imaging of mammalian tissues. While
teaching others I was learning myself.
During the PhD years it was great to work and share ideas
with inspiring people within academia, and at the same time
I found it extremely important to have activities outside of
science in order to maintain a versatile and balanced life. Finally, after several years of PhD studies, I was happy when
graduation day came!
Life after graduation often raises the question of shall one
continue in academia or pursue a non-academic career? The
general statistics for continuing as a postdoc are actually discouraging. For instance, in the USA, 65% of PhDs choose to
go for postdoctoral training, but later only 15–20% of them
obtain academic positions. In the UK only about 3.5% of
science doctorates move into permanent research staff positions1. From a financial point of view, the ones that pursue
an academic career have a more than 40% lower income than
those outside academia1. It is good to be aware of the academic bottleneck that we are also about to face, as a shorter
PhD training period has been adopted and more PhDs are
graduating every year.
In my case, I knew that I wanted to continue with postdoc
research despite all those alarming facts. I started to search for
a postdoc position. In order to sort this out I enrolled on an
extensive course on a broad range of subjects. I aimed to find
out if during the years of focusing on a particular subject I’d
missed out on something else that was interesting. This turned
out to be useful, as I could meet other postdocs and principal investigators to discuss and explore possible subjects and
topics. After deciding the topic for my postdoc research and
being accepted by the lab of interest, I started the marathon
of grant writing.
Now, being a postdoctoral fellow at Baylor College of Medicine in Houston, Texas, I am opening another page of scientific education, getting exposed to new ideas, subjects and
methods. It is stimulating to be exposed to a different research
and living environment, an invaluable experience and a joy to
interact with people from around the globe.
In conclusion; there is no right choice, just as there is no
wrong choice, but it will be your own choice and the life that
you choose to live.
I extend my gratitude to the Academy of Finland and the Sigrid Jusélius foundation for support.
Renata Prunskaité-Hyyryläinen
Reference
1
Powell K. (2015) The future of the postdoc. Nature
( http://www.nature.com/news/the-future-of-the-postdoc-1.17253)
CORE FACILITIES
PROTEIN ANALYSIS CORE FACILITY
The BCO Protein Analysis core facility is part of the BF Proteomics and Metabolomics infrastructure network and technology platform services. It offers a wide range of technologies
to study protein expression, modification, interaction and
function from single proteins up to complex protein extracts
(proteomes). The main technologies are 1) biophysical protein
analysis, 2) mass spectrometry, 3) proteomics and 4) protein
amino acid analysis. The facility has its focus on biophysical
protein analysis and gel-based proteomics. Services (consultation, training, service, research) range from in-depth study
of single proteins up to large-scale screening of proteomes. In
2015 the EMBO practical course “Modern Biophysical Methods for protein-ligand interactions” was organized for the third
time at BCO, reflecting the recognition the core facility has
gained within the scientific community. In addition to basic
funding from the University of Oulu (UO), the BCO Protein
Analysis Core Facility has been supported by funds from UO
to Biocenter Finland operations and strategic infrastructure
investments, and the European Regional Development Fund.
Biophysical protein analysis
CORE FACILITIES 2015
The BCO core facilities with their expert personnel represent a crucial part of
the research milieu that has been built within the University of Oulu by Biocenter
Oulu. BCO core facilities are part of the Biocenter Finland (BF) Infrastructure
networks and technology platform services (www.biocenter.fi). BF is a distributed national research infrastructure providing research services in life sciences
and biomedicine to the entire Finnish research community. BF was selected to
the National Research Infrastructure Roadmap in 2014. The BCO profile in BF
technology platforms focuses on mouse models, structural biology and electron
microscopy. BCO coordinates the Finnish participation in the Infrafrontier ESFRI
projects and participates in the EuroBioImaging and Instruct ESFRI projects.
The BCO core facilities are introduced here in brief. All BCO core facilities operate
on an open access principle and the latest information concerning contact persons, services and prices can be found at www.oulu.fi/biocenter/core-facilities.
This facility offers in-depth analyses of specific protein parameters, protein–protein as well as protein–ligand interactions,
with a spectrum of biophysical approaches which is unique
in Finland.
Services
• Study of protein secondary structures and stability (circular
dichroism)
• Spectroscopic analysis at 165–1150 nm, slow kinetics
(Stopped-Flow unit)
• Analysis of thermodynamic parameters of interactions, e.g.
enzymatic reactions or interactions with drugs (isothermal
titration calorimetry)
• Study of biomolecular interactions (association, dissociation), e.g. a complex of a pharmaceutical molecule and its
receptor (surface plasmon resonance)
Main equipment
• Isothermal titration calorimetry (ITC): VP-ITC and
ITC200 (Malvern Instruments)
• Surface plasmon resonance (SPR): Biacore 3000 and T200
(GE Healthcare), SPR Navi220A (Bionavis)
• Circular dichroism (CD): Chirascan Plus with a StoppedFlow unit and an autotitrator unit (Applied Photophysics)
pH 4
7
Mass spectrometry
Mass spectrometry (MS) allows the identification and characterization of metabolites, peptides, proteins and proteomes.
Current applications are protein identification, characterization of protein modifications, and lipid analysis (lipidomics).
Services
• Protein identification and characterization of protein
modifications
• Protein identification and label-free quantification
(MS-based proteomics)
• Analysis of small molecules (e.g. lipids or metabolites)
• Characterization of full-length proteins for structural
biology and enzymology
• Quality control
Main equipment
• Waters Synapt G2 HDMS Q-Tof (with nano Aquity
UPLC for MS-proteomics, proteins)
• Bruker UltrafleXtreme MALDI Tof/Tof (for protein
identification, proteomics)
• Waters Synapt G1 Q-Tof (with Aquity UPLC for
full-length proteins and small molecule work)
Proteomics
Two-dimensional gel electrophoresis (2-DE) allows the largescale screening of protein levels in complex protein extracts.
High sensitivity in protein detection is achieved with Difference Gel Electrophoresis (DIGE) as the current main application.
Services
• Study of protein levels dependent on drugs, environmental
factors, growth or developmental stages, regulators, etc.
• Analysis of disease mechanisms and biomarker search
• Quality control
Main equipment
• Protein separation: IPGphor3, MultiphorII, Ettan DALTII
• Protein detection: Typhoon 9400, MolecularImager FX
Protein amino acid analysis
The amino acid analysis service is carried out by combining
Shimadzu Prominence HPLC and Waters AccQ•Tag chemistry. Amino acid composition can be analyzed of purified
proteins and protein extracts from cell and tissue lysates. The
system has a special setup for protein hydroxylation studies.
CONTACTS
Kalervo Hiltunen, Professor, Coordinator of the BCO Protein Analysis
core facility, [email protected]
Ulrich Bergmann, Docent, PhD, Mass spectrometry,
[email protected]
Steffen Ohlmeier, Docent, PhD, Proteomics, [email protected]
Hongmin Tu, PhD, Biophysical protein analysis and Protein amino acid
analysis, [email protected]
www.oulu.fi/biocenter/proteomics-and-protein-analysis
CORE FACILITIES
CORE FACILITIES
BCO TISSUE IMAGING CENTER (BCO-TIC)
The Biocenter Oulu Tissue Imaging Center (BCO-TIC) is
part of the BF Biological imaging infrastructure network and
technology platform services, where its national spearhead expertise is mesoscopic-scale imaging (high resolution visualization of microscopically large 3D objects). BCO-TIC is also a
founder of the Oulu Bioimaging network (www.oulu.fi/obi).
BCO-TIC consists of light and electron microscopy units,
optical projection tomography and IVIS Spectrum devices. A
joint infrastructure project of BCO-TIC and the Center for
Machine Vision Research (CMV) at Infotech Oulu and the
Faculty of Technology provides imaging and analysis tools for
microscopic imaging. In addition to basic funding from the
UO, the BCO-TIC has been supported by funds from UO
to Biocenter Finland operations and strategic infrastructure
investments, Academy of Finland FIRI funding (2014 and
2016), and in 2016–2017 by the European Regional Development Fund. Together with Åbo Akademi (coordinator), University of Helsinki and University of Turku, BCO-TIC forms
the Finnish advanced light microscopy node for the EuroBioImaging ESFRI that is funded by the Academy of Finland
FIRI funding in 2016–2020.
• Microscopes and image analysis software are open to all
users after user training.
Equipment
The light microscopy unit is equipped with an Olympus
FluoView 1000 confocal microscope, Olympus Cell^M and
Olympus cellSens fluorescence microscopes, a Zeiss fluorescence microscope with an Eppendorf microinjection/Kibron
microprobe intender device, a Zeiss Cell Observer spinning
disc confocal microscope, a Zeiss LSM 780 confocal microscope and a custom-built OpenSPIM light-sheet microscope.
The Zeiss confocals and OpenSPIM are equipped with environmentally controlled (O2, CO2, temperature) on-stage
incubators. These imaging modalities allow high-resolution
spectral imaging, sensitive and fast 4D imaging and imaging
of protein dynamics in planar surfaces and 3D environments.
Special techniques such as FRAP, TIRF and confocal interference reflection are also available. In 2016 the BCO-TIC will
purchase two-photon and photoacoustic microscopy systems.
CONTACTS
Lauri Eklund, Docent, PhD, Coordinator of the BCO LM core facility,
[email protected]
Veli-Pekka Ronkainen, PhD, [email protected]
www.oulu.fi/biocenter/lm
Light Microscopy
The light microscopy (LM) unit offers state-of-the-art imaging
equipment and education for researchers. In addition to traditional fluorescence and advanced confocal microscopes, the
LM core facility offers light-sheet microscopy as an imaging
modality for thick samples and for sensitive high-speed 4D
imaging of various samples in 3D cultures and matrices. In
the EuroBioImaging ESFRI the LM unit provides pan-European services in mesoscopic scale imaging and image analysis
together with the CMV.
Services
• Light, fluorescent, confocal and light-sheet microscopy,
live-cell imaging, cell and tissue manipulations and measurements, computer-based demonstration and analysis of
image data and morphology.
• Assistance and training in sample preparation, choosing
appropriate techniques/instruments, trouble-shooting and
data analysis.
Optical Projection Tomography
Optical projection tomography (OPT) allows 3D imaging of
mesoscopic biological specimens up to ~1 cm across. It is a
fast method for viewing 3D anatomy and gene and protein
expression patterns during embryo development, and in organs and biopsy samples. In particular, it has two important
advantages over confocal microscopy: it can image much larger specimens, and it can image non-fluorescent specimens.
This means that in situ hybridisation and LacZ staining can be
mapped in 3D. Fluorescent signals can also be imaged, allowing the mapping of multiple protein distributions within the
same tissue. OPT scans are performed by using a Bioptonics
3000 device equipped with white, infrared, GFP and TXR
illumination systems. Further 3D analysis and morphometric
measurements are carried out by using 3D analysis software
(Imaris, Dirshti etc.).
trans-illumination and epi-illumination modes are available.
The instrument is located in the Laboratory Animal Centre.
CONTACT
Veli-Pekka Ronkainen, PhD, [email protected]
Electron Microscopy
The Biocenter Oulu Electron Microscopy core facility (BCOEM) is a nationally and internationally acknowledged service
laboratory. The areas of expertise are ultrastructural analysis
of gene-modified mouse tissues and immunoelectron microscopy. The BCO-EM core facility is part of the BF electron
microscopy technology platform.
The BCO-EM facility provides services and training in various
electron microscopy techniques for the analysis of biological
specimens. It also offers technical and scientific consultation
on the planning of experiments and provides training for sample preparation and operation of electron microscopes. The
laboratory is equipped with modern and up-to-date instrumentation for sample preparation and analysis and highly experienced and skilful personnel provide excellent service for
both national and international users from academia and industry. Currently, the following services are offered:
Plastic embedding and thin sectioning. With transmission electron microscopic analysis of post-stained ultrathin sections,
ultrastructural details at the nanometre scale can be resolved.
Immunoelectron Microscopy (IEM). For localization of molecules at the ultrastructural level we apply the Tokuyasu cry-
osectioning technique, where cryosections are labelled with
specific antibodies and conjugated gold markers are used to
visualize antibody binding sites.
High-pressure freezing and freeze substitution (HPF-FS). One
of the best available methods of biological sample preparation
for electron microscopy as regards structural preservation is
cryoimmobilization by high-pressure freezing (HPF) followed
by freeze-substitution (FS). After substitution, samples can be
embedded in plastic or rehydrated and processed by way of the
Tokuyasu technique for IEM.
Negative staining. This is a rapid technique to analyse detailed
structures of macromolecules, isolated organelles, and microorganisms. Negative staining can be combined with immunostaining. In this case antibody incubations are carried out
prior to the application of negative stain.
Tomography. Electron microscopic tomography is a technique
where image series are collected by imaging samples at various
tilt angles. The tilt series is then processed to reconstruct a
three-dimensional image which can be segmented to model
the structure of interest.
Scanning electron microscopy (SEM) analysis. SEM analysis of
biological samples started at the BCO premises in the beginning of 2015. Our FE-SEM enables versatile analysis of surface structures of cells and tissues. Due to good performance
using low acceleration voltage and a variable pressure mode
option, we can readily analyse beam sensitive and nonconductive samples. The instrument is also equipped with Shuttle &
Find software, facilitating its use in correlative light and electron microscopy applications.
CONTACT
Ilkka Pietilä, PhD, [email protected]
www.oulu.fi/biocenter/opt
IVIS Spectrum In Vivo Imaging System
The IVIS system can be used non-invasively to follow and
image disease progression and therapy, cancer metastasis, cell
trafficking, microbial infection and gene expression patterns in
living animals. It is capable of luminescence and fluorescence
signal detection and filters are covering the blue to near infrared wavelength region, enabling the scanning of reporters over
the range of 480–850 nm. In addition, the software provides
tools that allow separation of signals from multiple reporters within the same animal. For fluorescence imaging, both
CORE FACILITIES
CORE FACILITIES
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM).
With this method it is possible to obtain a series of images
from a conventionally prepared resin-embedded tissue block.
Material is removed layer by layer from the specimen surface
by using an ion beam and subsequently imaged via a scanning
electron beam. An aligned high-resolution image series can
then be used for volume reconstruction and 3D analysis of
cellular structures.
Instrumentation
Scanning electron microscope
(Sigma HD VP, Carl Zeiss AB, Germany)
Transmission electron microscope
(Tecnai Spirit, 120 kV, FEI Company)
CCD cameras Quemesa and Veleta
(Olympus Soft Imaging Solutions, GMBH)
Tissue processor (Lynx)
High pressure freezing device
(EM Pact, Leica, Vienna, Austria)
Freeze substitution device (Leica EM AFS 2, Leica)
Block trimmer (Leica)
Ultramicrotome (EM UC7 with EM FC7, Leica)
Ultramicrotome (Leica Ultracut UCT with EM FCS, Leica)
Leica EMKMR2 glass knife maker
Critical Point Dryer (K850, Quorum Technologies)
Sputter coater and carbon evaporator
(Q150T ES, Quorum Technologies)
CONTACTS
Raija Sormunen, Docent, PhD, Coordinator of the BCO EM core facility,
[email protected]
Ilkka Miinalainen, PhD, Coordinator of the BCO EM core facility from
2016 onwards [email protected]
www.oulu.fi/biocenter/em
TRANSGENIC ANIMALS – GENERATION AND ANALYSIS
OF GENETICALLY MODIFIED MICE
The BCO Transgenic (BCO-Tg) Core Facility coordinates
the Finnish participation in the Infrafrontier ERSFRI project
and serves as the Finnish node of the European Mouse Mutant Archive (EMMA). In addition, it coordinates the activities of the BF Model Organisms infrastructure network &
technology platform services, the BF FinGMouse network
(www.fingmice.org) and the NordForsk-funded Nordic Infrastructure for Mouse Models, NorIMM. EMMA is a decen-
tralized mouse archive involving 14 partners in 11 European
countries. The partners have established common operating
procedures and the highest-quality standards. Since 2013,
EMMA has been part of the Infrafrontier ESFRI research infrastructure (www.infrafrontier.eu), where large-scale primary
phenotyping is also included. From the EC Infrafrontier-I3
(FP7-Infrastructures-2012) grant BCO receives funding to
provide repository services: cryopreservation of germ cells and
embryos, generation of mouse lines from embryonic stem
(ES) cells, and re-derivation and distribution of mouse lines.
In addition to the EMMA services, the BCO-Tg core facility
provides a wide range of other services related to genetically
modified (GM) mice. All necessary information concerning
the transgenic core facility services, including the prices, can
be found at www.oulu.fi/biocenter/tgcore. Consultation, education of researchers, and following and being involved in the
development and set-up of new methods are also important
activities of the BCO-Tg core facility.
In addition to basic funding from the UO, the BCO-tg core
facility has been supported by funds from UO to Biocenter
Finland operations and strategic infrastructure investments.
The BCO-tg core facility equipment has been upgraded with
funds obtained from Biocenter Finland and Academy of Finland FIRI grants, which has also made it possible to purchase
an animal database and furnish the quarantine unit in the animal house with up-to-date equipment. Close collaboration
with the Laboratory Animal Centre is essential in GM mouse
work.
The following technologies and services are currently available
at the BCO-Tg core facility:
Gene targeting and culture of embryonic stem (ES) cells. All steps
requiring specific expertise – ES cell culture, selection, isolation and cryopreservation of ES cell clones, as well as isolation
of DNA from the clones – are performed by the core facility
staff. The customer is asked to plan the experiment, design the
targeting construct, provide the linearized targeting construct
DNA in a sufficient quantity, and screen the clones for correct
targeting.
Generation of chimeric mice. Cells from clones carrying the targeted allele, either originating from the customer’s laboratory
or generated elsewhere (e.g. in large knock-out mouse consortia) are injected into blastocyst- or morula-stage embryos to
generate chimeric mice. All steps are performed by the core
facility staff, but the customer has to obtain a license for generation of the GM mouse line before injections can be started.
Generation of transgenic and genetically modified mice by DNA
or RNA injection into zygotes. The isolation of fertilized eggs,
injections, and embryo transfers are performed by the core
facility staff. The customer prepares the DNA or RNA and
supplies it to the core facility. The customer has to have a license for generation of the mouse line before injections can
be started and is responsible for the analysis of pups born and
further breeding.
Re-derivation of contaminated mouse lines. Re-derivation of
mouse lines into specific pathogen-free (SPF) conditions is
performed via embryo transfer by the core facility staff.
Cryopreservation and recovery of mouse lines. A mouse line is
cryopreserved as germ cells (usually sperm) or embryos, and
the material is stored in liquid nitrogen. Recovery of the cryopreserved material includes thawing and transfer of cryopreserved embryos, or, if sperm is the cryopreserved material, the
core facility staff perform in vitro fertilization and transfer resulting embryos.
PROTEIN CRYSTALLOGRAPHY CORE FACILITY
The BCO Protein Crystallography core facility is one of the
major structural biology centers in Finland. It is the coordinator of the BF Structural Biology infrastructure network and
its X-ray technology platform (FIX-UP), and offers services
related to expertise in protein crystallographic structure determinations. The BF Structural Biology Infrastructure network
is an EU-Instruct-NAC (national affiliated center) platform
(Instruct-FI), which is also on the Finnish research infrastructure roadmap. The BCO Protein Crystallography core facility
participates in the EU-Biostruct-X and EU-iNEXT networks
as a Training, Implementation and Dissemination (TID) centre for organising international courses on protein crystallographic methods. In 2015 such a course was organised, using
the linux class room and the data collection unit as well as
Generation and isolation of pluripotent stem cells. ES cells are
isolated from morula-stage embryos using the 2i method
(Nicholls et al. 2009), whereas induced pluripotent stem (iPS)
cells are generated from fibroblasts by induction via retrovirus
infection. All steps are performed by the core facility staff.
Phenotype analysis. The BCO-Tg core facility personnel assist
and instruct scientists in mouse necropsy and tissue sectioning.
CONTACT
Raija Soininen, Docent, PhD, Coordinator of the BCO transgenic animal
core facility, [email protected]
www.oulu.fi/biocenter/tgcore
Physiological analyses of transgenic mice. The analyses available
are targeted to cardiac physiology, metabolic performance/
energy expenditure and haematology. For more information:
professor Risto Kerkelä, [email protected] (Scisense and
Vevo 2100, telemetry), professor Karl-Heinz Herzig, [email protected] (Labmaster) and docent Raisa Serpi, [email protected] (Exigo).
The services and equipment available
• Langendorff perfused heart protocol
• telemetry monitoring
• Scisense ADVantage system for measurement of haemodynamic parameters
• high frequency echocardiography technique
• high resolution in vivo micro-imaging system (Visualsonics
Vevo 2100)
• Flexcell FX-5000 for mechanical stretching of cultured
cells
• TSE LabMaster System® for analysis of metabolic performance/energy expenditure in cages for mice and rats
• haematological analysis with Exigo
CORE FACILITIES
CORE FACILITIES
offering a remote data collection session at Diamond. 17 students (6 from Finland) out of 43 applicants were selected to
the course. In addition to basic funding from UO, the BCO
Protein Crystallography core facility has been supported by
funds from UO to Biocenter Finland operations and strategic
infrastructure investments, European Regional Development
funding and EU funding.
BCO core facility provides also support for researchers and
professionals not familiar with structural studies so that they
can get the structure determination process started. As a national data collection center, the BCO X-ray data collection
facility, with its Bruker Microstar X8 Proteum diffractometer,
is available for academic users as well as for industrial partners.
Services
As highlighted on the BCO X-ray homepage (http://www.
oulu.fi/biocenter/protein-crystallography), the provided services cover all aspects of the structure determination pipeline.
They include advice and help with bioinformatics approaches as well as with several protocols for the characterization
of protein samples, for example using static light scattering
(SLS) and dynamic light scattering (DLS). Support for (CD)
spectroscopy, other biophysical characterization methods and
enzymology is also provided. Crystallization screens, using a
range of screens, can be set up and support is provided for
preliminary X-ray analysis of the crystals obtained. Help with
data collection from crystals (grown either in Oulu or elsewhere) and support with structure determination are also provided.
Facilities and equipment for X-ray studies
The available equipment and expertise is documented on the
internet pages. For protein crystallization there are dedicated
temperature-controlled laboratory facilities equipped with a
TTP Lab Tech Mosquito LCP nanodispenser for setting up the
crystallization screens (sitting drops of at least 75nL+75nL), a
Tecan Freedom EVO pipetting robot for liquid handling (can
also be used for setting up sitting drops of at least 1mL+1mL)
and two Formulatrix Rock Imager (RI54 and RI27) crystallization plate hotels for imaging of the crystallization drops
according to a user-set imaging schedule. The RI54 is at 22oC
and the RI27 is at 4oC, in a cold room. For viewing (on-campus and off-campus) and tracking of crystallization experiments, the xtalPiMS software is used, which is being developed in-house by Ed Daniel, in collaboration with Diamond
Light Source in Oxford, UK. Together with the other X-ray
centers in Finland this software is further developed. Ed Daniel has also made software for tracking the usage of the Formulatrix devices. Altogether in 2015 753 crystallization plates
have been setup. The total capacity is about 1350 plates per
year, assuming that the crystallisation results are monitored for
three weeks. With each plate 96 conditions can be screened.
The most popular plate type has three drops per well, therefore
at least 100 000 crystallisation conditions have been tested
and evaluated in 2015. Dedicated space, equipment and tools
are available for crystal storage, handling, shipment to synchrotrons and in-house data collection. For the in-house data
collection there is the Bruker Microstar X8 Proteum diffractometer with Helios MX focusing optics, a kappa goniometer and a Platinum 135 CCD detector, allowing for optimal
diffraction data collection from protein crystals of 100–200
micrometer in size. The required equipment software is integrated in the Linux and Windows computing environment of
the Faculty of Biochemistry and Molecular Medicine.
CONTACTS
Rik Wierenga, Professor, Coordinator of the Protein Crystallography
core facility [email protected]
Tiila-Riikka Kiema, PhD, Data collection, [email protected]
Kristian Koski, PhD, Remote data collection, courses, DLS, SLS, protein
purification, [email protected]
Ville Ratas, Technician, Crystallization/crystal imaging, [email protected]
Ed Daniel, Software-application designer of the xtalPiMS package,
[email protected]
Ari-Pekka Kvist, PhD, Windows and Linux computing environment,
[email protected]
www.oulu.fi/biocenter/protein-crystallography
BIOCOMPUTING CORE FACILITY
The Biocenter Oulu Biocomputing core facility is part of the
BF Bioinformatics infrastructure network and technology
platform services. In addition to basic funding from the UO,
the BCO Biocomputing core facility has been supported by
funds from UO to Biocenter Finland operations.
The following services are provided
• In silico modeling and simulation of biological systems, in
particular molecular systems such as enzymes and membranes
• Method and software development
• Special calculation services:
Analysis of the similarity between non-sequential protein
residue segments
Emphasis is given to the BEHAVIOR of physical (biological)
processes or systems on the basis of a theoretical model representing the given process or system, to provide quantitative
insight into the properties of the process or system.
Scientific computing facilities
For its operation, the facility has access to local and national scientific computing facilities, the latter at the Center of
Scientific Computing (CSC). The latest addition to the local
computing resources is ‘Tagyeta’ (funded by the Academy of
Finland FIRI2010 program), which consists of over 300 cores.
It was delivered in December 2011, bringing the total number
of computing cores to around 600. It is part of the Finnish
Grid Infrastructure (FGI2) set up to enable access to shared
computing resources throughout Finland. The FGI is coordinated by Kai Nordlund (University of Helsinki), and Pekka
Lehtovuori and Jura Tarus (CSC).
The scientific computing facilities Tagyeta and those at the
CSC are accessible to all trained users to perform in silico
modeling of various molecular systems. Software is available
for various types of modeling and simulation, including molecular dynamics, coarse-grained molecular dynamics, quantum mechanics/molecular mechanics, protein modelling, protein–ligand docking, and the like.
CONTACTS
BIOCENTER OULU VIRUS CORE LABORATORY
André H. Juffer, Coordinator of the BCO Biocomputing core facility,
[email protected]
Pierre Leprovost, [email protected]
www.oulu.fi/biocenter/biocomputing-and-bioinformatics
DNA SEQUENCING AND EXPRESSION
ANALYSIS CENTER
The center is located at Biocenter Oulu premises in the main
building of the medical campus and offers services and equipment for research projects with different needs for DNA/RNA
analyses. In addition to basic funding from the UO, the core
facility has been supported by funds from UO to strategic
infrastructure investments. Researchers can benefit from the
following services:
• DNA and RNA extraction from a wide range of starting
materials can be performed using QIAsymphony SP. The
instrument is connected to a QIAsymphony AS cabinet,
which enables automated PCR assay setup.
• Researchers can use QIAxcel electrophoresis to obtain fast
results, for example when checking a large number of PCR
products or when in need of checking RNA quality. With
QIAxcel there is no need for time-consuming gel preparation. It is a fully automated DNA fragment and RNA
analysis system that uses ready-to-run gel cartridges. Twelve
samples are processed simultaneously in 3–10 minutes.
• Services for DNA sequencing and genotyping are performed
using an ABI3500xL Genetic Analyzer. Researchers can
choose either full-service sequencing (preparation, purification, capillary electrophoresis) or ready-to-run sequencing
(electrophoresis only). In addition, DNA fragment analyses
such as microsatellite marker and SNaPshot analyses can be
performed. We offer help in experimental design and problem solving.
•The microarray facility is equipped with an Affymetrix
GeneChip Operating System. The facility aims to provide
a complete Affymetrix microarray service which includes
participating in planning the experiments, synthesis of the
probes from customer-provided RNA, hybridization, staining and scanning of the arrays, initial analysis of the data,
and basic training in using the dChip and/or Chipster data
analysis software, as well as help in choosing and operating
programs suitable for further analysis of the data.
More detailed information about our instrumentation and
services, including prices, can be found at http://www.oulu.
fi/biocenter/sequencing-center. Note that this core facility was
reorganized in the end of 2015 and a new service portfolio
including next generation sequencing was launched in 2016.
The Biocenter Oulu Virus Core laboratory is part of the BF
Viral Gene Transfer and Cell Therapy (VGTCT) infrastructure network and technology platform services. The BCO
Virus Core laboratory is a fully equipped Biosafety Level 2
cell-culture laboratory whose mission is 1) to provide training
and facilities for researchers who want to use viral vectors in
their research, and 2) to provide virus vector production as a
service for research groups. In addition to basic funding from
the UO, the core facility has been supported by funds from
UO to Biocenter Finland operations and strategic infrastructure investments.
Services
• Technical training, assistance, scientific consultation and
service-based production of viral vectors from constructs
• Easy entry and a cost-effective jump-start to learn how to
use viral vectors for gene expression and gene-silencing
studies
All the necessary reagents, consumables and molecular tools
are made available for retroviral (MuLV), lentiviral and adenoviral gene transfer approaches to transduce cell lines from
different species. We can also help in designing custom-made
viral RNA interference (RNAi) gene-silencing constructs and
CRISPR-based gene KO constructs and in planning genetic
manipulation of mouse embryonic stem cells and/or generation of lenti-transgenic mouse models.
BCO Virus Core laboratory facilities
The Virus Core laboratory is accessible to trained users at any
time. The laboratory contains two laminar flow hoods which
can be reserved by users. One out of three cell culture incubators is reserved for experiments with primary cell cultures and
tissues while the other two are for tested cell lines.
• Cells can be observed and imaged using a high-quality Zeiss
inverted fluorescence microscope equipped with a sensitive
camera. The laboratory also hosts a standard stereomicroscope.
• Purification and concentration of viral vectors can be carried out by ultracentrifugation or FPLC chromatography.
• An Ultra-low-temperature freezer is provided for users to
allow storage of viral preparations at the BCO Virus Core
laboratory.
CONTACTS
Aki Manninen, Docent, PhD, Coordinator of the Virus Core laboratory,
[email protected]
Jaana Träskelin, Technician, [email protected]
www.oulu.fi/biocenter/virus-core
CONTACTS
Katri Pylkäs, Docent, PhD, Coordinator of BCO DNA sequencing center,
[email protected]
Marko Suokas, MSci, NGS sequencing with Ion Torrent,
[email protected]
Aira Erkkilä, Technician, DNA sequencing and expression analysis
center, [email protected]
Anu Myllymäki, Technician, DNA sequencing and expression analysis
center, [email protected]
www.oulu.fi/biocenter/sequencing-center
FUNDING OF BIOCENTER OULU 2014
FUNDING OF BIOCENTER OULU IN 2015
TOTAL FUNDING OF BIOCENTER OULU 2015
SOURCE
SALARIES
EQUIPMENT
OTHER COSTS
TOTAL EUR
1 725 993
4 007 449
Biocenter Oulu:
University of Oulu*
2 281 456
Targeted funding for the Doctoral Programme
1 036 000
Academy of Finland
10 917
EU
18 889
Foundations
29 599
1 036 000
9 377
Others
Total
3 376 861
9 377
2 029 272
7 700
134 546
154 840
45 860
64 749
30 063
59 662
154 638
154 638
2 091 100
5 477 338
University of Oulu:
Depts. of Project Leaders
Academy of Finland
2 053 676
Foundations
598 632
EU
240 015
EVO**
7 000
47 000
Industry
266 333
Others
246 591
2 500
519 366
2 556 338
1 667 706
3 721 382
418 358
1 023 990
1 988 122
2 228 137
65 000 112 000
104 617
373 450
151 116
397 707
Total
5 481 519
17 200
4 914 285
10 413 004
Total EUR
8 858 380
26 577
7 005 385
15 890 342
* includes strategy funding for Biocenter Finland technology platform activities 468 555 EUR
** special research money for the University Hospital provided by the state
BREAKDOWN OF BIOCENTER OULU FUNDING 2014
SOURCE
SALARIES
Project funding*
1 615 500
Core facilities**
1 475 599
Course funding***
EQUIPMENT
9 377
103 139
Rents
Administration
Total EUR
182 623
3 376 861
* includes UO targeted funding for BCO Doctoral Programme 1 036 000 EUR
** includes UO strategy funding for Biocenter Finland technology platform activities 468 555 EUR
*** includes BCODP coordinator
9 377
OTHER COSTS
TOTAL EUR
620 600
2 236 100
588 515
2 073 491
99 900
203 039
744 568
744 568
37 517
220 140
2 091 100
5 477 338
RESEARCH
PROJECTS
RESEARCH PROJECTS
BACKGROUND AND SIGNIFICANCE
A long-standing problem in the biology of mammalian peroxisomes is the role of their membrane as a permeability barrier to metabolic intermediates. Studies have identified an
ATP transporter and the three human peroxisomal half-ABC
transporters are considered to participate in peroxisomal lipid
transport. Our contribution to solving the puzzle on material
exchange between the cytosol and peroxisomes is identification of the peroxisomal membrane protein Pxmp2 as a channel allowing transmembrane traffic for molecules less 500 Da.
Among the recently recognized features of mitochondrial
function is their ability to synthesize fatty acids in an acyl carrier protein (ACP)-dependent manner. Failure of mitochondrial fatty acid synthesis (mtFAS) in yeast leads to loss of mitochondrial respiratory function and incapability of growing on
non-fermentable carbon sources, and to defective mitochondrial RNA processing. A striking finding is that mtFAS is also
operational in mammals, and there is also mounting evidence
pointing to indispensable functions of mtFAS for the well-being of mammals. This notion is in line with observations that
disruption of mtFAS results in embryonic lethality in mice.
From left: Laura Pietikäinen, Tero Piltonen (trainee from OAMK), Leena Ollitervo, Juha Kerätär, Remya R. Nair, Geoffray Monteuuis, Anne Mäkelä, Alexander Kastaniotis,
Kalervo Hiltunen.
PEROXISOMES AND MITOCHONDRIA
IN LIPID METABOLISM OF CELLS
ABSTRACT
Our research addresses lipid biology and biochemistry, with a tight link to biomedicine and nutrition. We use mouse, yeast and cellular models with perturbations in mitochondrial and peroxisomal metabolism and employ genomics, physiological, biochemical, biophysical and imaging approaches to identify signalling
pathways that respond to disturbances in order to understand the molecular
mechanisms by which fatty acids and their metabolites affect cellular function.
The work includes three interlinked research areas: (i) mitochondria and regulation of metabolism, (ii) modified fatty acid metabolites and peroxisomes, and (iii)
intracellular solute channels.
PROJECT LEADER
Prof. Kalervo Hiltunen, M.D., Ph.D.
University of Oulu
The enzyme α-methylacyl-CoA racemase (Amacr) catalyses
interconversion of the R and S forms of the 2-methyl groups
of various 2-methylacyl-CoA molecules, a reaction required
for degradation of methyl-branched fatty acids and other isoprenoid-derived compounds such as cholesterol and its metabolites. Patients with Amacr deficiency have been described.
Amacr has also recently been identified as a tissue biomarker
of prostate and colon cancer tissues.
RECENT PROGRESS
More than 30 proteins (Pex proteins) are known to participate
in the biogenesis of peroxisomes – ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. Our work on yeast Pex11 revealed
an unexpected novel function of Pex11 as a non-selective
channel responsible for transfer of metabolites across peroxisomal membranes. The amino acid sequence shows sequence
similarity to transient receptor potential (TRP) cation-selective channels. In vivo, detection of the rate of β-oxidation revealed involvement of the Pex11 channel in transmembrane
transfer of metabolites and in regulation of peroxisomal metabolic processes. As a whole, the data show that Pex11 is a multipurpose protein performing distinct functions in peroxisome
biogenesis and metabolism.
Based on sequence similarity between Pxmp2 and other members of the family we suggest that all these proteins are transmembrane channels. In line with this hypothesis, we turned
to human mitochondrial Mpv17p and tested its function as
a potential channel-forming protein. Mutations in human
Mpv17 cause hepatocerebral mitochondrial DNA (mtDNA)
depletion syndrome (MDDS) that progresses at an early age
and is characterized by developmental delay, sensory and motor neuropathy, and metabolic abnormalities. Mice with deleted Mpv17 show signs of premature aging: grey coat early
in adulthood, high blood pressure, glomerulosclerosis, senso-
rineural deafness, depletion of mtDNA and a decrease in mitochondrial cytochromes. We expressed human mitochondrial
Mpv17p in yeast (Pichia pastoris) cells, and the data obtained
with the purified recombinant protein indicated that Mpv17
forms a non-selective channel with a pore size of 1.8 nm and
we located the channel’s selective filter. Voltage-dependent
gating of the channel was found to be regulated by redox conditions and pH. The results suggest that the function of the
channel is modulation of membrane potential to preserve homeostasis in mitochondria and to conduct quality control of
these organelles.
In contrast to humans, Amarc-deficient mice are clinically
symptomless on a standard laboratory diet, but fail to thrive
when fed phytol-enriched chow. We have studied the effect
and the mechanism behind the phytol-feeding-associated disease state in Amarc-deficient mice. All Amarc−/− mice died
within 36 weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death, accompanied by kidney and brain abnormalities. Histological analysis
of liver showed inflammation, fibrotic and necrotic changes,
Kupffer cell proliferation and fatty changes in hepatocytes,
and serum analysis confirmed the hepatic disease. A phytol
diet for two weeks in this study resulted in 60-fold elevation
of phytanic and pristanic acid in the liver tissue of Amacr−/−
mice. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after
two weeks of the phytol diet compared with a control diet.
This indicates that detoxification of phytol metabolites in the
liver is accompanied by activation of multiple pathways at the
molecular level and Amacr−/− mice are not able to respond
adequately. The results of this study show that the primary
physiological function of Amacr is detoxification of α-methyl
branched chain fatty acids. A secondary role of Amacr is in
the bile acid synthesis pathway, which can be compensated
for by alternative pathways when Amacr activity is missing.
The findings in this study also suggest that strict dietary intervention, avoiding phytol and its metabolites, might restrain
the emergence of pathological symptoms in patients suffering
from Amacr deficiency.
FUTURE GOALS
Several mouse models to investigate mtFAS deficiency have
been established and a priority of our work is characterization
of our mouse mtFAS dysfunction model. Complete knockout
of the Mecr enoyl reductase gene in mice causes early embryonic lethality. We have replaced this gene with a construct encoding a mitochondrially localized bacterial enoyl reductase.
Embryos carrying this transgene survive two days longer than
complete KO lines, and are currently under study to determine the effects of this construct on the development of the
embryo, mitochondrial markers and respiration. Data collection on mouse lines with tissue-specific deletions (forebrain
and cerebellum) is ongoing. A second goal is characterization
and possibly reconstruction of the mitochondrial lipoylation
pathway in yeast to help our understanding of this process.
A large number of studies have demonstrated the significance
of polyunsaturated fatty acids (PUFAs) for human health.
In spite of intensive international research, the molecular
mechanisms translating PUFA sensing into changes in gene
RESEARCH PROJECTS
RESEARCH PROJECTS
expression have remained largely enigmatic. As one approach
to shed light on the responses of animals to PUFAs we have
recently generated a mouse line defective in mitochondrial dienoyl-CoA reductase (Decr), which is a key enzyme required
for mitochondrial breakdown of PUFAs. These mice developed severe hypoglycemia, as do many other animal models
of fatty acid oxidation disorders during metabolic stress. In
contrast to other models, where hypoglycemia is associated
with hypoketonemia, absence of Decr activity did not alter the
ketogenic response to fasting. We will expand our investigation of the molecular mechanisms leading to the physiological
consequences of Decr deletion in mice.
RESEARCH GROUP MEMBERS
Project Leader:
Kalervo Hiltunen, M.D., Ph.D., Professor
(University of Oulu)
NATIONAL AND INTERNATIONAL ACTIVITIES
Group Members Who Spent More Than Two Weeks
in Foreign Laboratories During 2015
Kaija Autio, Ph.D. University of Arizona at Tucson, Tucson,
Arizona, USA (1.9.2015–)
Visiting Researchers in 2015 (over two weeks)
Carol Dieckman, University of Arizona, Tucson, AZ, USA
(6.7.–1.8.2015)
Han Ding, Jilin University, Changchun, China
(7.1.2015–31.5.2015)
Joana Schröder, Hamburg School of Life Sciences, Hamburg
(5.1.2015–30.4.2015)
Thimo Meyer, Hamburg School of Life Sciences, Hamburg,
Germany (1.9.2015–31.12.2015)
Karina Kürpick, Friedrich‐Schiller‐University Jena, Jena,
Germany (3.10.–20.12.2015)
PUBLICATIONS 2015–
Antonenkov VD, Isomursu A, Mennerich D, Vapola MH, Weiher H, Kietzmann T, Hiltunen JK.
The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective
Channel That Modulates Membrane Potential. J
Biol Chem 290(22):13840-61, 2015.
Jokinen R, Lahtinen T, Marttinen P, Myöhänen M, Ruotsalainen P, Yeung N, Shvetsova
A, Kastaniotis AJ, Hiltunen JK, Öhman T, Nyman TA, Weiler H, Battersby BJ. Quantitative
changes in Gimap3 and Gimap5 expression
modify mitochondrial DNA segregation in mice.
Genetics 200: 221-235, 2015.
Liu P, Wang X, Hiltunen K, Chen Z. Controllable
drug release system in living cells triggered by
enzyme-substrate recognition. ACS Appl Mater
Interfaces 7:26811-26818, 2015.
Liu PC, Wang H, Hiltunen JK, Chen ZJ, Shen
JC. Cross-linked proteins with gold nanoclusters: A dual-purpose pH-responsive material for
controllable cell imaging and antibiotic delivery.
Part Part Syst Char 32:749-755, 2015.
Selkälä EM, Nair RR, Schmitz W, Kvist A-P,
Baes M, Hiltunen JK, Autio KJ. Phytol is lethal for Amacr-deficient mice. Biochim Biophys
Acta 1851: 1394-13405, 2015.
Senior and Post-doctoral Investigators:
Vasily Antonenkov, M.D., Ph.D., Visiting Professor
Alexander Kastaniotis, Ph.D., Docent, group leader
(Academy of Finland)
Kaija Autio, Ph.D. (Academy of Finland)
Antonina Shvetsova, Ph.D. (Foundation, a part of the year)
Ph.D. Students:
Jahangir Alam, B.Sc. (Biocenter Oulu)
Guangyu Jiang, M.Sc. (Foundations)
Juha Kerätär, M.Sc. (Biocenter Oulu)
Geoffray Monteuuis, M.Sc. (Academy of Finland)
Anne Mäkelä, M.Sc. (Academy of Finland)
Laura Pietikäinen, M.Sc. (Biocenter Oulu)
Remya R. Nair, M.V.Sc. (Academy of Finland)
Eija Selkälä, M.D., M.Sc. (Foundations)
Foreign Scientists, 7
Mindthoff S, Grunau S, Steinfort L, Girzalsky
W, Hiltunen JK, Erdmann R, Antonenkov VD.
Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels. BBA – Molecular
Cell Research 1863: 271-283, 2016.
RESEARCH PROJECTS
From left: Verena Zoller, Daniela Mennerich, Maire Jarva, Franklin Tabughang Chi, Nina Kozlova, Anja Konzack, Kati Richter, Thomas Kietzmann.
INTRACELLULAR REDOX
COMPARTMENTS IN
HYPOXIA-SIGNALLING
AND ASSOCIATED DISEASES
ABSTRACT
Hypoxia-inducible factor-1a (HIF-1a) plays a key role in the response to hypoxia.
Our previous work showed reactive oxygen species (ROS), the EGF receptor
(Her1) adaptor protein CIN85, and the GSK-3/Fbw7/USP28 pathway contribute
to oxygen-independent regulation of HIF-1a protein stability. Therefore, in our
project we aim i) to determine the role of compartment-specific ROS generation
in HIF signalling, ii) to understand the mechanism by which CIN85 induces HIF-1a
levels, and iii) to identify the role of USP28 in cancerogenesis.
PROJECT LEADER
Prof. Thomas Kietzmann, M.D., Ph.D.
Faculty of Biochemistry and Molecular Medicine,
University of Oulu
Adaptation to changes in ambient O2 tension is essential for
adequate energy supply in humans and all other aerobic living organisms. Disturbances in O2 and nutrient supply have
profound effects not only on cellular but also whole body
function and may contribute to the development of various
diseases such as cancer, diabetes mellitus, metabolic syndrome,
artherosclerosis and thrombosis. The a-subunits of HIFs play
key roles in cellular responses to hypoxia and the pathogenesis
of these diseases. Major regulation of HIFs occurs at the level
of protein stability; the actions of HIF proline hydroxylases
allow subsequent recruitment of the pVHL ubiquitin ligase
complex, which enables proteasomal degradation of HIFs.
Moreover, compelling evidence has been obtained for the existence of other, non-pVHL-mediated HIF-1a degradation
mechanisms. One example is our finding showing degradation
of HIF-1a via a new pathway involving Fbw7 and USP28. In
addition, a number of reports including our own have shown
that HIFs also respond to growth and coagulation factors,
hormones, cytokines and stress factors under non-hypoxic
conditions by using ROS as mediators. Although these data
show that ROS levels have an impact on HIF-1a levels, there
are diverse opinions about the enzymes and cellular compartments participating in ROS generation. Thus, different cellular compartments such as the endoplasmic reticulum, the
Golgi complex and the mitochondria may have different roles
in redox signalling.
RECENT PROGRESS
Our research in the past has shown that HIFs respond to
growth and coagulation factors (PDGF, IGF-1, thrombin),
hormones (insulin) and cytokines (TNFa) in normoxic conditions by using ROS as mediators. Thereby, different cellular compartments and therein localized enzymes such as
NOX4, an ER-based member of the NADPH oxidases, and
a ROS-generating Fenton reaction at the ER can modulate
HIF-1a levels. At the same time hypoxia and ROS can also
activate the HIF-1a promoter by involving phosphatidylinositol 3-kinase and a functional NFkappaB site in the hif-1a
gene promoter.
Moreover, we were the first to discover the molecular mechanisms by which hypoxia activates transcription of plasminogen activator inhibitor-1 (PAI-1), which is a breast cancer
marker as well as being associated with obesity. Resveratrol, a
constituent of grapes and berries, was proposed to improve a
number of health problems. However, we demonstrated that
the effects of resveratrol on PAI-1 induction depend on the
cell type. Thus, resveratrol may be beneficial in non-tumour
cells but promote carcinogenesis in tumour cells.
In addition to the established finding that PAI-1 serves as a
breast cancer marker indicating a bad prognosis, we found
that it acts via low-density lipoprotein receptor-related protein 1 (LRP1), which was proposed to modulate the b-catenin pathway. Therefore, we used wild-type mouse embryonic fibroblasts (MEFs) and MEFs deficient in LRP1 to study
PAI-1 as a modulator of the b-catenin pathway. We found
that PAI-1 influences MEF proliferation and motility in a
LRP1-dependent manner and that b-catenin is important for
that response. In addition, expression of b-catenin and b-catenin-dependent transcriptional activity were induced by PAI1 in wild-type MEFs, but not in LRP1-deficient cells. Moreover, PAI-1-induced ERK1/2 activation was more prominent
in the LRP1-deficient cells and, interestingly, knockdown of
b-catenin abolished this effect. Together, these data show that
PAI-1 can promote cell migration via LRP1-dependent activation of the b-catenin and ERK1/2 MAPK pathway, which
may be important in stage-specific treatment of human diseases associated with high PAI-1 levels.
In addition to the above, we showed that HIF-1 signalling is
dysregulated in the livers of mice lacking manganese superoxide dismutase (MnSOD). Lack of MnSOD and the subsequent
production of ROS contributed to cellular conversion toward
a more malignant phenotype, affecting all cell properties generally associated with metabolic transformation and tumorigenesis. In line with impaired HIF a-subunit regulation, we
showed that the PKB/Akt target GSK-3 initiates VHL-independent HIF-1a degradation. In line with these observations,
we found that GSK-3 induced phosphorylation and recruitment of ubiquitin ligase and the tumour suppressor F-box and
WD protein Fbw7. Further, GSK-3b- and Fbw7-dependent
HIF-1a degradation can be antagonized by ubiquitin-specific
protease-28 (USP28). Altogether, we identified a new pathway
which can influence HIF-1a-dependent processes.
FUTURE GOALS
We defined three major goals: i) to determine the role of compartment-specific ROS generation in HIF signalling, ii) to understand the mechanism by which MnSOD regulates HIF-1a and
iii) to identify the role of USP28 in cancerogenesis.
Our first goal is to investigate how compartment-specific concentration changes of ROS modulate the activity of the HIFa
family and expression of their target genes. In particular, we will
address the question of whether or not disturbances of ROS balance in mitochondria, peroxisomes or in the ER have an impact
on HIFa expression and degradation in vivo and in vitro. To
investigate the impact of the different compartments in vivo we
will first use our mice in which the mitochondrial antioxidative
enzyme MnSOD (SOD2) has been knocked out specifically in
hepatocytes. These mice can be placed in special hypoxia chambers and induction of HIFa proteins can be investigated. These
studies will be accompanied by cell culture experiments with either immortalized hepatocytes or mouse embryonic fibroblasts
derived from knockout mice. To extend these analyses we also
aim to investigate HIFa levels upon knockdown of other components of various compartments (e.g. Gpx, Mpv17, catalase,
ERp72). In addition, it is necessary to monitor ROS changes with respect to different cellular compartments, something
which has not yet been done. We will realize this by combining
two-photon confocal laser scanning microscopy together with
redox-sensitive green fluorescent proteins that are targeted specifically to different cellular compartments.
As regards our second goal we will continue to unravel the
mechanisms by which MnSOD interferes with HIF degradation and investigate whether or not it interferes with PHD
function. So far, it is unknown how these molecules interact
and which domains would be involved.
RESEARCH PROJECTS
Work on the third goal will include systematic screening for
further USP28-interacting proteins with the help of tandem
affinity chromatography and mass spectrometry. In these analyses we will greatly benefit from the expertise and equipment
available in the Biocenter Oulu Proteomics and Protein Analysis core facility. The impact of the identified USP28 interactors on HIFs, PAI-1 expression, insulin-regulated metabolism
as well as angiogenesis will then be studied.
Novel outcomes can be expected by combining the work of
our group with expertise on mitochondria, peroxisomes and
ER available in Oulu.
Verena Zoller, M.Sc., University of Ulm, Germany
EU Projects (present and progress)
EU-ROS; COST Action: BM1203; MC member substitute
Project Leader:
Thomas Kietzmann, M.D., Ph.D., Professor
Antonina Shvetsova, Ph.D. (University of Oulu)
Daniela Mennerich, Ph.D. (Biocenter Oulu)
Ph.D. Students:
Nina Kozlova, M.Sc. (Biocenter Oulu)
Kati Richter, M.Sc. (University of Oulu)
Anja Konzack, M.Sc. (University of Oulu)
Franklin Tabughang Chi, M.Sc. (Sigrid Jusélius Foundation)
Laboratory Technicians:
Maire Jarva (University of Oulu)
Main source of salary in brackets.
Antonenkov VD, Isomursu A, Mennerich D, Vapola MH, Weiher H, Kietzmann T, Hiltunen JK.
The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective
Channel That Modulates Membrane Potential. J
Biol Chem 290(22):13840-61, 2015.
Deschoemaeker S, Di Conza G, Lilla S, MartínPérez R, Mennerich D, Boon L, Hendrikx S,
Maddocks OD, Marx C, Radhakrishnan P,
Prenen H, Schneider M, Myllyharju J, Kietzmann T, Vousden KH, Zanivan S, Mazzone M.
PHD1 regulates p53-mediated colorectal cancer
chemoresistance. EMBO Mol Med 7(10):135065, 2015.
Espinosa-Diez C, Miguel V, Mennerich D,
Kietzmann T, Sánchez-Pérez P, Cadenas S,
Lamas S. Antioxidant responses and cellular
adjustments to oxidative stress. Redox Biol
6:183-97, 2015.
Ganjam GK, Chi TF, Kietzmann T, Dimova EY.
Resveratrol: beneficial or not? Opposite effects
of resveratrol on hypoxia-dependent PAI-1 ex-
pression in tumour and primary cells. Thromb
Haemost 115(1), 2015.
Görlach A, Dimova EY, Petry A, Martínez-Ruiz
A, Hernansanz-Agustín P, Rolo AP, Palmeira
CM, Kietzmann T. Reactive oxygen species, nutrition, hypoxia and diseases: Problems solved?
Redox Biol 6:372-85, 2015.
Horbach T, Götz C, Kietzmann T, Dimova EY.
Protein kinases as switches for the function of
upstream stimulatory factors: implications for
tissue injury and cancer. Front Pharmacol 6:3,
2015.
Kietzmann T. Myocardial infarction in elderly patients: How to assess their bleeding risk?
Thromb Haemost 114(5):869-71, 2015.
Konzack A, Jakupovic M, Kubaichuk K, Görlach
A, Dombrowski F, Miinalainen I, Sormunen R,
Kietzmann T. Mitochondrial Dysfunction Due to
Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis. Antioxid Redox
Signal 23(14):1059-75, 2015.
Kozlova N, Jensen JK, Chi TF, Samoylenko A,
Kietzmann T. PAI-1 modulates cell migration in
a LRP1-dependent manner via b-catenin and
ERK1/2. Thromb Haemost 113(5):988-98, 2015.
Kozlova N, Samoylenko A, Drobot L, Kietzmann T. Urokinase is a negative modulator of
Egf-dependent proliferation and motility in the
two breast cancer cell lines MCF-7 and MDAMB-231. Mol Carcinog 55(2):170-81, 2015.
Richter K, Konzack A, Pihlajaniemi T, Heljasvaara R, and Kietzmann T. Redox-fibrosis:
Impact of TGFb1 on ROS generators, mediators and functional consequences. Redox Biol
28:344-352, 2015.
Spohrer S, Dimova EY, Kietzmann T, Montenarh M, Götz C. The nuclear fraction of protein
kinase CK2 binds to the upstream stimulatory
factors (USFs) in the absence of DNA. Cell Signal 28(2):23-31, 2015.
Zagotta I, Dimova EY, Debatin KM, Wabitsch
M, Kietzmann T, Fischer-Posovszky P. Obesity
and inflammation: reduced cytokine expression
due to resveratrol in a human in vitro model of
inflamed adipose tissue. Front Pharmacol 6:79,
2015.
From left: Arne Raasakka, Petri Kursula, Maryna Chukhlieb, Matti Myllykoski, Saara Laulumaa, Srinivas Kumar Ponna, Weisha Luan.
PROJECT LEADER
Prof. Petri Kursula, Ph.D., Docent
& Biocenter Oulu, University of Oulu
Department of Biomedicine,
University of Bergen
STRUCTURAL BIOLOGY
OF THE MYELIN MEMBRANE
ABSTRACT
The myelin sheath enables the rapid transmission of nerve impulses in the vertebrate nervous system. The multilayered myelin membrane, tightly wrapped
around axons, contains a number of specific proteins involved in myelin formation and neurological diseases. We are interested in the structure–function
relationships in this diverse set of proteins, in order to better understand the tight
packing of the myelin membrane and the factors governing its function in nervous system development, function, and disease. We also use these molecules as
general models for studying protein–membrane interactions and the formation
of stacked membrane multilayers. In addition, we work on a number of other
neurobiologically relevant structural biology projects.
RESEARCH PROJECTS
RESEARCH PROJECTS
The myelin sheath is a specialized membrane structure in the
vertebrate nervous system, enabling the fast ‘saltatory’ conduction of nerve impulses. Myelin is formed by the differentiated
plasma membrane of a myelinating glial cell (Schwann cell or
oligodendrocyte), which wraps itself tightly around the axon,
forming a highly ordered, compact, multilayered membrane
complex with a very low solvent content. As a biochemical
membrane, myelin is unique. Essentially all of the myelin-specific proteins interact intimately with lipid bilayers, being either integral or peripheral membrane proteins. Despite a large
volume of literature on myelin proteins, little is still known
about their 3D structures and their complexes with other
molecules. In addition to being specific to myelin, myelin
proteins, in general, share little homology with proteins from
other tissues or lower organisms.
structure–dynamics relationships, whereby conformational
changes at the portal region of the protein are linked to its
binding to lipid bilayers and monomeric fatty acid binding
inside the barrel structure (Laulumaa et al. 2015). Most of this
long-term work, combining sub-atomic resolution crystallography with complementary methods, is in progress and includes significant multidisciplinary aspects. The first neutron
crystal structure of P2 is also currently under refinement. The
overall structure of human P2 is shown in Figure 1.
We also finalized our study on the structure of the cytoplasmic
domain of type III neuregulin 1, a protein regulating peripheral nerve myelination. This domain was shown to be intrinsically disordered; however, local folding could be induced by
the presence of metal ions and a membrane-mimicking environment (Chukhlieb et al. 2015). A typical model for the
structure of the NRG1 cytoplasmic domain in solution, obtained by synchrotron small-angle X-ray scattering, is presented in Figure 2.
The project will also expand in the direction of obtaining an
understanding at the structural level of mutations and sequence variants in proteins and complexes involved in neurological disease conditions, including psychiatric disorders. In
addition, one future project will involve studies on neurobiologically relevant protein kinases, as well as the molecular details and possible pharmacological intervention of important
regulatory proteins in neurons and myelinating cells, such as
calmodulin and CRMP2.
Long-standing questions exist in myelin biology, specifically
related to the relationships between structure, dynamics, and
function of individual myelin proteins and multilayered membranes, as well as the factors contributing to these properties.
Specific points of interest include how these molecules interact
with membranes and the cytoskeleton, how they are arranged
on the membrane, and how they contribute to the formation
and maintenance of the compact structure of myelin. More
esoteric questions are related to the dynamics of the protein
and lipid components, both separately and when in the setting
of an artificial multilayer resembling myelin.
Detailed structure–function information will be crucial to
understanding the physiological function of myelin proteins.
Neurological demyelinating diseases, including, for example,
multiple sclerosis and peripheral neuropathies, occur upon autoimmune attack against myelin or because of inherited mutations in myelin protein genes. Understanding of such diseases
will be enhanced by accurate 3D structural data on myelin
molecules and their interactions with each other and with ligands, including lipid membranes. Mutagenesis can be used to
mimic disease mutations in vitro, and peptides corresponding
to autoimmune epitopes can be used in addition to full-length
myelin proteins. In addition to strictly demyelinating diseases,
we have also shifted some of our current focus slightly towards
other neurobiological disorders and the proteins involved in
these diseases, including autism, ALS, and neuropsychiatric
conditions.
RECENT PROGRESS
During 2015, a lot of effort was invested in studying protein-membrane interactions. Different myelin peripheral
membrane proteins were used as model systems in these experiments. The studied proteins have included folded proteins,
intrinsically disordered proteins, and peptides representing
membrane-binding segments. Novel methodologies were also
heavily implemented, including neutron reflectometry, neutron crystallography and scattering, atomic force microscopy,
electron microscopy, and atomistic membrane simulations,
for example. The work links protein structure and dynamics
to protein–membrane binding, with detailed characterization
of protein insertion into the membrane. Extensive studies on
the myelin protein P2 have revealed important details of its
We will also use myelin proteins as general models for protein–membrane interactions. Detailed analyses of protein and
membrane structure and dynamics will be carried out. A number of highly multidisciplinary state-of-the-art methods will
be used to reach such goals. The results will not only be relevant to myelination, but also to peripheral membrane protein
function in general. High-resolution imaging of the molecular
organization of a multilayered myelin-mimicking membrane
is one major goal of our research in the future. The first steps
in this direction have already been taken, and the results are
exciting. It is likely that many myelin proteins act as linkers
between the plasma membrane and the cytoskeleton; we will
focus on elucidating the structural basis of such functions.
Project Leader:
Petri Kursula, Ph.D. (University of Bergen, Norway)
Salla Ruskamo, Ph.D. (Academy of Finland)
Matti Myllykoski, Ph.D. (Foundations, Biocenter Oulu)
Huijong Han, Ph.D. (Academy of Finland)
Weisha Luan, Ph.D. (Foundations)
Jussi Tuusa, Ph.D. (Foundations)
Figure 1. Crystal structure of the human P2 peripheral membrane protein.
Upon membrane binding, conformational changes occur in the helical lid
and the portal region adjacent to it (at the top in this view).
One of our long-term projects concerns the myelin enzyme
CNPase. We have recently examined its reaction mechanism
at atomic resolution (Raasakka et al. 2015), and studies focusing on its evolutionary aspects have been initiated (Myllykoski
et al. 2015). We now have purified CNPase homologues from
several organisms, and structure–function studies are being
carried out. These studies will highlight details of evolution
in the 2H phosphoesterase family, and will shed light on the
relationship between CNPase and myelination.
Our earlier work resulting in the crystallization of the N-terminal PDZ-like dimerization domain of the myelin protein
periaxin (Han & Kursula 2015) is being continued on several fronts. The projects include structural characterization of
further domains in periaxin, the formation of physiologically
relevant protein–protein complexes involving periaxin, the
dynamics of the formation of periaxin dimers, as well as analysis of disease-linked mutations at the structural level.
Figure 2. A model for the solution structure of the type III NRG1 cytoplasmic domain. The protein is intrinsically disordered.
Projects focusing on a number of other myelin proteins have
also advanced, including the production of major myelin integral membrane proteins for structural studies. Furthermore,
other neurobiologically relevant projects have been started.
These include, for example, structural characterization of protein complexes formed by the post-synaptic density protein
Shank and small molecule-mediated interference of protein
interactions of the central axonal pathfinding protein CRMP2.
FUTURE GOALS
Ph.D. Students:
Arne Raasakka, M.Sc. (Foundations, University of Bergen)
Saara Laulumaa, M.Sc. (Foundations)
Maryna Chukhlieb, M.Sc. (Biocenter Oulu)
Srinivas Kumar Ponna, M.Sc. (Biocenter Oulu)
Petri Kursula, DESY, Hamburg, Germany & University
of Bergen, Norway
Huijong Han, DESY, Hamburg, Germany
Saara Laulumaa, ILL, Grenoble, France
Arne Raasakka, University of Bergen, Norway
We will continue to obtain high-resolution structural data
from various myelin proteins and their complexes, and by
combining the structures with complementary experiments,
we will get detailed information on the structure–function relationships in myelin proteins. This information can be used
to understand myelin protein function in normal and diseased
myelin. Together with international collaborators, the structural data will further be linked to in vivo functions.
RESEARCH PROJECTS
Bhargav SP, Vahokoski J, Kallio JP, Torda A,
Kursula P, Kursula I. Two independently folding
units of Plasmodium profilin suggest evolution
via gene fusion. Cell Mol Life Sci 72:4193-4203,
2015.
Han H, Kursula P. The olfactomedin domain
from gliomedin is a beta-propeller with unique
structural properties. J Biol Chem 290:36123621, 2015.
Onwukwe GU, Kursula P, Koski MK, Schmitz
W, Wierenga RK. Human Δ3, Δ2-enoyl-CoA
isomerase, type-2: a structural enzymology
study on the catalytic role of its ACBP-domain
and helix-10. FEBS J 282:746-768, 2015.
Laulumaa S, Blakeley MP, Raasakka A, Moulin
Chukhlieb M, Raasakka A, Ruskamo S, Kursula P. The N-terminal cytoplasmic domain of
neuregulin 1 type III is intrinsically disordered.
Amino Acids 47:1567-1577, 2015.
Freischmidt A, Wieland T, Richter B, Ruf W,
Schaeffer V, Müller K, Marroquin N, Nordin F, Hübers A, Weydt P, Pinto S, Press R,
Millecamps S, Molko N, Bernard E, Desnuelle
C, Soriani MH, Dorst J, Graf E, Nordström U,
Feiler MS, Putz S, Böckers TM, Meyer T,
Winkler AS, Winkelman J, de Carvalho M, Thal
DR, Otto M, Brännström T, Volk AE, Kursula
P, Danzer KM, Lichtner P, Dikic I, Meitinger T,
Ludolph AC, Strom TM, Andersen PM,
Weishaupt JH. Haploinsufficiency of TBK1
causes familial amyotrophic lateral sclerosis
and fronto-temporal dementia. Nat Neurosci
18:631-636, 2015.
M, Härtlein M, Kursula P. Production, crystallization, and neutron diffraction of fully deuterated human myelin peripheral membrane protein
P2. Acta Cryst F Struct Biol Commun 71:13911395, 2015.
Piirainen H, Hellman M, Tossavainen H, Permi
P, Kursula P, Jaakola VP. Human adenosine
A2A receptor binds calmodulin with high affinity in a calcium-dependent manner. Biophys J
108:903-917, 2015.
Laulumaa S, Kursula P, Natali F. Neutron scattering studies on protein dynamics using the
human myelin peripheral membrane protein P2.
EPJ Web of Conferences 83:02010, 2015.
Raasakka A, Myllykoski M, Laulumaa S, Lehtimäki M, Härtlein M, Moulin M, Kursula I,
Kursula P. Determinants of ligand binding and
catalytic activity in the myelin enzyme 2’,3’-cyclic nucleotide 3’-phosphodiesterase. Sci Rep
5:16520, 2015. Laulumaa S, Nieminen T, Lehtimäki M,
Aggarwal S, Simons M, Koza MM, Vattulainen
I, Kursula P*, Natali F* (*joint senior authors).
Dynamics of the peripheral membrane protein
P2 from human myelin measured by neutron
scattering – a comparison between wildtype protein and a hinge mutant. PLoS ONE
10:e0128954, 2015.
Myllykoski M, Seidel L, Muruganandam G,
Raasakka A, Torda AE, Kursula P. Structural
and functional evolution of 2´,3´-cyclic nucleotide 3´-phosphodiesterase. Brain Res, doi:
10.1016/j.brainres.2015.09.004, 2015, Epub
ahead of print.
From left sitting: Raisa Serpi, Anna Laitakari, Ann-Helen Rosendahl, Riikka Pietilä, Anu Laitala, Irina Raykhel, Peppi Karppinen, Mia Raasakka, Kari Kivirikko, Johanna Myllyharju. From left standing: Minna Siurua, Eeva Lehtimäki, Riitta Polojärvi, Tanja Aatsinki, Anne Kokko, Raija Salmu, Kati Drushinin, Teemu Ollonen, Antti Railo, Nadiya Byts,
Karim Ullah, Joni Mäki, Antti Salo, Tuomas Laukka, Mikko Myllykangas, Fazeh Moafi.
PROJECT LEADER
Prof. Johanna Myllyharju, Ph.D.
Oulu Center for Cell-Matrix Research,
University of Oulu
KEY ENZYMES IN THE SYNTHESIS
OF COLLAGENS AND THE RESPONSE
OF CELLS TO HYPOXIA
ABSTRACT
We are studying enzymes that are necessary for two important biological processes, 1) synthesis of the major extracellular matrix (ECM) proteins, collagens
and 2) regulation of the hypoxia response. Detailed information on the specific
in vivo roles of these enzymes in normal development and in pathological conditions will have a major impact in the utilization of these enzymes as drug
development targets to treat diseases characterized by abnormal ECM synthesis
(e.g. fibrosis) and insufficient O2 levels (e.g. anaemia, ischaemic diseases). To
achieve this, we use a wide repertoire of methods ranging from analysis of the
catalytic and inhibitory properties of recombinant enzymes, to cell biology and
animal models.
RESEARCH PROJECTS
RESEARCH PROJECTS
Figure 1. Collagen synthesis.
Collagen synthesis involves many co-translational and
post-translational modifications that require at least eight
specific enzymes with multiple isoenzymes (Figure 1). We are
focusing especially on three of them, the collagen prolyl 4-hydroxylases (C-P4Hs), lysyl hydroxylases (LHs) and lysyl oxidases. 4-Hydroxyproline generated by the C-P4Hs is essential
for the formation of stable triple-helical collagen molecules
and C-P4Hs are thus regarded as potential targets for the development of drugs that inhibit collagen formation in fibrotic
diseases. All vertebrate C-P4Hs are a2b2 tetramers in which
the b subunit is identical to the enzyme and chaperone protein
disulphide isomerase.
Hypoxia-inducible transcription factor (HIF), an αβ dimer,
is the key inducer of ~ 300 hypoxia-responsive genes involved
in erythropoiesis, angiogenesis and metabolism, for example.
HIF is accumulated in hypoxic conditions, whereas it is rapidly degraded in normoxic cells. The oxygen-sensing mechanism
behind this phenomenon is provided by HIF prolyl 4-hydroxylases (HIF-P4Hs) (Figure 2). Hypoxia inhibits HIF-P4Hs,
leading to stabilization of HIF-a and formation of the active
HIF dimer (Figure 2). Like collagen hydroxylases, the HIFP4Hs belong to the family of 2-oxoglutarate-dependent dioxygenases, which, in addition to oxygen, require iron, 2-oxoglutarate and ascorbate.
Figure 2. Oxygen-dependent regulation of the stability and transcriptional
activity of HIF.
Detailed information on the specific mechanistic and in vivo
roles of these enzymes will have a direct translational impact
in drug development for pathological conditions characterized by fibrosis, severe anaemias and ischaemic conditions,
for example. Clinical trials on the use of a small-molecule
HIF-P4H inhibitor to treat anaemia in patients with chronic
kidney disease are currently in progress by our collaborator
FibroGen Inc.
RECENT PROGRESS
One of our major approaches to understand the individual
roles of the above enzymes is to utilize gene-modified mouse
lines that we have generated.
We have produced knockout mice for C-P4Hs I and II (all our
knockout mouse work is carried out in collaboration with Dr.
Raija Soininen, Biocenter Oulu). The C-P4H-I-/- embryos
died after E10.5, showing an overall developmental delay and
abnormal basement membrane (BM) assembly. C-P4H-II is
the major C-P4H isoenzyme in chondrocytes, but unexpectedly, C-P4H-II null mice do not have any obvious abnormalities in skeletogenesis (collaboration with Prof. E. Schipani,
University of Michigan). In contrast, C-P4H-I+/-;C-P4H-II-/mutant mice were smaller than their littermates, had moderate chondrodysplasia and developed kyphosis. A transient
inner cell death phenotype was detected in their developing
growth plates. The columnar arrangement of proliferative
chondrocytes was impaired, the amount of 4Hyp and the Tm
of collagen II were reduced and the extracellular matrix was
softer in the growth plates of newborn C-P4H-I+/-;C-P4HII-/-mice. Interestingly, no signs of uncompensated ER stress
were detected in the mutant growth plate chondrocytes. Our
data thus show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone
development, but their combined partial and complete inactivation, respectively, leads to moderate chondrodysplasia and
kyphosis. Our mouse data also explain why no human skeletal
disorders have been associated with mutations in the human
C-P4H-I and C-P4H-II genes, as simultaneous mutations in
both genes would most probably be required to generate a disease phenotype.
The main lysyl oxidase (LOX) has an important role in the
synthesis of extracellular matrix, as it cross-links collagens and
elastin. We have previously shown that LOX null mice die perinatally due to cardiovascular dysfunction, aortic aneurysms
and impaired lung development. In collaboration with Dr. P.
Hasson, Israel Institute of Technology, we have shown that
muscle composition is regulated by a LOX-TGFβ feedback
loop. We found that deletion of LOX uncouples the balance
between the amount of myofibers and that of muscle connective tissue (MCT). LOX secreted from the myofibers attenuated TGFβ signaling, an inhibitor of myofiber differentiation
and promoter of MCT development. We further demonstrated that a LOX-TGFβ feedback loop between the MCT and
myofibers maintains the dynamic developmental homeostasis
between muscle components while also regulating MCT organization. Our results allow a better understanding of diseases such as Duchenne Muscular Dystrophy where LOX and
TGFβ signaling have been implicated and balance between
muscle constituents is disturbed.
HIF-P4H-2 is the major regulator of HIF. We have previously
shown that HIF-P4H-2 hypomorph (HIF-P4H-2gt/gt) mice
are protected against cardiac ischemia-reperfusion and infarct
injuries, they have improved glucose and lipid metabolism
and are protected against obesity and metabolic dysfunction.
In addition, we found out that HIF-P4H-2gt/gt mice are protected against skeletal muscle ischemia-reperfusion injury. The
mechanisms involved were mediated via normoxic HIF1 and
HIF2 stabilization, increased capillary size but not number in
the muscle and improved maintenance of skeletal energy metabolism during ischemia-reperfusion. Furthermore, we have
studied the effect of pharmacologic and genetic HIF-P4H-2
inhibition on the development of atherosclerosis. We utilized
low-density lipoprotein receptor (LDLR)-/- mice treated with
a HIF-P4H inhibitor, FG-4497, and HIF-P4H-2 gt/gt/LDLRC669Y double mutant mice, all fed a high-fat diet (HFD).
FG-4497 administration to LDLR-/- mice reduced the area of
atherosclerotic plaques by about 50% and also reduced the
weight gain, insulin resistance, liver, and white adipose tissue
weights, adipocyte size, number of inflammation-associated
WAT macrophage aggregates and the HFD-induced increases
in serum cholesterol levels in these mice. The levels of certain
atherosclerosis-protecting circulating autoantibodies were increased and the inhibitor treatment stabilized HIF1 and HIF2
and altered the expression of several glucose and lipid metabolism and inflammation-associated genes in liver and WAT.
The HIF-P4H-2 gt/gt/LDLRC669Y mice likewise had about 50%
reduction in the atherosclerotic plaque areas, reduced WAT
macrophage aggregate numbers, and increased autoantibodies
against oxidized LDL, but did not have reduced serum cholesterol levels. These data indicate that HIF-P4H-2 inhibition
may be as a novel strategy for protection against development
of atherosclerosis.
FUTURE GOALS
Our goal is to understand the individual in vivo roles of the
key enzymes that regulate collagen synthesis and hypoxia response. Detailed information on these enzymes will have a major impact in furthering our understanding of cell–extracellular
matrix interplay and how cells and tissues survive and respond
to situations of acute or chronic hypoxia. These findings will
be valuable in the development of therapeutics against several
common diseases such as fibrosis, anaemia, ischaemia and metabolic disorders.
DOCTORAL THESES 2015
Lea Rahtu-Korpela: Hypoxia-inducible factor prolyl 4-hydroxylase-2 in glucose and lipid metabolism and atherosclerosis. Faculty of Biochemistry and Molecular Medicine, University of Oulu, FBMM 005, ISBN 978-952-93-5161-9
Sara Karsikas: Hypoxia-inducible factor prolyl 4-hydroxylase-2 in cardiac and skeletal muscle ischemia and metabolism.
Acta Universitatis Ouluensis, Series D, Medica 1288, ISBN
978-952-62-0780-3
Project Leader:
Johanna Myllyharju, Ph.D., Professor
(University of Oulu and Biocenter Oulu)
Kari I. Kivirikko, M.D., Ph.D., Professor
(Academy of Finland, emeritus)
Peppi Karppinen (née Koivunen), M.D., Ph.D., Professor
Nadiya Byts, Ph.D. (Academy of Finland)
Elitza Dimova, Ph.D. (Jane & Aatos Erkko Foundation)
Minna Komu, Ph.D. (Academy of Finland)
Anu Laitala, Ph.D. (Academy of Finland)
Joni Mäki, Ph.D. (University of Oulu)
Antti Railo, Ph.D. (Academy of Finland)
Irina Raykhel, Ph.D.
(Biocenter Oulu and Academy of Finland)
Antti Salo, Ph.D. (Biocenter Oulu)
Raisa Serpi, Ph.D. (Aaltonen Foundation),
leave of absence until May
Ph.D. Students:
Kati Drushinin, M.Sc.
(Biocenter Oulu Doctoral Programme)
Sara Karsikas, M.D. (University of Oulu, Biocenter Oulu
Doctoral Programme and Academy of Finland), until June
Anna Laitakari, M.Sc. (Jane & Aatos Erkko Foundation)
from February
Tuomas Laukka, Med.Cand.
(Sigrid Jusélius Foundation and University of Oulu)
Fazeh Moafi, M.Sc. (Biocenter Oulu Doctoral Programme)
Mikko Myllymäki, Med.Cand.
Jenni Määttä, Med.Cand.
Teemu Ollonen, Odont.Stud. (Sigrid Jusélius Foundation
and University of Oulu), from May
Lea Rahtu-Korpela, M.Sc.
(Biocenter Oulu Doctoral Programme), until June
Mia Raasakka, Med.Cand. (Sigrid Jusélius Foundation
and University of Oulu) from May
Ann-Helen Rosendahl, Med.Cand.
RESEARCH PROJECTS
Jussi-Pekka Tolonen, Med.Cand.
(Academy of Finland and University of Oulu)
Karim Ullah, M.Sc. (Academy of Finland and
Biocenter Oulu Doctoral Programme)
Laboratory Technicians, 6 (University of Oulu, Biocenter
Oulu, Academy of Finland and FibroGen Inc.)
Centre of Excellence in Cell-Extracellular Matrix Research,
Academy of Finland Program for 2012–2017
Taina Pihlajaniemi, Director, Johanna Myllyharju, Vice
director, other Group leaders Lauri Eklund, Aki Manninen,
Seppo Vainio and Robert Winqvist
Kari I. Kivirikko, M.D., Ph.D.: FibroGen Inc.,
San Francisco, CA, USA
Johanna Myllyharju, Ph.D.: University of Ulm, Ulm,
Germany
Co-operation With Finnish and Foreign Companies
FibroGen Inc., San Francisco, CA, USA
FibroGen Europe, Helsinki, Finland
Aro E, Salo AM, Khatri R, Finnilä M, Miinalainen I, Sormunen R, Pakkanen O,Holster T,
Soininen R, Prein C, Clausen-Schaumann H,
Aszódi A, Tuukkanen J, Kivirikko KI, Schipani
E, Myllyharju J. Severe Extracellular Matrix
Abnormalities and Chondrodysplasia in Mice
Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme IIin Combination with a Reduced Amount
of Isoenzyme I. J Biol Chem 290(27):16964-78,
2015.
Deschoemaeker S, Di Conza G, Lilla S, MartínPérez R, Mennerich D, Boon L, Hendrikx S,
Maddocks OD, Marx C, Radhakrishnan P, Prenen H, Schneider M, Myllyharju J, Kietzmann
T, Vousden KH, Zanivan S, Mazzone M. PHD1
regulates p53-mediated colorectal cancer
chemoresistance. EMBO Mol Med 7(10):135065, 2015.
Komulainen T, Lodge T, Hinttala R, Bolszak M,
Pietilä M, Koivunen P, Hakkola J, Poulton J,
Morten KJ, Uusimaa J. Sodium valproate induces mitochondrial respiration dysfunction in
HepG2 in vitro cell model. Toxicology 331:4756, 2015.
Kutchuk L, Laitala A, Soueid-Bomgarten S,
Shentzer P, Rosendahl AH, Eilot S, Grossman
M, Sagi I, Sormunen R, Myllyharju J, Mäki
JM, Hasson P. Muscle composition is regulated by a Lox-TGFb feedback loop. Development
142(5):983-93, 2015.
KY, Myllyharju J, Vainio SJ. Wnt5a Deficiency
Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and
Basement Membrane Integrity Pointing to a Role
in Kidney Collecting Duct Patterning. PloS ONE,
11(1):e0147171, 2016.
Myllyharju J. Collagen Hydroxylases. In: 2-Oxoglutarate-Dependent Dioxygenases. RSC Metallobiology Series No. 3., p. 149-168. Editors
Hausinger RP , Schofield CJ. The Royal Society
of Chemistry, 2015.
Karsikas S, Myllymäki M, Heikkilä M, Sormunen
R, Kivirikko KI, Myllyharju J, Serpi R, Koivunen
P. HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury. J Mol
Med (Berl), 2015, Epub ahead of print.
Siegert I, Schödel J, Nairz M, Schatz V, Dettmer K, Dick C, Kalucka J, Franke K, Ehrenschwender M, Schley G, Beneke A, Sutter J,
Moll M, Hellerbrand C, Wielockx B, Katschinski
DM, Lang R, Galy B, Hentze MW, Koivunen
P, Oefner PJ, Bogdan C, Weiss G, Willam C,
Jantsch J. Ferritin-mediated iron sequestration
stabilizes hypoxia-inducible factor-1a upon LPS
activation in the presence of ample oxygen. Cell
Rep 13:2048-2055, 2015.
Laukka T, Mariani CJ, Ihantola T, Cao JZ, Hokkanen J, Kaelin WG Jr, Godley LA, Koivunen
P. Fumarate and succinate regulate expression
of hypoxia-inducible genes via TET enzymes.
J Biol Chem 2016, pii: jbc.M115.688762, Epub
ahead of print.
Pietilä I, Prunskaite-Hyyryläinen R, Kaisto S, Nicolaou N , van Eerde AM , Salo AM,
Garma L, Miinalainen I, Feitz WF, Bongers
EMHF, Juffer AH, Knoers NVAM, Renkema
Rahtu-Korpela L, Määttä J, Dimova EY, Hörkkö S, Gylling H, Walkinshaw G, Hakkola J, Kivirikko KI, Myllyharju J, Serpi R, Koivunen P.
Hypoxia-inducible factor prolyl 4-hydroxylase-2
inhibition protects against development of atherosclerosis. Arterioscler Thromb Vasc Biol pii:
ATVBAHA.115.307136, Epub ahead of print.
By the railing from left: Jaana Peters, Lauri Eklund, Sabrina Santoleri, Hongmin Tu, Sirkka Vilmi, Anne Heikkinen, Riitta Jokela, Valerio Izzi, Marjut Nätynki, Minna Kihlström,
Mia Rinta-Jaskari, Sanna Karppinen, Heli Ruotsalainen. From left: Inderjeet Kaur, Ritva Heljasvaara, Taina Pihlajaniemi, Riku Kallunki, Tiina Petäjistö, Juho Lakkala, Zarin
Zainul, Aila White, Heli Härönen, Charlotta Henriksson, Maija Seppänen, Päivi Tuomaala, Jaakko Kangas, Jaana Träskelin, Raman Devarajan, Jarkko Koivunen, Harri Elamaa.
PROJECT LEADER
Prof. Taina Pihlajaniemi, M.D., Ph.D.
University of Oulu
CONSERVED COLLAGENS
IN CELL–MATRIX HOMEOSTASIS
ABSTRACT
The extracellular matrix (ECM) provides structural support and guidance cues
for cells, and is centrally involved in dynamic processes in organogenesis, organ
function and tissue repair. We investigate the structural and regulatory roles
of collagens, a key group of ECM proteins in the pericellular environment, in
supporting homeostasis and function of cells and tissues. We also study the
mechanisms whereby vascular endothelial cells interact with the ECM, and aim
for better understanding of pathological mechanisms responsible for vascular
anomalies, especially focusing on Tie/angiopoietin signalling system. The project
is part of the Finnish Centre of Excellence Programme funded by the Academy
of Finland.
RESEARCH PROJECTS
RESEARCH PROJECTS
We address the physiological and pathological significance
of two evolutionarily conserved subgroups of collagens, the
basement membrane (BM) collagens XV and XVIII forming
the multiplexin subfamily, and the structurally homologous
collagens XIII and XXIII, which belong to the subfamily of
transmembrane collagens. These collagens have important
regulatory roles by virtue of affecting the structure and properties of the ECM, and by binding growth factors and cellular
receptors, and they are associated with malignant processes.
Studies on our unique mouse models have led to identification of collagen XIII as a novel muscle-derived regulator of
motor synapse differentiation, maturation and functional efficacy. Recently the first human disease was identified with
mutations in COL13A1 – congenital myasthenic syndrome
type 19 with impaired neuromuscular transmission (Logan et
al., Am. J. Human Genetics, 2015). Our mouse models also
suggest functional roles for collagen XIII in bone, skin and the
cardiovascular system.
We have shown that genetic inactivation of Col15a1 or Col18a1 affects the integrity of several organs and tissues such
as the eyes, brain ventricles, skeletal muscle, and heart and
blood vessels. Mutations in COL18A1 are known to lead to
Knobloch syndrome, characterized by severe ocular abnormalities and in some cases also a spectrum of other defects. Collagen XVIII has also gained much attention on account of its
anti-angiogenic and anti-tumourigenic C-terminal endostatin
domain.
Structural and functional alterations in the vasculature contribute to many common human diseases and certain inherited and sporadic mutations in genes regulating blood vessel
morphogenesis cause vascular anomalies. Angiopoietin growth
factors (Ang1–4) and tyrosine kinase receptors Tie1 and Tie2
form an endothelial signalling system. We have found that
Tie2 shows unique, ligand-mediated translocation into endothelial cell (EC)–ECM contact sites. Disturbed Ang/Tie
signalling in cultured cells and in vasculature causes changes
in EC–ECM interactions and in the structure of the perivascular ECM, suggesting important roles for Ang/Tie signalling
in regulating the vasculature via the EC–ECM axis.
RECENT PROGRESS
We have investigated the evolution and conserved properties
of the unique subgroup of transmembrane collagens XIII,
XXIII and XXV, and we have named them MACITs (membrane-associated collagens with interrupted triple-helices) to
distinguish them from the structurally different transmembrane collagen type XVII. Through identification of MACITs
in multiple metazoan phyla and in collaboration with Prof.
Josephine Adams, University of Bristol, UK, we developed a
model for the evolution of MACITs. Invertebrates were shown
to encode a single MACIT, which went through two rounds
of en-bloc genome duplication in early vertebrate evolution
to generate the collagen XIII/XXIII/XV gene family. MACITs were shown to have conserved domain architectures at a
plasma membrane-adjacent furin-cleavage site and at the short
C-terminal part, but the N-terminal cytoplasmic domain is
only weakly conserved between species. We also demonstrated
that the C. elegans MACIT, COL-99, is plasma membrane-associated, undergoes furin-dependent ectodomain cleavage and
shedding, and is present mostly in motor neurons and muscle
of the body wall, tail, pharynx and mouth, thus corroborating
the conservation of MACIT’s molecular and cellular properties and tissue localization from invertebrates to mammals.
Our recent work has also resulted in the development of light
microscopic, tissue culture and image analysis methods to
monitor cells in 3D cultures mimicking tissue environments
and for quantitative analysis of tumour and stroma interactions using computer vision and computer learning-based
analysis methods in collaboration with Prof. Janne Heikkilä
(University of Oulu) .
Collagen XVIII is expressed under two promoters, resulting
in three isoforms that differ in their N-terminal regions and
expression profiles. Using our mutant mice lacking either promoter 1- or 2-derived transcription, we have begun to unravel
the significance of the isoforms in different tissues and biological processes. We have previously shown that the short isoform is critical for retinal angiogenesis and the BM of kidney
tubules, and that the medium and long isoforms determine
the number of adipocyte precursors committing to adipocyte
differentiation.
FUTURE GOALS
We have studied the expression and roles of collagens XV
and XVIII in the skin and skin cancer of humans and mice,
and identified them as potential biomarkers in cutaneous
squamous cell carcinoma (cSCC). We have shown that while
collagen XVIII expression is significantly up-regulated in malignant human cSCC cells in vitro and in vivo in comparison
with normal keratinocytes, collagen XV appears as deposits
in the tumour stroma, in particular in the vascular BM zone.
Interestingly, collagen XVIII – but not collagen XV or other
common BM proteins collagen IV and laminin – is selectively
reduced in tumour vasculature and this reduction is associated
with cancer progression. Our data suggest that the two homologous BM collagens may contribute in a distinct manner
to processes related to cSCC tumourigenesis. In collaboration
with Prof. Tuula Salo’s group, University of Oulu, we have
studied collagen XVIII-derived endostatin in oral SCC, and
showed that endostatin may directly promote SCC proliferation, but its effects on cancer cell invasion are modulated by
the tumour microenvironment. In collaboration with Prof.
Veli-Matti Kähäri, University of Turku, we have reported that
the receptor tyrosine kinase ephrin B2 promotes progression
of cSCC by inducing expression of several genes associated
with tumour cells’ viability, migration and invasion, including collagen-degrading matrix metalloproteinases MMP1 and
MMP13.
Together with Prof. Miikka Vikkula’s group (de Duve Institute,
Brussels, Belgium) we have made breakthroughs in unravelling
genetic, molecular and cellular alterations that characterize a
large proportion of venous malformations (VMs). More than
50% of VMs are positive for mutations in TIE2 and our recent
analysis of a comprehensive collection of 22 TIE2 VM mutations in cultured ECs and in a novel VM mouse model revealed
cellular and molecular features that separate VM ECs from
normal endothelium. Our studies also led to identification of
mutations in PIK3CA encoding the p110a catalytic subunit of
PI3K in a significant proportion of TIE2 mutation-negative
VMs. Importantly, recurrent mutations in TIE2 and PIK3CA
resulted the same cellular abnormalities in ECs, indicating that
the TIE2 receptor and the PIK3CA signal transducer participate in the same VM signalling pathway. Identification of
PI3K/Akt signalling in VMs resulted in a pilot clinical study
using the first molecular therapy against VMs.
We will expand the current view of multiplexins to entirely
new functions with high medical relevance and potential for
novel therapies. In the case of collagen XVIII this includes unravelling its isoform-specific roles and mechanisms of action
in bone marrow, skin and adipose tissue stem cell microenvironments, and in the case of collagen XV defining its roles
in mesenchymal stem cells and in bone. We will pursue the
roles of collagen XIII in the motor synapse, the cardiovascular
system, and in bone formation and properties.
We will assess the significance of multiplexins and MACITs
in epithelial cancers by using mutant mice as experimental
breast and skin cancer models, and study their mechanisms
of action in cultured tumour cells. The medical significance
of cell–ECM homeostasis will be addressed in terms of target
identification and validation in order to develop new diagnostic, prognostic and therapeutic measures. We will characterize
mechanistic details of how Tie receptor signalling and angiopoietin ligands regulate cell–ECM interplay in the vasculature. Furthermore, we aim to better characterize the altered
Tie2/PI3K signalling pathway responsible for VMs and other
venous and lymphatic diseases, and to further develop advanced microscopic imaging and image analysis methodology
in cell and tissue studies.
Ann-Marie Auvinen, Modeling the roles of collagen XIII in
cardiac integrity and function, and in cutaneous wound healing. ISBN 978-952-93-6348-3.
Riikka Pietilä, Angiopoietin-1 and -2 regulated Tie2 receptor
translocation in endothelial cells and investigation of angiopoietin-2 splice variant 443. Acta Universitatis Ouluensis Series D, Medica 1295, 2015. ISBN 978-952-62-0796-4.
Project Leader:
Taina Pihlajaniemi, M.D., Ph.D., Professor, Vice President
(Vice Rector) Provost for Science and Research
Lauri Eklund, Ph.D. (Academy of Finland,
University of Oulu and Biocenter Oulu)
Harri Elamaa, Ph.D. (Academy of Finland)
Anne Heikkinen, Ph.D. (Biocenter Oulu)
Ritva Heljasvaara, Ph.D. (University of Oulu)
Valerio Izzi, Ph.D.
Mika Kaakinen, Ph.D. (Academy of Finland)
Sanna-Maria Karppinen, Ph.D. (Academy of Finland)
Jarkko Koivunen, Ph.D. (Biocenter Oulu,
50% September–December 2015)
Heli Ruotsalainen, Ph.D. (Academy of Finland,
University of Oulu and Finnish Cultural Foundation)
Sabrina Santoleri, Ph.D. (Academy of Finland)
Hongmin Tu, Ph.D. (Biocenter Oulu)
David Vicente, D.V.M., Ph.D. (Academy of Finland),
until February 2015
Ph.D. Students:
Miki Aho, Med.Cand., leave of absence
Ann-Marie Auvinen, M.D.
Raman Devarajan, M.Sc. (Academy of Finland)
Charlotta Henriksson, Med. Stud. (University of Oulu)
Heli Härönen, Med. Cand.
Riku Kallunki, Med. Cand.
(University of Oulu and Sigrid Jusélius Foundation)
Jaakko Kangas, M.D.
Inderjeet Kaur, Ph.D. (chemistry) (Biocenter Oulu)
Antti Kemppainen, Med.Cand.
Minna Kihlström, M.Sc. (Adacemy of Finland)
Juho Lakkala, Med. Cand.
(University of Oulu and Sigrid Jusélius Foundation)
Guillermo Martinez-Nieto, M.Sc. (CAFFEIN,
EU Marie Curie Actions – Initial Training Networks)
Marjut Nätynki, M.Sc. (Finnish Cultural Foundation)
Tiina Petäistö, M.Sc.
(Sigrid Jusélius Foundation, University of Oulu)
Riikka Pietilä, M.Sc. (Academy of Finland)
Mia Rinta-Jaskari, Med. Cand.
Zarin Zainul, M.Sc. (Biocenter Oulu)
Saad Ullah Akram, M.Sc.
(Biocenter Oulu Doctoral Programme)
Laboratory Technicians, 6
(University of Oulu and Academy of Finland)
Taina Pihlajaniemi, Director, Johanna Myllyharju, Vice director, other Group leaders Lauri Eklund, Aki Manninen, Seppo
Vainio and Robert Winqvist
Ph.D. Mari Aikio, Harvard Medical School, USA
Ph.D. Mika Kaakinen, University of Queensland, Brisbane,
Australia
RESEARCH PROJECTS
M.Sc. Hengshuo Liu, University of Bergen, Norway
EU Projects
EC Infrafrontier-I3, FP-7-Infrafrontier-2012, grant for
2013–2016. Partner.
Initial training network “Cancer associated fibroblasts (CAF)
function in tumor expansion and invasion”. FP7 grant no.
316610. 2012–2016. Partner.
NordForsk Projects
Researcher network project “Nordic Infrastructure in Mouse
Models (NorIMM)” NordForsk project no. 69005, 2014–
2016. Coordinator Taina Pihlajaniemi.
Researcher network project “Bridging Nordic Imaging – Enabling Discoveries from Atoms to Anatomy”. 2014–2016.
Partner Lauri Eklund.
Co-operation with Finnish and Foreign Companies
Paras Biopharmaceuticals Finland Oy, Oulu, assistance in developing diagnostics and therapeutic products
Biocenter Publications:
Alahuhta I, Aikio M, Väyrynen O, Nurmenniemi
S, Suojanen J, Pihlajaniemi T, Heljasvaara R,
Salo T, Nyberg P. Endostatin induces proliferation of oral carcinoma cells but its effect on
invasion is modified by the tumor microenvironment. Exp Cell Res 336:130-140, 2015.
Bayramoglu N, Kannala J, Åkerfelt M, Kaakinen
M, Eklund L, Nees M, Heikkilä J. A novel feature descriptor based on image statistics. IEEE
International Conference on Image Processing
(ICIP) 2015.
Boscolo E, Limaye N, Huang L, Kang KT, Soblet
J, Uebelhoer M, Mendola A, Nätynki M, Seront
E, Dupont S, Hammer J, Legrand C, Brugnara
C, Eklund L, Vikkula M, Bischoff J, Boon LM.
Rapamycin improves TIE2-mutated venous
malformation in murine model and human subjects. J Clin Invest 125:3491-3504, 2015.
Farshchian M, Nissinen L, Siljamäki E, Riihilä
P, Toriseva M, Kivisaari A, Ala-aho R, Kallajoki
M, Veräjänkorva E, Honkanen HK, Heljasvaara
R, Pihlajaniemi T, Grénman R, Peltonen J, Peltonen S, Kähäri VM. EphB2 promotes progression of cutaneous squamous cell carcinoma. J
Invest Dermatol 135:1882-1892, 2015.
Fuoco C, Rizzi R, Biondo A, Longa E, Mascaro
A, Shapira-Schweitzer K, Kossovar O, Benedetti S, Salvatori ML, Santoleri S , Testa S, Bernardini S, Bottinelli R, Bearzi C, Cannata SM,
Seliktar D, Cossu G, Gargioli C. In vivo generation of a mature and functional artificial skeletal
muscle. EMBO Mol Med 7:411-22, 2015.
Hakanpää L, Sipilä T, Leppänen VM, Gautam P,
Nurmi H, Jacquemet G, Eklund L, Ivaska J, Alitalo K, Saharinen P. Endothelial destabilization
by angiopoietin-2 via integrin b1 activation. Nat
Commun 6:5962, 2015.
Limaye N, Kangas J, Mendola A, Godfraind C,
Schlögel MJ, Helaers R, Eklund L, Boon ML,
Vikkula M. Somatic activating PIK3CA mutations cause venous malformation. Am J Hum
Genet 97:914-921, 2015.
Nkizinkiko Y, Suneel Kumar BV, Jeankumar
VU, Haikarainen T, Koivunen J, Madhuri C,
Yogeeswari P, Venkannagari H, Obaji E, Pihlajaniemi T, Sriram D, Lehtiö L. Discovery of
potent and selective nonplanar tankyrase inhibiting nicotinamide mimics. Bioorg Med Chem
23:4139-4149, 2015.
Nätynki M, Kangas J, Miinalainen I, Sormunen
R, Pietilä R, Soblet J, Boon LM, Vikkula M,
Limaye N, Eklund L. Common and specific effects of TIE2 mutations causing venous malformations. Hum Mol Genet 24:6374-6389, 2015.
Richter K, Konzack A, Pihlajaniemi T, Heljasvaara R, Kietzmann T. Redox-fibrosis: Impact
of TGFb1 on ROS generators, mediators and
functional consequences. Redox Biol 28:344352, 2015.
Salo T, Sutinen M, Sundquist E, Cervigne NK,
de Oliveira CE, Akram SU, Ohlmeier S, Apu
HE, Suomi F, Eklund L, Juusela P, Åström P,
Bitu CC, Korvala J, Coletta RD. A Novel Human
Leiomyoma Tissue Derived Matrix for Cell Culture Studies. BMC Cancer 15:981, 2015.
Tu H, Huhtala P, Lee H-M, Adams JC, Pihlajaniemi T. Membrane-associated collagens with
interrupted triple-helices (MACITs): evolution
from a bilaterian common ancestor and functional conservation in C. elegans. BMC Evol Biol
15:281, 2015.
Åkerfelt M, Bayramoglu N, Robinson S, Toriseva M, Schukov H-P, Härmä V, Virtanen J,
Sormunen R, Kaakinen M, Kannala J, Eklund
L, Heikkilä J, Nees M. Automated tracking of
tumor-stroma morphology in microtissues
identifies functional targets within the tumor
microenvironment for therapeutic intervention.
Oncotarget 6:30035-30056, 2015.
Karppinen SM, Honkanen HK, Heljasvaara R,
Riihilä P, Autio-Harmainen H, Sormunen R,
Harjunen V, Väisänen M-R, Väisänen T, Hurskainen T, Tasanen-Määttä K, Kähäri V-M, Pihlajaniemi T. Collagens XV and XVIII show different expression and localization in cutaneous
squamous cell carcinoma: type XV appears in
tumour stroma while XVIII becomes up-regulated in tumour cells and lost from microvessels. Exp Dermatol, doi: 10.1111/exd.12913, Epub
ahead of print.
Other Publications:
Bárcena C, Stefanovic M, Tutusaus A, Martinez-Nieto GA, Martinez L, García-Ruiz C, de
Mingo A, Caballeria J, Fernandez-Checa JC,
Mari M, Morales A. Angiogenin secretion from
hepatoma cells activates hepatic stellate cells
to amplify a self-sustained cycle promoting liver
cancer. Sci Rep 5:7916, 2015.
In front: Antti Moilanen, Zhang Chi, Jiro Ogura. From left: Kati Korhonen, Ekaterina Biterova, Mirva Saaranen, Yuko Ushida, Heli Alanen, Lisette van Tassel, Lloyd Ruddock,
Johanna Veijola, Anna Gaciarz.
PROJECT LEADER
Prof. Lloyd Ruddock, Ph.D.
University of Oulu
MECHANISMS AND APPLICATIONS
OF DISULFIDE BOND FORMATION
ABSTRACT
There are hundreds of types of protein post-translational modification (PTM),
which alter protein structure, function, stability, etc. In eukaryotes, two of the
most common PTMs occur in the endoplasmic reticulum; disulfide bond formation and N-glycosylation. Disulfide bond formation is a complex, essential, multi-factorial process, which is often the rate limiting step in protein folding. The
major limitation in expansion of protein based therapeutics and analytics is protein production on an industrial scale, especially the generation of homogenous
disulfide bonded proteins. The same limitation severely inhibits academic studies
into a very wide range of biological processes and associated disease states.
RESEARCH PROJECTS
RESEARCH PROJECTS
One major limitation in the study of protein structure-function relationships is the production and purification of homogenously folded proteins with appropriate post-translational modifications (PTM). This is also the major limitation
in the effective and economic expansion of the use of protein-based therapeutics and analytics.
250 mg/litre culture. Recent results indicate that this can be
extended into batch and fed-batch fermentation in defined
minimal media even for proteins as complicated as full length
human antibodies, with yields in excess of 0.5g/L of soluble
folded protein being achieved.
Protein folding for both soluble and membrane proteins is
an essential and complex multi-factorial process. Around onethird of all human proteins, including outer membrane and
secreted proteins fold in the endoplasmic reticulum (ER) and
have the added complications associated with disulfide bond
formation. The formation of these covalent bonds is often the
rate limiting step of protein folding in vivo and in vitro.
During this year we have gone back to redesign the fundamentals of the system and in parallel to introduce new factors into
the system – again based on decades of fundamental studies
on the mechanisms of protein folding. New variants that are
more efficient in native disulfide bond formation as well as
variants which aid other rate limiting steps in protein folding have been introduced. These new variants can increase the
yield of folded protein obtained up to 4x. Two proteins have
been produced in fed-batch fermentation in yields exceeding
2.5g/L.
There are two distinct steps for disulfide bond formation, oxidation of two cysteines and subsequent isomerization of the
disulfide bonds formed to the native functional state. Native
disulfide bond formation in the ER can occur via multiple
pathways and this significantly complicates the interpretation
of in vivo data.
Application of CyDisCo
The CyDisCo system was originally developed due to frustration with the inability to produce proteins involved in, or suspected to be involved in, protein folding and quality control
in the ER in sufficient yields to undertake molecular characterization and structural studies.
Due to the complexities of their formation the production of
proteins that contain disulfide bonds is difficult. This severely inhibits scientific progress in understanding a myriad of
mechanistic processes and imposes limitations on the biotechnology industry for the production of therapeutic proteins.
Other PTMs impose similar limitations, for example there are
no methods available for the rapid or large scale production of
homogenous N-glycosylated proteins.
This year one focus has been on increasing the breadth of
proteins tested, both for academically interesting and industrially relevant proteins. Successful production of a range of
scFv and Fab antibody fragments have been achieved in the
cytoplasm, along with human N- and O-glycosyltransferases,
gastrointestinal mucosa proteins, signalling molecules – including growth factors and hormones, proteins involved in
development, angiogenesis and wound healing and a number of disulfide bond containing proteins involved in protein
folding, quality control and ERAD - including high-yields of
homogeneously folded human Ero1a/b.
RECENT PROGRESS
Development of CyDisCo
Based on more than two decades of studies by the PI on understanding the mechanisms for disulfide bond formation we
have developed systems which allow efficient disulfide bond
formation in the cytoplasm of E. coli. Unlike previously published systems, our systems do not require disruption of the
reducing pathways naturally found in the cytoplasm, and they
work in any media and in any E.coli strain tested to date.
Our system, known as CyDisCo (cytoplasmic disulfide bond
formation in E.coli), has a variety of formats, but those most
widely used share the common feature of co- or pre-expression
of a sulfhydryl oxidase and a protein disulfide isomerase i.e.
catalysts of the two steps of native disulfide bond formation.
The first patent has been granted and the system has been
commercialized.
Studies with the CyDisCo system shows that the system is
very successful and high yields of active, correctly folded, eukaryotic proteins can be obtained. CyDisCo can be combined
with other technologies, such as systems that secrete folded
proteins from the cytoplasm and N-glycosylation in the cytoplasm to generate Gen2Co, 2nd generation E.coli cell factories.
The patented variants of the system allow production of homogeneously folded human proteins with multiple disulfide
bonds in E.coli grown in shake flasks with yields of up to
As well as human Ero1a/b and range of other proteins involved in protein folding, quality control and ER-associated
degradation (ERAD) have been made with the aim of increasing mechanistic understanding of the system and then the application of this knowledge to make our production systems
more efficient.
Collaborative studies have commenced on several secreted
proteins for functional and structural studies. During the
past year two crystal structures have been solved for secreted
proteins involved in angiogenesis and in wound healing and
structural studies have commenced on a number of proteins
including perlecan (a major structural protein in the extracellular matrix) as well as proteins playing a role in development,
lung function and a novel chaperone family.
Molecular and structural characterization of these continue to
be a major focus in the coming year.
In parallel to these studies utilizing CyDisCo to understand
the mechanisms of protein folding, we have also continued
work on the mechanisms of action of key enzymes in oxidative protein folding such as PDI. It is known that PDI must
be able to trigger conformational changes in bound non-native protein subtrates to allow access to buried thiols and disulfides. This is an area that has been poorly pursued due to the
extreme difficulty in isolating/identifying intermediates and in
studying the process. We have data from collaborative NMR
studies that conformational exchange we had previously identified within PDI is linked to the ability to trigger change in
the conformation of folding intermediate mimics (produced
using CyDisCo). These studies feed into the development of
the CyDisCo system.
Finally, we have used our knowledge of the mechanisms of
oxidative folding in collaborative studies to elucidate the role
of variants in a PDI-family members in Amyotrophic Lateral
Sclerosis (ALS) and motor dysfunction.
FUTURE GOALS
The overall aim of the group is to provide a complete molecular
description of the processes by which protein folding occurs within the ER and the application of this knowledge for the efficient
production of disulfide bond containing proteins of scientific, medicinal or biotechnological importance.
With the final stages of development of CyDisCo in sight the
primary foci of the group will switch towards:
• The use of CyDisCo to obtain mechanistic understanding
of the pathways and synergy of protein folding, quality control and ER-associated degradation.
• The development of Gen2Co for efficient secretion of disulfide bonded proteins
• The development of systems analogous to CyDisCo for the
production of other PTMs in the cytoplasm of E.coli
Nguyen Van Dat. Mechanisms and Applications of Disulfide
bond formation. Acta Universitatis Ouluensis. D, Medica,
2015, no 1282, ISBN: 978-952-62-0724-4.
Project Leader:
Lloyd Ruddock, Ph.D., Professor
Ekaterina Biterova (Academy of Finland)
Mirva Saaranen, Ph.D.
Johanna Veijola (Biocenter Oulu)
Jiro Ogura (Uehara Memorial Foundation)
Ph.D. Students:
Zhang Chi, M.Sc. (Academy of Finland and Biocenter Oulu)
Anna Gaciarz, M.Sc. (Biocenter Oulu)
Kati Korhonen, M.Sc. (ISB, Academy of Finland)
Antti Moilanen (Academy of Finland)
Lisette Van Tassel, M.Sc. (Biocenter Oulu)
Heli Alanen (University of Kent)
Kati Korhonen (University of Kent)
Co-operation With Finnish and Foreign Companies
Paras Biopharmaceuticals (Oulu, Finland; R&D)
Biosilta Oy (Oulu, Finland; R&D)
PATENTS 2015
US Patent 9238817, Method for producing natively folded
proteins in a prokaryotic host
Alanen HI, Walker KL, Lourdes Velez Suberbie
M, Matos CF, Bönisch S, Freedman RB, et al.
Efficient export of human growth hormone,
interferon a2b and antibody fragments to the
periplasm by the Escherichia coli Tat pathway
in the absence of prior disulfide bond formation.
Biochim Biophys Acta 1854:756-63, 2015.
Psioni GB, Ruddock LW, Bulleid N and Mollinari
M. Division of labor among oxidoreductases:
TMX1 acts preferentially on transmembrane
polypeptides. Mol Biol Cell 26:3390-400, 2015.
Gaciarz A, Veijola J, Uchida Y, Saaranen MJ,
Wang C, Hörkkö S, Ruddock LW. Systematic
screening of soluble expression of antibody
fragments in the cytoplasm of E.coli. Micro Cell
Factories 15:22, 2016.
Woehlbier U, Colombo A, Saaranen MJ, Perez
V, Ojeda J et al. ALS-linked protein disulfide
isomerase variants cause motor dysfunction.
EMBO J pii: e201592224, Epub ahead of print,
2016.
RESEARCH PROJECTS
From left sitting: Toni Karhu, Saranya Palaniswamy, Laura Niiranen, Remi Kamakura, Shivaprakash Jagalur Mutt, Sari Pyrhönen, Tuire Salonurmi, Marja-Leena Kytökangas,
Sylvain Serbet, Kari Mäkelä. From left standing: Karl-Heinz Herzig, Juhani Leppäluoto, Raza Ghulam, Antti Nissinen, Saeid Haghighi, Markku Savolainen.
MOLECULAR DETERMINANTS
OF METABOLIC SYNDROME (MetSyn)
PROJECT LEADERS
Prof. Markku Savolainen, M.D., Ph.D.,
Research Unit of Internal Medicine,
Faculty of Medicine
Prof. Karl-Heinz Herzig, M.D., Ph.D.,
Research Unit of Metabolism, Nutrition and Environment,
Institute of Biomedicine, Faculty of Medicine
Prof. Marjo-Riitta Järvelin, M.D., MSc, Ph.D.,
Centre For Life-Course Health Research,
Oulu University Hospital,
Faculty of Medicine, University of Oulu
Cardiovascular disease (CVD) is the leading cause of death
globally, representing around 30% of all deaths worldwide
and nearly half of all deaths in Europe (WHO Statistics,
2008). Over the past 30 years mortality from CVD has declined steadily in the developed western economies but recent
dramatic increases in the prevalence of obesity, type 2 diabetes (T2D) and metabolic syndrome (MetSyn) are, however,
reversing this positive trend. Multiple factors through the
life-course have been related to the risk of MetSyn (Fig. 1).
Our recent work shows that determinants of early growth and
growth patterns are associated with inflammatory markers and
MetSyn phenotypes. There is increasing evidence that inflammatory mechanisms have an important role in the initiation
and progression of CVDs, and there may be an important link
between CVDs and clustering of MetSyn phenotypes (Fig. 1).
Altogether, our work suggests that systemic low-grade inflammation may lie on the causal pathway involving impaired foetal growth, growth patterns and body mass from childhood,
and adverse cardiometabolic health. However, the disease
mechanisms and its components are still poorly understood.
Consequently, there is urgent need for further exploration of
human life-course data, and for molecular-level studies on the
underlying pathophysiological mechanisms. These approaches
are key to the identification of risk groups, for early disease
prevention and treatment. This work is enormously enhanced
by our recent 46- to 48-year follow-up data collection in the
Northern Finland Birth Cohort (NFBC) 1966, and extended
experimental work.
Our life-course studies on the Northern Finland Birth Cohort
are strongly supportive of the fact that systemic low-grade inflammation may lie on the causal pathway towards MetSyn
(Sovio et al. 2013). Key mechanisms behind obesity-related
disorders include NFkB-dependent production of pre-inflammatory adipokines, Toll-like receptor (TLR) expression,
increased oxidative stress and inflammasome activation.
Macrophages can invade from the blood stream and induce
inflammatory responses through pathogen- or damage-associated molecular patterns (PRRs) (Rathinam et al. 2012).
Inflammatory cells can represent up to 40% of all cells in adipose tissue (Weisberg et al. 2003). In addition to macrophages, mast cells are present in adipose tissue, playing a central
role in the inflammatory process (Zhang & Shi 2012, Theoharides et al. 2011).
Impairment of the high-density lipoprotein cholesterol
(HDL-C) level and the quality of HDL particles is another
hallmark in the definition of MetSyn. Alteration of HDL metabolism and that of other lipoproteins exerts a strong and
independent risk as regards the onset of coronary heart disease
(CHD), and, in particular, early-onset and familial CHD. Inflammation has a substantial effect on the quality and quantity
of HDL. A recent study based on genetic instrumental variables is now raising an important debate on the causal pathways
to explain the role of HDL and HDL-related biomarkers in
the prevention of atherosclerosis and warrants further studies. These include not only the other major lipid and apolipoprotein components but also minor bioactive lipid molecules
residing in the HDL particles. Furthermore, a large number
of molecules circulating more or less firmly bound to HDL
particles may contribute to the anti-atherogenic potential of
HDL via antioxidative and anti-inflammatory effects or cholesterol transport capacity. Therefore, we urgently need novel
ways to assess the anti-atherosclerotic potential of HDL. One
of these concerns reverse cholesterol transport (RCT), where
HDL particles remove cholesterol from the arterial wall and
transport it to the liver. The first important step in RCT is
the outflow (efflux) of cholesterol from cells into the HDL
particles. The composition of HDL particles seems be a more
important determinant of their anti-atherogenic properties
than the amount of HDL-C. However, the effects of different HDL particle compositions on cholesterol efflux have not
been studied in detail.
RECENT PROGRESS
Our consortium posits a primary role of pro-inflammatory
pathways (Fig. 1) in setting up the risk of a myriad of disorders
associated with metabolic syndrome. In 2015 the group made
major advances in each of our three projects.
ABSTRACT
The project of the Savolainen, Herzig, Järvelin consortium involves teams in
clinical sciences, medical physiology and life-course epidemiology and we investigate new molecular factors associated with metabolic syndrome (i.e. obesity,
dyslipidaemia, high blood pressure and insulin resistance). We are focusing on
unravelling the roles played by molecular factors in pro-inflammatory pathways
and their potential as targets in therapeutic and preventive strategies. The consortium works in three main research areas: i) genetics and molecular determinants of atherosclerosis, ii) physiology of the inflammatory response as a path
for inter-organ communication, and iii) life-course and molecular epidemiology
of long-term metabolic health. We aim at defining intermediate and modifiable
phenotypes and subgroups of the population to implement personalised medicine
approaches. Following successful research into the genetics of common metabolic traits, the group is now developing approaches implementing exposomics,
exomics and epigenomics.
Figure 1. Systemic low-grade inflammation
might negatively influence foetal growth,
growth patterns and body mass from childhood,
to adverse adult cardiometabolic health.
RESEARCH PROJECTS
RESEARCH PROJECTS
Project 1: Life-course Epidemiology.
We have i) acquired new data to characterise further the pathways, ii) performed research in the genomic determinants
of the early risk for obesity and T2D, and iii) explored the
genetics and the pathophysiology of HDL composition and
function. Briefly, while analysing the factors associated with
the changes in metabolic health in mid-age in the NFBC, we
have further intensified our work on phenotyping the cohort
deeply. An essential aspect of the life-course origin of health
and diseases is interplay between genetic build-up and the
epigenetic program. DNA methylation is suggested to play a
major role in modulating the individual risk of obesity and
T2D (Chambers et al. 2015). It might at the same time biomark environmental insults and be informative as regards new
discoveries. In 2015, we were able to intensify the measure
of DNA methylation in the NFBC and we further organised
our teams to analyse the role of early stress in setting up the
intermediary risk factor (inflammation, metabolomics) and
clinical end-points (obesity, type 2 diabetes) (Academy project
EGEA). In the Horizon 2020 call, our application PHC-012014, “Understanding the dynamic determinants of glucose
homeostasis and psychosocial capability to promote healthy
and active ageing (DynaHEALTH)” was approved for funding at the beginning of 2015. In addition to this effort we have
intensified our research into the genomics of early growth, exemplified by our track record with the EGG (early growth
genetics) and GIANT consortium.
Our previous genome-wide linkage scan revealed six loci
showing suggestive evidence of linkage in connection with
HDL-C level regulation in families collected from Northern
Finland (Kangas-Kontio et al. Eur. J. Hum. Genet 2010).
Whole-exome sequencing was used to perform detailed analysis of these linkage regions and also additional genomic regions associated with HDL-C, other lipid traits and CHD
in large genome-wide association studies. Deeper genotyping
of these regions uncovered multiple inherited alleles possibly
contributing to the increased risk of CHD in these families.
Large population-specific cohorts will enable reliable replication of the results. It is estimated that protein-coding genes
harbour about 85% of the mutations with great effects on disease-related traits (Majewski et al. J Med Genet. 2011), which
makes whole-exome sequencing an effective tool to screen
rare genetic variants contributing to disease risk. Finland is
ideal for the first generation of large-scale medical sequencing
studies, focused on discovering high-impact rare variants and
demonstrating their impact on disease (Zuk et al. Proc Natl
Acad Sci USA 2014). Finnish allele frequency distribution
shows a paucity of singleton variants, compared with other
European populations (Lim et al. PLoS Genet 2014), and a
relative overabundance of variants in the 0.5–5% range. The
latter is particularly marked for those variants that are most
likely deleterious. We have reported that plasma levels of antibodies against oxidized LDL are inherited but not associated with HDL-cholesterol levels in families with early-onset
CHD (Paavola et al. Atherosclerosis 2012; Makinen et al. J
Intern Med 2013). To study the functional role of HDL in
reverse cholesterol transport (RCT), a mechanism presumed
to protect against CHD, we have investigated how different
HDL subclasses derived from CHD patients vs. control subjects mediate RCT from THP-1 cells. In this functional study,
early-onset CHD, metabolic syndrome and low HDL-C levels
were associated with impaired HDL2-mediated RCT (Paavola
et al, submitted).
comparison with total and LDL-cholesterol. Muscle biopsy
samples are currently being investigated.
Project 3: Obesity, adipogenesis and the role of Fto
Project 2: Contribution of inflammation to features of
metabolic syndrome.
Inflammatory markers (CD40L, CRP, TNF, etc) play an
important role in both the initiation and progression of atherosclerosis as well as reflecting the progression of complications in cases of T2D. In addition to its activity in blood, the
CD40-CD40L system probably executes cell-type signalling
on atherosclerotic plaques. We found that sCD40L levels were
elevated in middle-aged subjects with impaired glucose tolerance, which may indicate an increased cardiovascular risk.
The “fused-toe” or the fat-mass and obesity-associated FTO
gene (Dina et al. 2007, Frayling et al. 2007) is the most prevalent genetic variant as regards human BMI (Speliotes et al.
2010, Yang et al. 2012). It may provide a missing link in the
developmental regulation of energy metabolism (Sebert et al.
2014). The question of whether or not the obesity-associated
Furthermore, we investigated dietary changes and explored
gene expression in abdominal subcutaneous adipose tissue
(SAT) under a healthy Nordic diet including berries. We
found that a healthy Nordic diet reduces inflammatory gene
expression in SAT when compared with a control diet, independently of body weight change in individuals with features
of metabolic syndrome.
Berries, especially Nordic wild blueberries (bilberries), represent an important source of dietary anthocyanins, a group of
polyphenols with potential beneficial effects to combat obesity-associated metabolic disturbances. In C57BL mice fed with
a high-fat diet (HFD) bilberries reduced the development of
systemic inflammation and prevented the development and
progression of chronic hypertension.
gene FTO is key in obesity development in humans, and the
underlying molecular mechanisms are very poorly understood.
Current knowledge suggests a role of the FTO protein in the
demethylation of RNA in relation to energy-sensing pathways.
The physiological function of the protein and the mechanisms
associating weight gain and RNA demethylation demand
further attention. Our group is conducting research in Fto
knock-out mice to further comprehend the process through
which FTO predisposes individuals to weight gain.
We have found that Fto deficiency protects mice from diet-induced obesity, which was confirmed by reduced adipocyte size
(Ronkainen et al. 2015). Metabolic and behavioural parameters were unaltered in Fto-deficient mice. The expression of
genes regulating adipogenesis was higher and adipokine production altered in the white adipose tissue of Fto-deficient
mice. The expression of Irx3 (iroquois homeobox) was elevated in Fto-deficient mice after high-fat feeding, which was not
seen in wild-type mice. Our study demonstrates that Fto has
a role in adipose tissue which modifies the response of white
adipose tissue to high-fat feeding. Fto deficiency increases the
expression of genes related to adipogenesis, preventing adipocytes from becoming hypertrophic after a high-fat diet. In
addition, in our current research we are analysing interaction
with the dietary environment to characterise precisely how
exposure to an obesogenic environment may condition the
activity of FTO (Fig. 3). Our recent findings support the role
of gene–gene interaction (i.e. Fto with Irx3) in adipose tissue
in connection with a high-fat diet.
We are also looking at additional animal models in order to investigate inflammation related to obesity (high-fat diet), glucose homeostasis (Mutt et al. 2014), thermogenesis (Vähätalo
et al. 2015) and energy storage (Kinnunen et al. 2015).
In collaborative work we investigated the development of
obesity in homozygous OE-NPYDβH mice and the pathophysiological mechanisms underlying the phenotype. These
mice overexpress NPY in the sympathetic nervous system and
brain noradrenergic neurons. Homozygous OE-NPYDβH
mice showed significantly increased adipose tissue mass and
body weight. In contrast, food intake, physical activity and
energy expenditure measured by indirect calorimetry were not
changed in these mice. Increased lipid uptake together with
decreased lipolysis seemed to promote weight gain. Synthesis
and release of catecholamines were lowered and may contribute to development of the obese phenotype in these mice. The
results support the role of NPY as a direct effector and as a
modulator of sympathetic activity in the pathogenesis of metabolic syndrome.
Intervention: Modulation of metabolic and inflammatory
responses
During the last year we have assessed inflammatory markers
in members of the Northern Finland Birth Cohort 1966 and
are currently investigating associations with particular phenotypes. Furthermore, we finalized a local intervention study
in pre-diabetic high-risk subjects, demonstrating that light
physical activity decreases the levels of fasting and 2-h insulin,
total cholesterol, LDL-cholesterol and visceral fat. Significant
reductions in the concentrations of extremely large, very large,
medium and small VLDL particles, extremely large VLDLand VLDL-triglycerides, small HDL particles and HDL3
cholesterol took place at even lower physical activity levels in
Figure 2. Pictures of whole organs dissected from wild-type (left) and
heterozygous Fto-G10 (right) mice stained with X-gal for β-gal expression. a) brain, b) muscle, c) bowel (with pancreas on the right-hand side),
d) epididymal adipose tissue, e) spleen, f) embryo at the age of 11.5 dpc
Figure 3. Proposal for the altered response of Fto-knockout white adipose
tissue to high-fat feeding. The physiological response to a high-fat diet in
wild-type mice causes Fto to decrease the levels of genes related to adipogenesis, such as Pparg, Rxra and Cebpa, promoting adipocyte hypertrophy
and obesity, with unfortunate consequences. When Fto is absent, levels of
Pparg, Rxra and Cebpa remain elevated even after a high-fat diet, which
prevents adipocytes from becoming hypertrophic and maintains adiponectin production.
RESEARCH PROJECTS
RESEARCH PROJECTS
FUTURE GOALS
We aim to identify inflammatory pathways from early life
leading to MetSyn, by using an integrated systems approach
in human epidemiological and clinical data, molecular mechanistic studies and application of causal analytical approaches.
We will continue to explore the genetic determinants of MetSyn phenotypes by considering life-course exposures (exposomics).
The new areas of research are to investigate whether the early exposome is associated with specific methylation marks, as
molecular-level indicators of the impact of environment, and
whether these marks are associated with later-life metabolic
outcomes. The results obtained will be further applied in experimental work.
We hypothesize that factors secreted from adipocytes and/or
other cells in adipose tissue are responsible for the inflammatory state and activated macrophages/mast cells. Low concentrations of 25(OH)D (as a model substance) are associated
with adverse metabolic phenotypes via increased cytokine/
altered adipokine production, which might be reversed by
high 25(OH)D levels. These mechanisms will be investigated
in vivo and in vitro, including under hypoxic conditions, and
the downstream targets elucidated.
The main aim of our work with lab animals will be to characterize the role of Fto in the early epigenetic programming of
obesity in the fetal and early postnatal period. We will define
the underlying molecular programming of inflammation- and
oxidative stress-related pathways in key organs, including the
hypothalamus and adipose tissues, to characterize the underlying pathways and the pathophysiological responses in terms
of regulation of body weight and other pro-inflammatory
functions. Furthermore, inflammation via stress responses
will affect crosstalk in insulin-sensitive tissues. Changes in
the environment, physical or perceived, are coordinated in
the body by the hypothalamic-pituitary-adrenal (HPA) axis
to maintain homeostasis which, via a coordinated neuroendocrine response communicates with all tissues modulating
metabolic responses.
Estelle Lowry, Ph.D., Post doctoral researcher
– Life-course epidemiology (DynaHEALTH)
Nina Rautio, Ph.D., Post doctoral researcher – Social
epidemiology (DynaHEALTH and Academy of Finland
Kari Mäkelä, Ph.D., Post doctoral researcher
– Experimental physiology (University of Oulu)
Juhani Leppäluoto, M.D., Ph.D.; Prof emeritus hc.,
endocrinology, physiology (Foundations)
Miia Kovalainen, Ph.D., Post doctoral researcher
– peptide and nutrient application (Foundations and
Academy of Finland)
Tuire Salonurmi, Ph.D., Post doctoral researcher
– molecular dietetic biology, transgenic mice
(Oulu University Hospital, University of Oulu)
Antti Nissinen, Ph.D., Post doctoral researcher
– immunological biology of lipids
(Foundations, University of Oulu)
Ph.D. Students:
Saranya Palaniswamy, M.Sc. (BCO)
Rozenn Nedelec, M.Sc. (CLCHR and Foundation)
Eeva Nevala, M.Sc. (UniOGS)
Ghulam Shere Raza, M.Sc. (Foundation)
Toni Karhu, M.Sc. (Foundation)
Shivaprakash J Mutt, M.Sc. (Foundation)
Laura Niiranen, M.Sc. (UniOGs and Foundation)
Justiina Ronkainen, M.Sc. (BCO)
Timo Paavola, Lic.Med., (Foundations)
Saeid Haghighi, M.Sc. (Foundations)
Laboratory Technicians,
Marja-Leena Kytökangas, Laboratory technician
Sari Pyrhönen, Laboratory technician
(Oulu University Hospital)
Saara Korhonen, Biomedical laboratory scientist
Statisticians:
Sauli Herrala (CLCHR, DynaHEALTH)
Ville Karhunen (CLCHR, DynaHEALTH)
Eeva Varamo (CLCHR, DynaHEALTH)
Foreign Scientists, 5 for the group Järvelin
Project Leaders:
Marjo-Riitta Järvelin M.D., Ph.D. (Center for Life-Course
Health Research (CLCHR) and Imperial College London)
Karl-Heinz Herzig, M.D., Ph.D., Professor
Markku Savolainen, M.D., Ph.D., Professor
Jouko Miettunen, Ph.D., Professor, Statistics
(CLCHR, Academy of Finland)
Sylvain Sebert, Ph.D., Docent – Early programming
(CLCHR, DynaHEALTH)
Minna Männikkö, Ph.D., Docent – Genetics (CLCHR)
Dylan Williams, Ph.D., Post doctoral researcher
– Life-course epidemiology (DynaHEALTH)
For group Järvelin:
Coordination and management of the DynaHEALTH
research action (dynahealth.eu)
Active members of:
Early Growth Genetics Consortium (EGG)
GIANT consortium
PACE consortium
CHARGE consortium
For Group Järvelin:
Ville Karhunen, multiple visits at the Department of Epidemiology and Biostatistics, Imperial College London.
For Group Savolainen:
Justiina Ronkainen, visits at the Development and Plasticity
of the Postnatal Brain, Jean-Pierre Aubert Research Center,
Inserm University of Lille, Lille, France.
EU H2020-HCO-2004, Prevention and treatment of type
2 diabetes, J Chambers (PI), M-R Järvelin (Co-PI) et al.
01.02.2015-31.01.2020. Team members Prof. K-H Herzig,
Prof. S Keinänen-Kiukaanniemi et al.
Principal investigator (MRJ) in EurHEALTHAgeing (European ResearcH on DevElopmentAL, BirTH and Genetic Determinants of Ageing).
Co-operation with Finnish and Foreign Companies
Co-operation with ABBOTT Nutrition and ORDESA laboratorios (as part of DynaHEALTH)
Principal investigator (MRJ) in EPI-MIGRANT (Identification of epigenetic markers underlying increased risk of T2D
in South Asians).
EU H2020: Understanding the dynamic determinants of
glucose homeostasis and social capability to promote Healthy
and active aging, Grant no. 633595, 01.04.2015-31.03.2019,
M-R Järvelin (Coordinator), Team members: Prof. K-H Herzig, Dr. S Sebert et al.
Aarlt A, Ilves I, Aloji L, Miettinen P, Paajanen H,
Brunke G, Häsler R, Ellrichmann M, Schreiber
S, Rosenstiel R, Ott S, Herzig KH. Characteristic changes in microbial community composition
and expression of innate immune genes in acute
appendicitis. Innate Immun 21(1):30-41, 2015.
Artigas MS, et al (incl Järvelin MR). Sixteen
new lung function signals identified through
1000 Genomes Project reference panel imputation. Nat Commun 6:8658. doi: 10.1038/
ncomms9658, 2015.
Cartwright R, Kirby AC, Tikkinen KA, Mangera A, Thiagamoorthy G, Rajan P, Pesonen
JS, Ambrose C, Gonzalez-Maffe J, Bennett P,
Palmer T, Walley A, Järvelin M-R, Chapple C,
Khullar V. Systematic review and meta-analysis
of genetic association studies of urinary symptoms and prolapse in women. Am J Obstet Gynecol 212:199.e1-24, 2015.
lin Resistance: Additional Support for the Novel
Heuristic Model in Perimenopausal Depression.
Am J Psychiatry 172(8):796-7, 2015.
Fall T, et al (incl Järvelin MR), on behalf of the
ENGAGE Consortium: Age- and Sex-Specific
Causal Effects of Adiposity on Cardiovascular
Risk Factors. Diabetes 64(5):1841-52, 2015.
Fung E, Järvelin MR, Doshi RN, Shinbane JS,
Carlson SK, Grazette LP, Chang PM, Sangha
RS, Huikuri HV, Peters NS. Electrocardiographic patch devices and contemporary wireless
cardiac monitoring. Front Physiol 6:149. doi:
10.3389/fphys.2015.00149, eCollection 2015.
Gharib SA, et al (incl Järvelin MR), CHARGE
Consortium, SpiroMeta Consortium. Integrative
pathway genomics of lung function and airflow
obstruction. Hum Mol Genet 24(23):6836-48,
2015.
Chambers JC, et al (incl Järvelin MR). Epigenome-wide association of DNA methylation
markers in peripheral blood from Indian Asians
and Europeans with incident type 2 diabetes: a
nested case-control study. Lancet Diabetes Endocrinol 3(7):526-34, 2015.
Graversen L, Sørensen TI, Gerds TA, Petersen
L, Sovio U, Kaakinen M, Sandbaek A, Laitinen J, Taanila A, Pouta A, Järvelin M-R, Obel
C. Prediction of adolescent and adult adiposity
outcomes from early life anthropometrics. Obesity 23(1):162-9, 2015.
EArly Genetics and Lifecourse Epidemiology (EAGLE) Eczema Consortium; Australian Asthma Genetics Consortium (AAGC),
et al (incl Järvelin MR). Multi-ancestry genome-wide association study of 21,000 cases
and 95,000 controls identifies new risk loci for
atopic dermatitis. Nat Genet 47(12):1449-56,
2015.
Hukkanen J, Puurunen J, Hyötyläinen T, Savolainen MJ, Ruokonen A, Morin-Papunen
L, Orešič M, Piltonen T, Tapanainen JS. The
effect of atorvastatin treatment on serum oxysterol levels and cytochrome P450 activity. Br
J Clin Pharmacol 80: 473-479, 2015.
Eskola PJ, Jokelainen J, Järvelin MR, Keinänen-Kiukaanniemi S, Ruokonen A, Puukka K,
Timonen M, Auvinen JP. Depression and Insu-
Hukkanen J, Rysä J, Mäkelä K, Herzig K-H,
Hakkola J, Savolainen MJ. The effect of pregnane X receptor agonists on postprandial incretin hormone secretion in rats and humans. J
Physiol Pharmacol 66(6):821-39, 2015.
Hägg S, et al (incl Järvelin MR), for the European Network for Genetic and Genomic
Epidemiology (ENGAGE) consortium. Adiposity
as a Cause of Cardiovascular Disease: A Mendelian Randomization Study, Int J Epidemiol
44(2):578-86, 2015.
Junno JA, Paananen M, Karppinen J, Niinimäki
J, Niskanen M, Maijanen H, Väre T, Järvelin MR, Nieminen MT, Tuukkanen J, Ruff C.
Age-related trends in vertebral dimensions. J
Anat 226(5):434-9, 2015.
Juvonen KR, Macierzanka A, Lille ME, Laaksonen DE, Mykkänen HM, Niskanen LK, Pihlajamäki J, Mäkelä K, Mills C, Mackie A, Mclcome
P, Herzig KH, Poutanen KS, Karhunen LJ.
Cross-linking of sodium caseinate structured
emulsion with transglutaminase alters the postprandial metabolism and appetite responses in
healthy young individuals. Br J Nutr 114(3):41826, 2015.
Jääskeläinen A, Kaila-Kangas L, Leino-Arjas P,
Lindbohm ML, Nevanperä N, Remes J, Järvelin
MR, Laitinen J. Association between occupational psychosocial factors and waist circumference is modified by diet among men. Eur J Clin
Nutr 69(9):1053-9, 2015.
Jääskeläinen A, Kaila-Kangas L, Leino-Arjas P,
Lindbohm ML, Nevanperä N, Remes J, Järvelin
MR, Laitinen J. Psychosocial Factors at Work
and Obesity Among Young Finnish Adults: A
Cohort Study. J Occup Environ Med 57(5):48592, 2015.
RESEARCH PROJECTS
RESEARCH PROJECTS
Kato N, et al (incl Järvelin MR). Trans-ancestry genome-wide association study identifies 12
genetic loci influencing blood pressure and implicates a role for DNA methylation. Nat Genet
47(11):1282-93, 2015.
Kinnunen S, Mänttäri S, Herzig KH, Nieminen P, Mustonen AM, Saarela S. Maintenance
of skeletal muscle energy homeostasis during
prolonged wintertime fasting in the raccoon dog
(Nyctereutes procynoides). J Comp Physiol B
185(4):435-45, 2015.
Kolehmainen M, Ulven SM, Paananen J, de
Mello V, Schwab U, Carlberg C, Myhrstad M,
Pihlajamäki J, Dungner E, Sjölin E, Gunnarsdottir I, Cloetens L, Landin-Olsson M, Åkesson B, Rosqvist F, Hukkanen J, Herzig K-H,
Dragsted LO, Savolainen MJ, Brader L, Hermansen K, Risérus U, Thorsdottir I, Poutanen
KS, Uusitupa M, Arner P, Dahlman I. Healthy
Nordic diet down-regulates the expression of
genes involved in inflammation in subcutaneous
adipose tissue in individuals with features of the
metabolic syndrome. Am J Clin Nutr 101: 228239, 2015.
Kovalainen M, Mönkäre J, Riikonen, Pesonen
U, Vlasova M, Salonen J, Lehto VP, Järvinen
K, Herzig KH. Novel Delivery Systems for Improving the Clinical Use of Peptides. Pharmacol
Rev 67(3):541-561, 2015.
Käräjämäki AJ, Pätsi OP, Savolainen MJ, Kesäniemi YA, Huikuri H, Ukkola O. Non-Alcoholic Fatty Liver Disease as a Predictor of Atrial
Fibrillation in Middle-Aged Population (OPERA
study). PLoS One 16; 10(11): e0142937, 2015.
Lampi J, Koskela H, Hartikainen AL,
Ramasamy A, Couto Alves A, Järvelin MR,
Pekkanen J. Farm environment during infancy
and lung function at the age of 31: a prospective birth cohort study in Finland. BMJ Open
22;5(7):e007350, 2015.
Lehne B, Drong AW, Loh M, Zhang W, Scott
WR, Tan ST, Afzal U, Scott J, Jarvelin MR, Elliott P, McCarthy MI, Kooner JS, Chambers JC.
A coherent approach for analysis of the Illumina
HumanMethylation450 BeadChip improves data
quality and performance in epigenome-wide
association studies. Genome Biol 16(1):37, 2015
Linneberg A, et al (incl Järvelin MR). Effect of
Smoking on Blood Pressure and Resting Heart
Rate: A Mendelian Randomisation Meta-Analysis in the CARTA Consortium. Circ Cardiovasc
Genet 8(6):832-41, 2015.
Locke AE, et al (incl Järvelin MR). Genetic
studies of body mass index yield new insights
for obesity biology. Nature 518(7538):197-206,
2015.
Morris RW, et al (incl Järvelin MR). Heavier smoking may lead to a relative increase in
waist circumference: evidence for a causal relationship from a Mendelian randomisation meta-analysis. The CARTA consortium. BMJ Open
5(8):e008808, 2015.
Männistö T, Vääräsmäki M, Sipola-Leppänen
M, Tikanmäki M, Matinolli HM, Pesonen AK,
Räikkönen K, Järvelin MR, Hovi P, Kajantie
E. Independent living and romantic relations
among young adults born preterm. Pediatrics
135(2):290-7, 2015.
Nevanperä N, Ala-Mursula L, Seitsamo J,
Remes J, Auvinen J, Hopsu L, Husman P, Karppinen J, Järvelin MR, Laitinen J. Long-Lasting
Obesity Predicts Poor Work Ability at Midlife:
A 15-Year Follow-Up of the Northern Finland
1966 Birth Cohort Study. J Occup Environ Med
57(12):1262-8, 2015.
Obeidat M, et al (incl Järvelin MR). Molecular
mechanisms underlying variations in lung function: a systems genetics analysis. Lancet Respir
Med 3(10):782-95, 2015.
Ruiz M, Goldblatt P, Morrison J, Kukla L,
Švancara J, Järvelin M-R, Taanila A, Saurel-Cubizolles M-J, Lioret S, Bakoula C, Veltsista A, Porta D, Forastiere F, van Eijsden M,
Vrijkotte TGM, Eggesbø M, White RA, Barros
H, Correia S, Vrijheid M, Torrent M, Rebagliato
M, Larrañaga I, Ludvigsson J, Olsen Faresjö Å,
Hryhorczuk D, Antipkin Y, Marmot M, Pikhart
H. Mother’s education and the risk of preterm
and small for gestational age birth: a DRIVERSQ1 meta-analysis of 12 European cohorts. J
Epidemiol Community Health 69(9):826-833,
2015.
Savolainen MJ. Epidemiology: Disease Associations and Modulators of HDL-Related Biomarkers. Handb Exp Pharmacol 224: 259-83, 2015.
Shungin D, et al (incl Herzig KH, Järvelin MR).
New genetic loci link adipose and insulin biology
to body fat distribution. Nature 518(7538):18796, 2015.
Ojelade SA, et al (incl Järvelin MR). Rsu1
regulates ethanol consumption in Drosophila and humans. Proc Natl Acad Sci U S A
112(30):E4085-93, 2015.
Sipola-Leppänen M, Karvonen R, Tikanmäki
M, Matinolli HM, Martikainen S, Pesonen AK,
Räikkönen K, Järvelin MR, Hovi P, Eriksson JG,
Vääräsmäki M, Kajantie E. Ambulatory Blood
Pressure and Its Variability in Adults Born Preterm. Hypertension 65(3):615-21, 2015.
Peet ED, McCoy DC, Danaei G, Ezzati M,
Fawzi W, Järvelin MR, Pillas D, Fink G. Early Childhood Development and Schooling Attainment: Longitudinal Evidence from British,
Finnish and Philippine Birth Cohorts. PLoS One
10(9):e0137219, 2015.
Sipola-Leppänen M, Vääräsmäki M, Tikanmäki
M, Matinolli HM, Miettola S, Hovi P, Wehkalampi K, Ruokonen A, Sundvall J, Pouta A, Eriksson JG, Järvelin MR, Kajantie E. Cardiometabolic risk factors in young adults who were born
preterm. Am J Epidemiol 181(11):861-73, 2015.
Pers TH, Karjalainen JM, Chan Y, Westra HJ,
Wood AR, Yang J, Lui JC, Vedantam S, Gustafsson S, Esko T, Frayling T, Speliotes EK.
Genetic Investigation of ANthropometric Traits
(GIANT) Consortium, Boehnke M, Raychaudhuri S, Fehrmann RS, Hirschhorn JN, Franke
L. Biological interpretation of genome-wide
association studies using predicted gene functions. Nat Commun 6:5890, 2015.
Solmi F, Sonneville KR, Easter A, Horton NJ,
Crosby RD, Treasure J, Rodriguez A, Järvelin
M-R, Field AE, Micali N. Prevalence of purging
at age 16 and associations with negative outcomes among girls in three community-based
cohorts. J Child Psychol Psychiatry 56(1):8796, 2015.
Puroila A, Paananen M, Taimela S, Järvelin
MR, Karppinen J. Lifestyle-Factors in Adolescence as Predictors of Number of Musculoskeletal Pain Sites in Adulthood: A 17-Year
Follow-Up Study of a Birth Cohort. Pain Med
16(6):1177-85, 2015.
Rodriguez A, Wang Y, Khan AA, Gissler M,
Cartwright R, Järvelin MR. Exposure to antenatal corticosteroid therapy is associated with
reduced size at birth: Evidence from Finnish
Medical Birth Register of 278,508 births. Psychoneuroendocrinology 61:37, 2015.
Ronkainen J, Huusko TJ, Soininen R, Mondini
E, Cinti F, Mäkelä KA, Kovalainen M, Herzig
KH, Järvelin MR, Sebert S, Savolainen MJ,
Salonurmi T. Fat mass- and obesity-associated
gene Fto affects the dietary response in mouse
white adipose tissue. Sci Rep 18, 5:9233, 2015.
Wang Q, Kangas AJ, Soininen P, Tiainen M,
Tynkkynen T, Puukka K, Ruokonen A, Viikari
J, Kähönen M, Lehtimäki T, Salomaa V, Perola M, Davey Smith G, Raitakari OT, Järvelin
MR, Würtz P, Kettunen J, Ala-Korpela M. Sex
hormone-binding globulin associations with circulating lipids and metabolites and the risk for
type 2 diabetes: observational and causal effect
estimates. Int J Epidemiol 44(2):623-37, 2015.
Warrington NM, Howe LD, Paternoster L,
Kaakinen M, Herrala S, Huikari V, Wu YY,
Kemp JP, Timpson NJ, Pourcain BS, Davey
Smith G, Tilling K, Jarvelin MR, Pennell CE,
Evans DM, Lawlor DA, Briollais L, PalmerLJ.
A genome-wide association study of body mass
index across early life and childhood. Int J Epidemiol 44(2):700-12, 2015.
Vimaleswaran KS, Cavadino A, Verweij N,
Nolte IM, Mateo Leach I; LifeLines Cohort
Study, Auvinen J, Veijola J, Elliott P, Penninx
BW, Snieder H, Järvelin MR, van der Harst
P, Cohen RD, Boucher BJ, Hyppönen E. Interactions between uncoupling protein 2 gene
polymorphisms, obesity and alcohol intake on
liver function: a large meta-analysed population-based study. Eur J Endocrinol 173(6):86372, 2015.
Vähätalo LH, Ruohonen ST, Kovalainen M,
Huotari A, Mäkelä K, Määttä JA, Mäkelä S,
Liisa Ailanen L, Ruohonen S, Röyttä M, Herzig KH, Savontaus E. Neuropeptide Y in the
noradrenergic neurons induces obesity and
inhibits sympathetic tone. Acta Physiol (Oxf)
213(4):902-19, 2015.
Kinnunen S, Mänttäri S, Herzig KH, Nieminen
P, Mustonen AM, Saarela S. Effects of wintertime fasting and seasonal adaptation on AMPK
and ACC in hypothalamus, adipose tissue and
liver of the raccoon dog (Nyctereutes procyonoides). Comp Biochem Physiol A Mol Integr
Physiol 192:44-51, 2016.
Äijälä M, Ronkainen J, Huusko T, Malo E,
Savolainen ER, Savolainen MJ, Salonurmi T,
Bloigu R, Antero Kesäniemi Y, Ukkola O. The
fat mass and obesity-associated (FTO) gene
variant rs9939609 predicts long-term incidence of cardiovascular disease and related
death independent of the traditional risk factors.
Ann Med 47(8):655-63, 2015.
Brauer R, Tureckova J, Kanchev I, Dziechciarkova M, Skarda J, Jirouskova M, Beck IM,
Zbodakova O, Kasparek P, Korinek V, Chalupsky K, Karhu T, Herzig KH, Haase M, Hajduch
M, Gregor M, Sedlacek R. MMP-19 deficiency
causes aggravation of colitis due to inefficient
fractalkine processing. Mucosal Immunol doi:
10.1038/mi.2015.117, 2015, Epub ahead of print.
Felix JF, et al (incl Järvelin MR), Bone Mineral
Density in Childhood Study (BMDCS) Consortium, Early Genetics and Lifecourse Epidemiology (EAGLE) consortium, Early Growth Genetics (EGG) Consortium, Bone Mineral Density
in Childhood Study BMDCS Consortium. Genome-wide association analysis identifies three
new susceptibility loci for childhood body mass
index. Hum Mol Genet 25(2):389-403, 2016.
Jacobsen KK, Nievergelt CM, Zayats T,
Greenwood TA, Anttila V, Akiskal HS, BiGS
Consortium, IHG Consortium. Genome wide
association study identifies variants in NBEA
associated with migraine in bipolar disorder. J
Affect Disorders 172C:453-461. doi: 10.1016/j.
jad.2014.10.004, 2015, Epub ahead of print.
Surakka I, et al (incl Järvelin MR). The impact
of low-frequency and rare variants on lipid levels. Nat Genet 47(6):589-97, 2015.
Tanner T, Päkkilä J, Karjalainen K, Kämppi A,
Järvelin MR, Patinen P, Tjäderhane L, Anttonen V. Smoking, alcohol use, socioeconomic background and oral health among young
Finnish adults. Community Dent Oral Epidemiol
43(5):406-14, 2015.
The Coffee and Caffeine Genetics Consortium,
et al (incl Järvelin MR). Genome-wide meta-analysis identifies six novel loci associated
with habitual coffee consumption. Mol Psychiatry 20(5):647-56, 2015.
van der Valk RJ, et al (incl Järvelin MR). A
novel common variant in DCST2 is associated
with length in early life and height in adulthood.
Hum Mol Genet 24(4):1155-68, 2015.
Ollila MM, Piltonen T, Puukka K, Ruokonen
A, Järvelin MR, Tapanainen JS, Franks S,
Morin-Papunen L. Weight Gain and Dyslipidemia in Early Adulthood Associate with Polycystic
Ovary Syndrome: Prospective Cohort Study. J
Clin Endocrinol Metab jc20153543, 2015, Epub
ahead of print.
RESEARCH PROJECTS
Understanding the genetic basis of phenotypic variation in
relation to environmental variation is crucial in many areas
of life sciences. Evolutionary geneticists search for the loci
governing quantitative genetic variation. We ask what is the
distribution of effects of individual variants (small, large, deleterious, beneficial)? What are their mutation rates, are the variant alleles common or rare, are they regulatory or structural
variants, or variants in non-coding regions? Does adaptation
arise from existing variation or new mutations? Answering
these questions requires identification of the loci responsible
for variation. We can then examine the phenotypic effects of
individual alleles. This also allows examination of the patterns
of sequence variation to answer questions about natural selection. Prediction of phenotypes based on DNA-level information is also important. Plant and animal breeders aim to
predict the genomic breeding value underlying phenotypes on
the basis of SNP (single nucleotide polymorphism) markers.
One of the goals of medical genetics is to predict disease phenotypes. A shared framework of population genetics theory
underlies these efforts in different fields.
From left: Jaakko Tyrmi, Sonja Kujala, Tuomas Hämälä, Soile Alatalo, Helmi Kuittinen, Mikko Kuismin, Outi Savolainen, Mikko Sillanpää, Emmi Aikio, Tiina Mattila,
Nader Aryamanesh, Margarita Takou.
GENETIC AND STATISTICAL
EVOLUTIONARY GENOMICS:
ANALYSIS OF ADAPTIVE PHENOTYPIC
VARIATION
ABSTRACT
The genetics of quantitative variation and the factors influencing the patterns
of this variation are important issues in evolutionary biology, plant and animal
breeding, and medicine. The Savolainen group, working on plant population genetics, and the Sillanpää group, working on statistical genetics, develop genomic
and statistical approaches to study the genetics of environmental adaptation,
with special emphasis on two plant systems, Scots pine and Arabidopsis. The
population genetic approaches as well as the related statistical methods will be
broadly applicable to other systems. The results can be used, for example, in
tree breeding. Further, they are applicable in managing plant populations facing
climate change. The statistical methods and tools are being developed for a broad
set of problems, in human, animal and plant populations, with specific applications here.
PROJECT LEADERS
Prof. Outi Savolainen, Ph.D.
Department of Biology, Faculty of Science Prof. Mikko J. Sillanpää, Ph.D.
Research Unit of Statistical Models and Data Analysis,
Department of Mathematical Sciences,
Faculty of Science
The possibilities of achieving the goals have increased rapidly.
This has been driven partly by advances in sequencing and
genotyping technologies. A major thrust has come from developments in population genetics theory, bioinformatics, and
genetic statistics.
Within the broad area of biometric and genetic analysis of
quantitative variation, the Savolainen group studies the genetics of local adaptation of plants. The Sillanpää group develops
statistical tools for analyzing the genetic basis of quantitative
genetic variation, using Bayesian approaches in particular.
RECENT PROGRESS
We have developed conceptually new methods for LASSO-based association mapping when phenotype distribution
is non-normal or includes outlier observations (Möttönen and
Sillanpää 2015; Li et al. 2015), mapping epistatic interactions
from genome-wide data sets (Kärkkäinen et al. 2015), estimating genetic parameters from pedigree data using multiple-trait
animal model and integrated nested Laplace approximation
(INLA) (Mathew et al., 2015a, 2015b), estimating rare variant
associations from sequence data (He et al., 2015), estimating
human disease phenotypes based on molecular markers (Bhattacharjee et al. 2015), and studying multilocus associations in
connection with dynamic traits (Li and Sillanpää, 2015a, b).
For estimation in these pieces of work, we have used Markov
Chain Monte Carlo sampling or iterative methods to find a
maximum point of target function.
In addition to the above, we have placed special emphasis on
procedures in decision-making concerning marker significance in association analysis (Pasanen et al., 2015). An adjustment procedure concerning a tuning parameter in Bayesian
LASSO is also of central importance (Pasanen et al., 2015).
More applied work has also been published (Khazaei et al.,
2015; Bari et al. 2015). Currently we are preparing a manuscript concerning meta-analysis of several European populations of Scots pine.
In plant genetics, we have finalized the estimation of associations between SNP markers in candidate and reference genes
in a European set of populations (Kujala 2015, thesis). This
allowed identification of associated and non-associated markers. An important finding was that the northern and central
European populations of pines seem to have different loci
governing variation in the timing of budset. This result will
require further study. The data allowed us to test two theories
predicting the genetic basis underlying clinal variation. Our
data set did not provide evidence of steep allele frequency
clines, as predicted by one of the theories. Surprisingly, we
also did not detect high covariances between populations of
allelic frequencies of associated loci across the cline, but power
may have been rather low. For our work on adaptation in Chinese pines, we completed the analysis of demographics related
to speciation using coalescent analyses. We also examined the
roles of selection and reproductive isolation in governing ecological speciation in two closely related pines (Zhou et al. submitted). In the EU-funded ProCoGen project, we completed
the European-scale exome capture of Scots pine populations
and also developed the related pipelines.
For population genetics of Arabidopsis lyrata, a large-scale sequencing analysis of range-wide flowering time sequence diversity was completed (Mattila et al., in press). We have been
developing the genetic mapping tools based on a variant of
RAD sequencing to obtain dense maps to be able to examine
the progress of chromosome segments in a hybrid zone. We
have also now analyzed the demographic patterns and signals
of selection using whole genome sequences of Arabidopsis
lyrata.
FUTURE GOALS
In the future, we will concentrate on developmental work on
robust variable selection tools which are not so sensitive to the
distributional assumptions concerning outlying observations
or missing data. We will also continue developmental work
on our Bayesian multiple locus method and Gibbs sampling
algorithms in joint analysis of association and linkage, using
pedigree data. This should provide us with a chance to extract
more information from the same amount of data than sole
association or linkage analysis. We will also continue our work
on Bayesian variable selection methods for semi-parametric
and Gaussian process models.
For both Arabidopsis lyrata and Pinus sylvestris, we are now
analyzing genome-wide sequence variation. For pines, we are
combining earlier data on phenotypes with the recently obtained exome sequence variation. For A. lyrata, we have started examining patterns of local adaptation on a smaller scale
than before by comparing two altitudinal clines in Norway,
at phenotype, gene expression and whole genome sequence
variation levels, aiming specifically at resolving the effects of
photoperiod in adaptation to northern conditions.
PH.D. THESIS
Kujala, Sonja 2015. Dissecting genetic variation in European
Scots pine (Pinus sylvestris L.) – special emphasis on polygenic
adaptation. Acta Univ. Ouluensis A661.
RESEARCH PROJECTS
Project Leaders:
Outi Savolainen, Ph.D., Professor (University of Oulu)
Mikko J. Sillanpää, Ph.D., Professor (University of Oulu)
Helmi Kuittinen, Ph.D. (University of Oulu)
Tanja Pyhäjärvi, Ph.D. (University of Oulu)
Yongfeng Zhou (University of Oulu, Eu-Project ProCoGen)
Päivi H. Leinonen, Ph.D.
Nader Aryamanesh
(starting October 2015, University of Oulu)
Zitong Li, Ph.D. (Biocenter Oulu)
Ph.D. Students:
Timo Knürr, M.Sc. (MTT Agrifood Research Finland)
Pinja Pikkuhookana, M.Sc. (University of Oulu)
Markku Kuismin (University of Oulu)
Bhattacharjee M, Rajeevan MS, Sillanpää MJ.
Prediction of complex human diseases from
pathway-focused candidate markers by joint
estimation of marker effects: case of chronic
fatigue syndrome. Human Genomics 9: 8, 2015.
He L, Pitkäniemi J, Sarin A-P, Salomaa V,
Sillanpää MJ, Ripatti S. Hierarchical Bayesian model for rare variant association analysis
integrating genotype uncertainty in human sequence data. Genet Epidemiol 39: 89-100, 2015.
Heikkinen M, Ruokonen M, Alexander M, Aspi
J, Pyhäjärvi T, Searle JB. Relationship between wild greylag and European domestic
geese based on mitochondrial DNA. Anim Genet
46: 485-497, 2015.
Khazaei H, O’Sullivan DM, Jones H, Pitts N,
Sillanpää MJ, Pärssinen P, Manninen O, Stoddard FL. Flanking SNP markers for vicine-convicine concentration in faba bean (Vicia faba L.).
Molecular Breeding 35: 38, 2015.
Kärkkäinen HP, Li Z, Sillanpää MJ. An efficient
genome-wide multilocus epistasis search. Genetics 201:865-870, 2015.
Sonja Kujala, M.Sc.
(University of Oulu, Emil Aaltonen Foundation)
Tiina Mattila (Doctoral Programme of Population Genetics)
Jaakko Tyrmi (EU-project ProCoGen)
Tuomas Hämälä (Biocenter Oulu)
Soile Finne (University of Oulu)
Päivi H. Leinonen, Ph.D.: Duke University, 12 mo
EU Projects (present)
Networking within EU Project Promoting Conifer Genetic
Resources (2012–2015)
Li Z, Möttönen J, Sillanpää MJ. A robust multiple-locus method for quantitative trait locus
analysis of non-normally distributed multiple
traits. Heredity 115:556-564, 2015.
Li Z, Sillanpää MJ. Dynamic quantitative trait
locus analysis of plant phenomic data. Trends in
Plant Sci 20:822-833, 2015.
Li Z, Sillanpää MJ. Efficient use of systems
mapping without expert knowledge: Comment
on “Mapping complex traits as a dynamic system” by L. Sun and R. Wu. Phys Life Rev 13:192193, 2015.
Mathew B, Holand AM, Koistinen P, Leon J,
Sillanpää MJ. Reparametrization-based estimation of genetic parameters in multi-trait
animal model using Integrated Nested Laplace
Approximation. Theor Appl Gen, 2015.
Mathew B, Leon J, Sillanpää MJ. Integrated
Nested Laplace Approximation inference and
cross-validation to tune variance components
in breeding value estimation. Molecular Breeding 35: 99, 2015.
Mattila T M, Aalto E A, Toivainen T , Niittyvuopio A, Piltonen S, Kuittinen H, Savolainen O.
Selection for population specific adaptation has
shaped patterns of variation in the photoperiod pathway genes in Arabidopsis lyrata during
post glacial colonization. Mol Ecol doi:10.1111/
mec.13489, 2015.
Möttönen J, Sillanpää MJ. Robust variable selection and coefficient estimation in multivariate
multiple regression using LAD-Lasso, Modern
Nonparametric, Robust and Multivariate Methods pp 235-247, Springer International Publishing, Switzerland, 2015.
PROJECT LEADER
Prof. Seppo Vainio, Ph.D.
Laboratory of Developmental Biology,
University of Oulu
GENETIC CONTROL
OF ORGANOGENESIS
Pasanen L, Holmström L, Sillanpää MJ. Bayesian LASSO, scale space and decision making in
association genetics. PLoS ONE 10:e0120017,
2015.
Bari A, Khazaei H, Stoddard FL, Street K, Sillanpää MJ, Chaubey YP, et al. In silico evaluation of plant genetic resources to search for
traits for adaptation to climate change. Climatic
Change, in press.
Lascoux M, Glémin S, Savolainen O. Local adaptation in plants. eLS Wiley, in press.
From left sitting: Florence Naillat, Ganna Reint, Qi Xu, Hannele Härkman, Melanie Roth, Genevieve Bart, Seppo Vainio, Paula Haipus, Ulla Saarela, Johanna Kekolahti-Liias,
Mirja Krause, Nsrein Ali, Aleksandra Rak-Raszewska. From left standing: Zenglai Tan, Khem Raj Giri, Ilya Skovorodkin, Ilkka Pietilä, Prateek Singh, Jingdong Shan, Timo
Pikkarainen, Anatoliy Samoylenko, Abhishek Sharma.
ABSTRACT
Emergence of form in living organisms in nature such as the one occurring during
morphogenesis is based on evolution and involves cell and tissue interactions.
The process occurs in three dimensions and leads gradually to the appearance of
specific structures via the process of pattern formation. This project targets the
fundamentals of morphogenesis and how terminally differentiated cells become
placed spatially and temporally so that the organ serves its function in the adult.
The kidney is used as a model for our studies. The capacity to program morphogenesis will open numerous avenues to develop novel and medically relevant
applications from reprogrammed cells.
RESEARCH PROJECTS
RESEARCH PROJECTS
Several emerging technologies and research lines have become available to study organogenesis. We can conduct genome-wide screening from single cells with the aid of microfluidistics. We can also establish stem cells from an adult
source or use pools of identified embryonic stem/progenitor
cells to generate specific cell types, spheroids or so-called organoids. The field benefits greatly from libraries of proteins and
extracellular matrix (ECM) components to identify conditions
that propagate progenitor cells such as those of the kidney.
Genetic technologies are available to map fates of specific cell
lineages. It is possible to purify specific primary cell types from
in vivo samples and reprogram them into progenitor states.
Such cells can be labelled by genetic means for fate mapping
(with time-lapse technology) during morphogenesis. Biomedical ICT has offered novel diagnostic avenues to be developed
for congenital disease diagnostics, for example. Via elegant
molecular imaging technologies virtual organ data banks can
be assembled from elementary regulatory components for in
silico modelling. The mechanical forces involved in organogenesis can be addressed in ex vivo organ cultures with the aid
of nanoparticles and microfluidistic platforms with cells that
have the capacity for complex interactions guiding morphogenesis. Finally, the emerging field of so-called Nanobiology
has opened great research avenues in organogenesis studies.
In the future organ assembly mechanisms can be expected to
offer ways to integrate cellular interactions to reach a better
view of how homeostasis is coordinated.
RECENT PROGRESS
We targeted the roles of the Wnt4 secreted signal in (mammalian) female Müllerian duct (MD) development. The MD is
the anlage of the oviduct, uterus and upper part of the vagina,
the main parts of the female reproductive tract. We found that
Wnt4 is critical for initiation and subsequent MD elongation.
Consistent with this, Wnt4 also promoted wound healing in
vitro. Some hypomorphic Wnt4mCherry/mCherry female mice
can survive to adulthood. Around 45% of cases acquired the
MD but demonstrated deficiencies in cell polarization and
basement membrane deposition relative to the controls. Indeed, the oviduct coils poorly, the endometrial glands remain
rudimentary and the myometrium undifferentiated. Some
mice had hydro-uterus as well. Our data indicate that Wnt4
is an important signal for female reproductive tract development.
Codon usage plays a crucial role when recombinant proteins
are expressed in different organisms. We described an opensource web-based application named ATGme. The program
can identify rare codons from most organisms, and provides
an opportunity to optimize the sequence. Collectively the developed application provides an interface with the following
optimization strategies: one-click optimization, bulk optimization, and individualized custom optimization. ATGme is
provided as an open-source application at http://atgme.org.
In collaborative work we developed certain Nano technological applications. These can be expected to offer power to
organogenesis studies as well. We synthesized and characterized gold Nano stars and their silica-coated derivatives with
7- to 50-nm shell thicknesses as contrast agents for optical
imaging. The scattering and absorption coefficients of these
nanoparticles (NPs) were estimated by means of collimated
transmittance and diffuse reflectance/transmittance analyses
and optical coherence tomography glass capillary imaging.
The silica-coated Nano stars have higher scattering ability than
the bare Nano stars. They were also weakly toxic for cells at
up to 200-μg/mL. Real-time visualization of Nano stars was
achieved in agarose and cell culture conditions. The intensity
of signal from the silica-coated NPs was higher in comparison
with bare Nano stars. The study was one of first ones where
laser confocal microscopy was applied to study combined scattering and light transmission modes and to reveal the backscattered signal from the gold Nano stars. These data should be
useful when considering analysis of uptake, translocation and
accumulation of NPs in living cells.
We targeted the roles of Wilms’ tumour protein (Wt1) functions, also in collaborative work. Wilms’ tumours are paediatric kidney cancers and biallelic loss of the Wt1 tumour suppressor gene is currently the best-characterized subgroup of
these tumours. A series of conditional knockout Wt1 mouse
models were developed. In these, Wt1 function was inactivated at different stages of nephrogenesis, before and after mesenchyme-to-epithelial transition (MET). The findings indicated
that Wt1 is functional throughout the developmental stages
in which it is expressed. With the aid of these novel models
a likely stage of origin of the corresponding human Wt1-mutant tumours was depicted. The results suggest that the noted
differences between human Wt1 tumour groups may be due
to their differential developmental origin during kidney ontogenesis.
We investigated the putative target genes of Wnt4 in the ovary by making use of murine knockout embryos. In the Wnt4
deficiency model the indifferent mammalian gonad becomes
determined to ovary or testis at mid-gestation, but the factors involved remain poorly characterized. Wnt4 represents
a female sex determinant, as its knockout leads to partial female-to-male sex reversal in mice. Notum, Phlda2, Runx1 and
Msx1 genes were identified as being typical of normal ovary,
while Osr2, Dach2, Pitx2 and Tacr3 genes were typical of embryonic testis. Interestingly, these latter genes were reversed in
their expression in the absence of Wnt4 function. Thus these
genes may normally have a role in control of ovarian development. Runx1 is considered as a putative Wnt4 target gene.
It is expressed in embryonic ovary and expression is reduced
in the case of Wnt4 knockout, while Wnt4 in turn induces
Runx1 expression. Wnt4 expression becomes down-regulated
when Runx1 function is impaired. The data suggests that the
transcription factor Runx1 may be a Wnt4 signalling target.
Runx1 and Wnt4 expression are mutually interdependent.
Thus Wnt4 signalling contributes to gonad development via a
complex gene network.
We addressed (in collaborative work) the roles of progesterone
on stem cell function by serially transplanting mouse mammary epithelial cells. Progesterone receptor (PR) knockout reduced mammary epithelial regeneration capacity. The PR target, receptor activator of Nf-κB ligand (RANKL), appears not
to be required for this function, but Wnt4 knockout was seen
to reduce mammary epithelial regeneration more than PR
knockout. Wnt4 appears to be perinatally independent of hormone signalling. However, in pubertal and adult stages, Wnt4
expression is localized to PR+ luminal cells and becomes dependent on PR signal transduction. Canonical Wnt signalling
in myoepithelium depends on PR and Wnt4 functions, but
the mammary bud and surrounding stroma are independent
of Wnt4. Together, the results suggest that progesterone and
Wnt4 control stem cell function via luminal–myoepithelial
crosstalk and that Wnt4 acts independently of PR function at
perinatal stages.
We developed a series of novel tissue-engineering methods in
connection with embryonic kidney mesenchyme (MM). We
used nephron segment-specific markers and showed that kidney tubule induction in the MM leads to assembly of highly
segmented nephrons ex vivo. The induction capacity could
be reconstituted when the MM tissue was dissociated into a
cell suspension and reaggregated (drMM) in the presence of
hBMP7 and hFGF2 for 24 hours prior to a tubule induction
stimulus. These growth factors promoted the capacity of the
ureteric bud to initiate branching with the reconstituted MM.
The data revealed that the nephron is apparently not derived
from a single progenitor cell, but a cluster of founder cells.
Transduction of green fluorescent protein (GFP) viruses was
efficient when the MM was dissociated and the nephrogenic competence of the GFP transduced was preserved. Viral
vector-mediated Lhx1 knockdown cells were excluded from
the nephric tubules, but control vector-containing cells were
incorporated. Together, the developed novel organ culture
methods enable detailed cellular and molecular studies of
nephrogenesis and kidney organogenesis in an ex vivo setting.
FUTURE GOALS
We have developed a series of technologies to study organ
morphogenesis in greater detail. These openings have made it
possible to target several key questions in the field. The technologies are based on use of primary embryonic progenitor
cells and will offer ways to make good use of other openings
in molecular biology, nanotechnology and imaging, to name
just a few. Moreover, these capacities offer new ways to integrate electronics, pharmacology and biophysics, to gain better
understanding of normal and dysfunctional development and
also diseases such as cancer. Our research can be expected to
lead to a better view of how morphoregulation during organogenesis occurs at the cellular and molecular level. Coupling
of nanotechnology and microfluidistic approaches to those of
genetic tools should offer great opportunities to develop organ
assembly-based therapies from programmed multipotent cells,
combined with skills in developmental programming and 3D
bio-printing. In summary, combination of several research
lines may be required to be able to engineer functional and
immunologically tolerated archetypes of organs and acquire
skills to construct transplants from reprogrammed stem cells.
DOCTORAL THESIS 2015
Ilkka Pietilä, The role of Dkk1 and Wnt5a in mammalian
kidney development and disease Acta Universitatis Ouluensis.
Series D, Medica 1280
Project Leader:
Seppo Vainio, Professor (University of Oulu)
Aleksandra Rak-Raszewska, Ph.D.
(FiDiPro, Academy of Finland)
Ilya Skovorodkin, Ph.D.
(Academy of Finland, Center of Excellence)
Timo Pikkarainen, Ph.D.
Anatoliy Samoylenko, Ph.D. (FiDiPro, Academy of Finland)
Nsrein Ali, Ph.D. (Tekes)
Genevieve Bart, Ph.D. (Tekes)
Mirja Krause, Ph.D. (Tekes)
Ilkka Pietilä, Ph.D. (Academy of Finland)
Jingdong Shan, Ph.D. (Bioncenter Oulu)
Renata Prunskaite-Hyyryläinen, Ph.D. (EURenomics)
Ph.D. Students:
Zenglai Tan, M.Sc. (EU, Marie Curie, RenalTract)
Prateek Singh, M.Sc. (Biocenter Oulu)
Susanna Kaisto, M.Sc. (Biocenter Oulu, Oulu-Ulm)
Abhishek Sharma, M.Sc. (Sigrid Jusélius Foundation)
Ulla Saarela, M.Sc. (FBMM)
Qi Xu, M.Sc. (Biocenter Oulu)
Tuomas Nurmi M.Sc. Student (Sigrid Jusélius Foundation)
Ganna Reint, M.Sc. (CIMO)
Kimmo Halt, M.D. (Oulu University Hospital)
FiDiPro:
Susan E Quaggin, M.D., Professor, Northwestern University
Feinberg School of Medicine, Director, Feinberg Cardiovascular Research Institute, Chief, Division of Medicine-Nephrology.
EURenOmics EU project
RenalTract Marie Curie ITN
Taina Pihlajaniemi, Director; Johanna Myllyharju, Vice
director; other Group leaders: Seppo Vainio, Robert Winqvist,
Lauri Eklund, Aki Manninen
Renata Prunskaite-Hyyryläinen, Baylor College of Medicine,
USA
Florence Naillat, University Cambridge, United Kingdom
RESEARCH PROJECTS
Ali N, Hosseini M, Vainio S, Taïeb A, CarioAndré M, Rezvani HR. Skin equivalents: skin
from reconstructions as models to study skin
development and diseases. Br J Dermatol
173:391-403, 2015.
Berry RL, Ozdemir DD, Aronow B, Lindström
NO, Dudnakova T, Thornburn A, Perry P, Baldock R, Armit C, Joshi A, Jeanpierre C, Shan
J, Vainio S, Baily J, Brownstein D, Davies J,
Hastie ND, Hohenstein P. Deducing the stage
of origin of Wilms’ tumours from a developmental series of Wt1-mutant mice. Dis Model Mech
8:903-17, 2015.
Bibikova O, Popov A, Bykov A, Prilepskii A,
Kinnunen M, Kordas K, Bogatyrev V, Khlebtsov N, Vainio S, Tuchin V. Optical properties
of plasmon-resonant bare and silica-coated
nanostars used for cell imaging. J Biomed Opt
20:76017, 2015.
Daniel E, Onwukwe GU, Wierenga RK, Quaggin
S E, Vainio S, Krause M. ATGme: Open-source
web application for rare codon identification
and custom DNA sequence optimization. BMC
Bioinformatics 16:303, 2015.
Junttila S, Saarela U, Halt K, Manninen A,
Pärssinen H, Lecca RM, Brandli AW, SimsLucas S, Skovorodkin I, Vainio SJ. Functional genetic targeting of the embryonic kidney
progenitor cells ex vivo. J Am Soc Nephrol
26(5):1126-37, 2015.
Krause M, Rak-Raszewska A, Pietilä I, Quaggin SE, Vainio S. Signaling during Kidney Development. Cells 4(2):112-32. doi: 10.3390/
cells4020112, 2015.
Krause M, Samoylenko A, Vainio SJ. Exosomes
as renal inductive signals in health and disease,
and their application as diagnostic markers and
therapeutic agents. Front Cell Dev Biol 3:65,
2015.
Naillat F, Yan W, Karjalainen R, Liakhovitskaia
A, Samoylenko A, Xu Q, Sun Z, Shen B, Medvinsky A, Quaggin S, Vainio SJ. Identification
of the genes regulated by Wnt-4, a critical signal for commitment of the ovary. Exp Cell Res
332:163-78, 2015.
Rajaram RD, Buric D, Caikovski M, Ayyanan
A, Rougemont J, Shan J, Vainio SJ, YalcinOzuysal O, Brisken C. Progesterone and Wnt4
control mammary stem cells via myoepithelial
crosstalk. EMBO J 34:641-52, 2015.
Rak-Raszewska A, Hauser PV, Vainio S. Organ
In Vitro Culture: What Have We Learned about
Early Kidney Development? Stem Cells Int doi:
10.1155/2015/959807, 2015.
Littunen K, Snoei de Castro J, Samoylenko A,
Xu Q, Quaggin S, Vainio S, Seppälä J. Synthesis of cationized nanofibrillated cellulose and its
antimicrobial properties. Eur Polymer J 75:116–
124, 2016.
Pietilä I, Prunskaite-Hyyryläinen R, Kaisto S,
Nicolaou N , van Eerde AM , Salo AM, Garma L,
Miinalainen I, Feitz WF, Bongers EMHF, Juffer
AH, Knoers NVAM, Renkema KY, Myllyharju J,
Vainio SJ. Wnt5a deficiency leads to anomalies
in ureteric tree development, tubular epithelial cell organization and basement membrane
integrity pointing to a role in kidney collecting
duct patterning. PloS ONE, 11(1):e0147171, 2016.
Prunskaite-Hyyryläinen R, Skovorodkin I, Xu
Q, Miinalainen I, Shan J, Vainio SJ. Wnt4 coordinates directional cell migration and extension
of the Müllerian duct essential for ontogenesis
of the female reproductive tract. Hum Mol Genet
25(6):1059-73, 2016.
Halt K, Pärssinen H, Junttila S, Saarela U,
Sims-Lucas S, Koivunen P, Myllyharju J,
Quaggin S, Skovorodkin I, Vainio SJ. The
CD146+ cells are essential for kidney vasculature development. Kidney Int, in press.
From left: Hanna Tuppurainen, Katri Pylkäs, Anna Tervasmäki, Meeri Otsukka, Annika Väntänen, Leena Keskitalo, Hellevi Peltoketo, Niina Laurila, Robert Winqvist, Muthiah Bose.
In front: Tuomo Mantere, Raman Devarajan.
PROJECT LEADER
Prof. Robert Winqvist, Ph.D.
Laboratory of Cancer Genetics and Tumour Biology,
Cancer and Translational Medicine Research Unit,
Faculty of Medicine, University of Oulu
FACTORS AND MECHANISMS
IN HEREDITARY BREAST CANCER
PREDISPOSITION
ABSTRACT
Breast cancer is the most common malignancy among women.
Accumulation of various genetic and epigenetic lesions plays
a major role both in initial development and in progression
of the disease. Constitutional mutations in high-penetrance
susceptibility genes such as BRCA1 and BRCA2 account for
approximately 5–10% of breast cancer cases, but recently
more commonly occurring mutations in various moderate- and
low-penetrance susceptibility genes have also been uncovered. Thus, a much greater proportion of breast cancer than
was previously anticipated is associated with hereditary risk
factors. Unfortunately, however, the mechanistic details of
how dysfunctional hereditary factors act to promote malignancy development has remained largely obscure. In addition,
a considerable fraction of the components involved in breast
cancer predisposition are still unknown. Consequently, besides studying more closely the effects of susceptibility gene
mutations already discovered in our previous studies, in our
research we are also trying to uncover additional, currently
unknown predisposing factors and to learn more about their
molecular interactions and biological effects as well as the
outcome of their functional inabilities. The functional consequences of observed genomic lesions will also be assessed
by using multiple disease modelling regimes. Ultimately, our
investigations should contribute to increased knowledge of key
molecular interactions, pathways and biological networks as
well as the impact and influence of various susceptibility gene
lesions on breast cancer risk, and in general, on the biology
of tumour development as well as on their effects on disease
behaviour.
RESEARCH PROJECTS
RESEARCH PROJECTS
Breast cancer is a heterogeneous disease and is the most common malignancy among women. At present, about one in
eight women will eventually develop breast cancer and despite
significant advances in detection and treatment, breast cancer
still remains a major cause of death in the Western world.
Accumulation of various genomic lesions plays a major role
both in the initial development and in the progression of
breast cancer. Increased genome instability is recognized as
a hallmark of cancer. Although most cases of female breast
cancer appear to be “sporadic”, between 5 and 10% of the
cases are estimated to have a strong hereditary background,
due to the transmission of certain harmful genetic risk factors, which make it more plausible for a mutation carrier
to develop a malignancy – often at a much earlier age than
in the general population. Unfortunately, however, the
known major susceptibility genes BRCA1 and BRCA2 explain only about 20% of cases, and altogether the known
high-penetrance heritable risk factors contribute to approximately 25% of familial breast cancer cases, leaving the remaining 75% unexplained. Therefore, the remaining highrisk genetic factors also need to be identified. In addition
to high-penetrance susceptibility factors, more recently multiple and more commonly occurring moderate- as well as
low-penetrance constitutional cancer-predisposing variants
have been identified. Together with various environmental
and life-style risk factors, all of these kinds of constitutional
disease-predisposing factors are likely to play very significant
roles in the aetiology of breast cancer.
Shared features of many of the presently known cancer susceptibility genes include the nature of their protein products,
which typically perform important functions in cell-cycle control, DNA double-strand damage response pathways, as well
as in other genome integrity control utilities. Therefore, it is
plausible that other genes influencing these essential cellular
pathways would also be targets as regards germline mutations
associated with an increased cancer risk.
The increased knowledge of the biology of tumour formation
and cancer progression can subsequently be utilized to develop
more effective tools for diagnostic, prognostic and therapeutic
purposes. Effective identification of individuals at significantly increased hereditary disease risk, together with intensified
surveillance of such individuals for early-stage malignancy detection is imperative for successful treatment and favourable
prognosis.
RECENT PROGRESS
The general aim of our research is to identify the most important genomic factors involved in the development and
also in the progression of breast cancer. Additionally, a better
understanding of the biological consequences of the various
aberrations observed in these genes will be helpful for creating more effective means of disease prevention, diagnosis and
treatment. Our investigations focus particularly on hereditary
predisposition to breast cancer and on unravelling novel susceptibility factors, mainly using a case-control study design,
supplemented by the utilisation of geographically matched
incident hospital-based breast cancer cases, unselected for a
family history of cancer.
By studying Northern Finnish breast cancer families that
had previously tested negative for germline mutations in
the BRCA1 and BRCA2 genes, during the past few years we
have been able to uncover several important and novel cancer susceptibility genes (e.g. PALB2, RAD50, NBS1, RAP80,
ABRAXAS, MCPH1). Furthermore, in studies that have also
included the ATM gene, we have been able to provide evidence of involvement of both haploinsufficiency and dominant-negative allele behaviour in heritable cancer risk. We
have also carried out some initial investigations to characterise
the molecular, physiological and clinical consequences of the
observed genetic aberrations, those of PALB2 in particular,
and more detailed assessments are currently in progress.
We reported in Nature in 2007 the discovery of PALB2, a
novel breast cancer susceptibility gene, the protein product of
which regulates key features of the physiological responses to
DNA damage mediated by the BRCA2 susceptibility product.
In addition, PALB2 directly interacts with the other major
breast cancer susceptibility gene product, BRCA1. In fact,
PALB2 bridges the interplay between these two biologically
essential proteins.
In a subsequent study published in Clinical Cancer Research
in 2008, we observed that carriers of this relatively common
Finnish PALB2 founder mutation on average displayed a
breast cancer risk of 40% by age 70, which is a substantially
higher risk estimate than had been originally anticipated. The
number is similar to that determined for BRCA2 (45%), but
somewhat lower than that for BRCA1 (76%). Consequently,
PALB2 germline mutations are likely to have important physiological as well as clinical implications. Interestingly, there is
now evidence for the existence of many different breast cancer
predisposition-related germline mutations in PALB2 in various populations worldwide (e.g. Finland, the UK, the Netherlands, Belgium, Italy, Germany, Poland, Canada, the USA,
South Africa, China and Australia), underlining the significant contribution of PALB2 dysfunction in hereditary-based
breast cancer development.
Testing for mutations in the BRCA1 and BRCA2 genes among
high-risk breast cancer patients has become routine practice in
clinical genetics. Unfortunately, however, at present the genetic background of the majority of cases coming to the clinics
remains currently unexplained, making genetic counselling
rather challenging. We have carried out a study evaluating
the need for routine clinical testing for the relatively common PALB2 c.1592delT founder mutation in BRCA-negative
Northern Finnish breast cancer families (BMC Med Genet
2013). The study revealed multiple PALB2 c.1592delT mutation carriers among the studied high-risk breast cancer cases. Subsequently, all of our exciting initial findings regarding
PALB2 have been further confirmed in a large comprehensive
international meta-analysis of families with various heterozygous loss-of-function PALB2 mutations, coordinated by the
PALB2 Interest Group (NEJM 2014). The observed breast
cancer risk of female PALB2 mutation carriers varied between
33 and 58%, and the greatest risk was seen for persons with
close relatives with breast cancer. All of the currently availa-
ble information supports the notion that besides BRCA1 and
BRCA2, PALB2 is also a high-risk susceptibility gene for female breast cancer, thus warranting its inclusion in the standard genetic counselling mutation screening protocol offered
to individuals at an increased risk of hereditary breast cancer.
In a study very recently published in PloS Genetics (2016), we
have discovered yet another novel gene, MCPH1, involved in
hereditary breast cancer susceptibility. As many as 21 cancer
families from Northern Finland have a founder germline mutation in this gene identified through massive parallel sequencing of hundreds of DNA damage response genes. Owing to
significantly elevated genomic instability, heterozygous mutation carriers exhibited a 3- to 8-fold increased risk of cancer.
Besides breast cancer, one third of the families with MCPH1
mutation also exhibited brain tumours and/or sarcomas. Next
it will be important to explore the role of MCPH1 mutations
in relation to cancer susceptibility in other populations.
Although females carrying a heterozygous germline mutation
in BRCA1, BRCA2, PALB2, MCPH1 or some other key gene
are associated with an increased propensity to develop breast
cancer, the mechanistic details of how this adverse effect is
brought about have remained largely obscure. For instance,
it has been previously shown that the BRCA1, BRCA2 and
PALB2 proteins together regulate cellular repair of DNA
breaks by homologous recombination. In order to investigate
the molecular basis and biological mechanisms that underlie
hereditable cancer risk we first focused on the PALB2 gene,
using lymphoblastoid cell lines derived from Finnish familial breast cancer patients that all harbour the same heterozygous c.1592delT truncation mutation. This mutation occurs
in as much as 1% of all Finnish breast cancer patients and
is expected to behave similarly to other PALB2 truncation
mutations worldwide that are associated with breast cancer
predisposition.
The mutation carrier cells still have one functional copy of the
PALB2 gene and contained approximately half the amount of
PALB2 protein compared with that in healthy control cells.
The carrier cells showed accelerated duplication of their DNA.
We discovered that PALB2 mutation carrier cells started replication twice as often as unaffected control cells, in this way
‘rushing’ through the process, utilizing part of the normally
dormant origins in the absence of an external disturbance. This
leads to problems, since DNA replication seems to stall more
regularly in the mutation carrier cells. What is more, these
cells are no longer able to react upon disturbances, since part
of the back-up of replication start sites is already exhausted
during normal growth. Furthermore, growing cells use multiple consecutive checkpoints to survey the genome for damage
and to ensure that one stage of the cell division cycle has been
completed successfully before the next stage is initiated. In
cases of DNA damage, PALB2 mutation carrier cells first show
a robust checkpoint response, but in contrast to healthy control cells, the carrier cells fail to maintain this checkpoint response and resume cell growth despite persisting damage. This
leads to genomic instability that is the driving force behind the
early stages of cancer development. The fact that metaphases
from short-term cultivation of primary blood lymphocytes
of heterozygous PALB2 mutation carriers show an increased
number of chromosome aberrations demonstrates that the ge-
nome destabilisation observed in our experiments is also operational at the organism level. As PALB2 haploinsufficiency
has already been found to cause aberrant DNA replication/
damage response resulting in increased genomic instability, it
would suggest that functional loss of the remaining wild-type
allele, as defined by the Knudson two-hit hypothesis, may not
always be a prerequisite for malignancy development. Altogether, these findings provide a new and exciting mechanism
for the early stages of breast cancer development that may also
apply to mutations in other genes indicated in hereditary cancer predisposition (Nat Commun 2013, Oncogene 2015).
We have also successfully carried out mouse modelling studies
in connection with constitutional PALB2 gene defects (Oncogene 2010). Mice lacking proper Palb2 function die as embryos and display defective mesoderm differentiation, suggesting
an essential role for Palb2 in embryogenesis, possibly in concert with extracellular matrix influences. Mice lacking Palb2
display failure in epithelial-mesenchymal transition (EMT),
a feature shared with many cancers. Follow-up investigations
concerning these important findings are on their way.
For several years we have actively participated in various
collaborative international studies via BCAC, TNBBC and
PALB2 Interest Group consortium initiatives. Many of these
investigations have been very successful, especially in revealing
major contributions of various novel low-penetrance susceptibility alleles in the aetiology of breast cancer, non-familial
disease in particular. Importantly, the same massive case-control studies have also uncovered the existence of a new class of
constitutional genetic variants, namely those that are helpful
in counteracting the development of cancer. Furthermore,
these consortium studies have revealed clinically important
associations between patient genotype and disease phenotype
and outcome.
FUTURE GOALS
The main aims of our ongoing studies include the following:
1. We will try to identify additional constitutional breast
cancer susceptibility alleles by multiple and partly parallel
approaches: screening of candidate genes for high- or moderate-penetrance mutations, and genome-wide SNP association
studies to identify relatively frequently occurring low-penetrance cancer risk alleles or risk haplotypes, including polyallelic clusters (mainly through international consortium collaboration). Furthermore, genome-wide exome sequencing and
targeted high-throughput deep-sequencing will be utilised in
order to obtain a more comprehensive view of the involved
germline defects in predisposition to breast cancer.
2. We will continue to use lymphoblastoid and fibroblast cell
lines derived from various germline mutation-positive individuals to characterise the molecular and physiological effects
of these cancer-associated genetic defects in yet greater detail.
Case and control cell lines will be challenged with various doses of γ-radiation, etoposide or hydroxyurea, for instance, and
compared as regards molecular responses and DNA damage
correction capacity and accuracy. In addition, in vitro 2D and
3D epithelial breast cancer modelling as well as disease modelling in mice will be used.
RESEARCH PROJECTS
RESEARCH PROJECTS
3. Various comprehensive next-generation sequencing approaches both at the DNA and RNA level will be used in order to define the high-resolution clonal cell population-specific aberration profiles of tumour specimens and other suitable
tissue specimens derived from individuals displaying various
constitutional gene aberrations originally identified in our laboratory in Oulu. In addition, CRISPR/Cas9 genome editing
will be used to replicate naturally occurring key breast cancer
susceptibility gene defects as well as somatic mutations and
to explore their molecular and biological consequences under
carefully monitored experimental conditions.
Kim Obermeier, M.Sc. (jointly with Prof. Lisa Wiesmüller,
International Graduate School in Molecular Medicine Ulm,
Germany), until dissertation in Ulm 7.1.2015.
Juliane Sachsenweger, M.Sc. (jointly with Lisa Wiesmüller,
International Graduate School in Molecular Medicine Ulm,
Germany, and Biocenter Oulu Doctoral Programme)
Ultimately, we hope to unravel as many novel hereditary risk
factors as we can and assess their possible combinatory effects on the development of breast cancer in particular. By
characterising the molecular, physiological and clinical effects
of these genetic and epigenetic lesions we aim to add to the
knowledge of critical biological pathways and mechanisms related to malignancy development, thus contributing towards
better means of assessing disease risk, and towards the prevention and improved diagnostics, monitoring and treatment of
cancer.
The International Graduate School in Molecular Medicine
Ulm, Germany: Joint Ph.D. project supervision
2010–1.2015
Prof. Lisa Wiesmüller and Robert Winqvist: M.Sc. Kim Obermeier’s Ph.D. project: Functional classification of cells derived
from patients carrying the Finnish PALB2 founder mutation.
Dissertation in Ulm, Germany, 7.1.2015.
Project Leader:
Robert Winqvist, Ph.D., Professor (University of Oulu)
Katri Pylkäs, Ph.D. (Academy of Finland, Biocenter Oulu)
Hellevi Peltoketo, Ph.D. (Academy of Finland)
Maria Haanpää, M.D., Ph.D. (from October 1, Sigrid
Juselius Post-doctoral Fellowship, Stanford University, USA)
Arja Jukkola-Vuorinen, M.D., Ph.D.
Mervi Grip, M.D. (Oulu University Hospital)
Saila Kauppila, M.D., Ph.D. (Oulu University Hospital)
Jukka Moilanen, M.D., Ph.D. (Oulu University Hospital)
Ph.D. Students:
Muthiah Bose, M.Sc. (Academy of Finland)
Tuomo Mantere, M.Sc.
(UniOGS, Biocenter Oulu Doctoral Programme)
Raman Devarajan, M.Sc.
(jointly with Taina Pihlajaniemi group, Academy of Finland)
Anna Tervasmäki, M.Sc.
Niina Laurila, M.Sc.
Hanna Tuppurainen, M.Sc.
(Biocenter Oulu, Finnish Cancer Foundation)
(Academy of Finland and Biocenter Oulu)
Academy of Finland Programme for 2012–2017
Taina Pihlajaniemi, Director; Johanna Myllyharju, Vice director; other Group leaders: Seppo Vainio, Robert Winqvist,
Lauri Eklund, Aki Manninen
The International Graduate School in Molecular Medicine
Ulm, Germany, and UniOGS/Biocenter Oulu Doctoral
Programme, University of Oulu: Joint Ph.D. project
supervision 10.2014–9.2017
Prof. Lisa Wiesmüller and Robert Winqvist: M.Sc. Juliane
Sachsenweger’s Ph.D project.
Muthiah Bose, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Niina Laurila, Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena, Germany
Visting Researchers in 2015 (over two weeks)
M.Sc. Franziska Feiertag, University of Jena, Germany
EU Projects
EU COGS collaborative initiative on hereditary susceptibility to cancer. The Oulu Breast Cancer Study, represented by
our team, is a member of the BCAC (breast cancer association consortium) sub-study of COGS that is coordinated by
Prof. Douglas Easton (Cambridge, UK), performing microarray-based, genome-wide SNP studies to reveal novel disease
association factors as well as patterns of occurrence and characteristic phenotypic and clinical features.
Other International Consortium Activities
The clinical parameter stratification approach also includes
participation in the Triple-Negative Breast Cancer Consortium (TNBCC) coordinated by Prof. Fergus Couch (Rochester,
USA). The studies of this consortium are focused on individuals with breast cancer negative for the oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, and will provide important insights into the aetiology of
different breast tumour subtypes.
The “PALB2 Interest Group” was established in 2007 through
the joint initiative of Prof. William Foulkes (coordinator;
Montreal, Canada), Prof. Melissa Southey (Melbourne, Australia) and Prof. Robert Winqvist for carrying out and coordinating comprehensive world-wide PALB2-related research
projects. These activities include further characterisation of
the occurrence and role of PALB2 defects in hereditary predis-
position to breast cancer as well as of the molecular and mechanistic details of how the defective gene function increases the
cancer risk of carrier individuals. A central goal of this activity,
based on the accumulated new information, is eventually to
establish some guidelines and recommendations for the clinical utilisation of PALB2 mutation data.
Darabi H, McCue K, Beesley J, Michailidou K,
Nord S, Kar S et al (Winqvist R, Jukkola-Vuorinen A of Oulu Breast Cancer Study). Polymorphisms in a Putative Enhancer at the 10q21.2
Breast Cancer Risk Locus Regulate NRBF2
Expression. Am J Hum Genet 97:22-34, 2015.
Day FR, Ruth KS, Thompson DJ, Lunetta KL,
Pervjakova N, Chasman DI et al (incl. Winqvist
R, Pylkäs K of Oulu Breast Cancer Study).
Large-scale genomic analyses link reproductive
aging to hypothalamic signaling, breast cancer
susceptibility and BRCA1-mediated DNA repair.
Nat Genet 2015 47:1294-303, 2015.
Glubb DM, Maranian MJ, Michailidou K, Pooley
KA, Meyer KB, Kar S et al (incl. Winqvist R,
Pylkäs K, Jukkola-Vuorinen A, Kauppila S of
Oulu Breast Cancer Study). Fine-scale mapping of the 5q11.2 breast cancer locus reveals at
least three independent risk variants regulating
MAP3K1. Am J Hum Genet 96:5-20, 2015.
Guo Q, Schmidt MK, Kraft P, Canisius S, Chen
C, Khan S et al (incl. Winqvist R, Pylkäs K,
Jukkola-Vuorinen A, Grip M of Oulu Breast
Cancer Study). Identification of novel genetic
markers of breast cancer survival. J Natl Cancer Inst 107 pii: djv081, 2015.
Guo X, Long J, Zeng C, Michailidou K, Ghoussaini M, Bolla MK et al (incl. Winqvist R of Oulu
Breast Cancer Study). Fine-Scale Mapping of
the 4q24 Locus Identifies Two Independent Loci
Associated with Breast Cancer Risk. Cancer
Epidemiol Biomarkers Prev 24:1680-1691, 2015.
Hartikainen JM, Tengström M, Winqvist R, Jukkola-Vuorinen A, Pylkäs K, Kosma VM, Soini Y,
Mannermaa A. KEAP1 Genetic Polymorphisms
Associate with Breast Cancer Risk and Survival
Outcomes. Clin Cancer Res 21:1591-1601, 2015.
Jamshidi M, Fagerholm R, Khan S, Aittomäki
K, Czene K, Darabi H et al (incl. Winqvist R,
Pylkäs K of Oulu Breast Cancer Study). SNPSNP interaction analysis of NF-kB signaling
pathway on breast cancer survival. Oncotarget
6:37979-37994, 2015.
Kabisch M, Lorenzo Bermejo J, Dünnebier T,
Ying S, Michailidou K, Bolla MK et al (incl. Winqvist R, Pylkäs K, Jukkola-Vuorinen A, Grip M
of Oulu Breast Cancer Study). Inherited variants in the inner centromere protein (INCENP)
gene of the chromosomal passenger complex
contribute to the susceptibility of ER-negative
breast cancer. Carcinogenesis 36:256-271,
2015.
cer Study). Common germline polymorphisms
associated with breast cancer-specific survival.
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Lin WY, Camp NJ, Ghoussaini M, Beesley J,
Michailidou K, Hopper JL et al (incl. Winqvist
R, Pylkäs K, Jukkola-Vuorinen A, Kauppila S
of Oulu Breast Cancer Study). Identification
and characterization of novel associations in
the CASP8/ALS2CR12 region on chromosome
2 with breast cancer risk. Hum Mol Genet
24:285-298, 2015.
Zhang B, Shu XO, Delahanty RJ, Zeng C,
Michailidou K, Bolla MK et al (incl. Winqvist R,
Pylkäs K, Jukkola-Vuorinen A, Grip M of Oulu
Breast Cancer Study). Height and Breast Cancer Risk: Evidence From Prospective Studies
and Mendelian Randomization. J Natl Cancer
Inst 107 pii: djv219, 2015.
Mantere T, Haanpää M, Hanenberg H,
Schleutker J, Kallioniemi A, Kähkönen M, Parto
K, Avela K, Aittomäki K, von Koskull H, Hartikainen JM, Kosma VM, Laasanen SL, Mannermaa A, Pylkäs K, Winqvist R. Finnish Fanconi
anemia mutations and hereditary predisposition
to breast and prostate cancer. Clin Genet 88:6873, 2015.
Mavaddat N, Pharoah PD, Michailidou K, Tyrer
J, Brook MN, Bolla MK et al (incl. Winqvist R,
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Dennis J, Wang Q, Barrowdale D et al (incl.
Winqvist R, Pylkäs K of Oulu Breast Cancer Study). BRCA2 Polymorphic Stop Codon
K3326X and the Risk of Breast, Prostate, and
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Michailidou K, Beesley J, Lindström S, Canisius S, Dennis J, Lush MJ et al (incl. Winqvist R,
Breast Cancer Study). Genome-wide association analysis of more than 120,000 individuals
identifies 15 new susceptibility loci for breast
cancer. Nat Genet 47:373-380, 2015.
Orr N, Dudbridge F, Dryden N, Maguire S, Novo
D, Perrakis E et al (incl. Winqvist R, Pylkäs K,
Jukkola-Vuorinen A, Grip M of Oulu Breast
Cancer Study). Fine-mapping identifies two
additional breast cancer susceptibility loci at
9q31.2. Hum Mol Genet 24:2966-2984, 2015.
Pirie A, Guo Q, Kraft P, Canisius S, Eccles DM,
Rahman N, (incl. Winqvist R, Pylkäs K, Jukkola-Vuorinen A, Grip M of Oulu Breast Can-
Lei J, Rudolph A, Moysich KB, Behrens S,
Goode EL, Bolla MK et al (incl. Winqvist R,
Grip M of Oulu Breast Cancer Study). Genetic
variation in the immunosuppression pathway
genes and breast cancer susceptibility: a pooled
analysis of 42,510 cases and 40,577 controls
from the Breast Cancer Association Consortium. Hum Genet 135:137-154, 2016.
Mantere T, Winqvist R*, Kauppila S, Grip M,
Jukkola-Vuorinen A, Tervasmäki A, Rapakko
K, Pylkäs K* (shared senior authorship). Targeted Next-Generation Sequencing Identifies a
Recurrent Mutation in MCPH1 Associating with
Hereditary Breast Cancer Susceptibility. PLoS
Genet 12:e1005816, 2016.
Dunning AM, Michailidou K, Kuchenbaecker
KB, Thompson D, French JD, Beesley J et al
(incl. Winqvist R, Pylkäs K of Oulu Breast
Cancer Study). Breast cancer risk variants at
6q25 display different phenotype associations
and regulate ESR1, RMND1 and CCDC170. Nat
Genet, in press.
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Johnatty SE, Aben KK, Agnarsson BA et al
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clinical utility of KRAS variant rs61764370 for
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PALB2 c.1592delT mutation channels DNA
double-strand break repair into error-prone
pathways in breast cancer patients. Oncogene
doi: 10.1038/onc.2015.448, 2015, Epub ahead
of print.
Diphtheria toxin-like human ADP-ribosyltransferases
(ARTDs), also known as poly (ADP-ribose) polymerases
(PARPs), are enzymes that catalyze a covalent modification
of target proteins – ADP-ribosylation. In the reaction NAD+
cofactor is cleaved to nicotinamide and ADP-ribose and an
ADP-ribosyl group is attached to the target protein or to the
growing polymer chain. The modification changes properties
of the target protein and the resulting ADP-ribose polymer
also interacts with various proteins. The polymer is metabolized by glucohydrolases that limit the time of the signalling
event.
The human ARTD enzyme family consists of 18 multidomain proteins all sharing a conserved catalytic domain. The
human enzyme family can be divided on the basis of activity to polymerases (ARTD1–6), mono ADP-ribosyltransferases (mARTDs; ARTD8–18) and the probably inactive
ARTD9/13.
From left: Lari Lehtiö, Yves Nkizinkiko, Alam Mahanudul, Harikanth Venkannagari, Sudarshan Murthy, Teemu Haikarainen, Alexander Ignatev, Ezeogo Obaji,
Mirko Maksimainen, Yashwanth Asok.
STRUCTURE, FUNCTION
AND SELECTIVE INHIBITION
OF ADP-RIBOSYLTRANSFERASES
PROJECT LEADER
Docent Lari Lehtiö, Ph.D.
University of Oulu
The widely studied human enzyme ARTD1/PARP1 is involved
in many processes, but it is mainly known for its function in
DNA repair mechanisms. On the other hand, ARTD1 is also
linked to death of the cell, as over-activation of ARTD1, due
to extensive DNA damage, leads to cell death. The involvement of ARTD1 in DNA repair implies that inhibitors could
be used to enhance the effects of anticancer drugs, whereas
the involvement in cell death suggests that inhibitors could
be used in cases of injury and inflammation. Currently many
ARTD1 inhibitors or “PARP inhibitors” are in clinical trials
and the first PARP inhibitor, Olaparib, was accepted in 2014
for the treatment of ovarian cancer. Notably, other members of
the ARTD family also have functions that could be potentially
utilized in therapeutics, especially for cancer, as many ARTD
enzymes are involved in DNA repair and cell proliferation.
RECENT PROGRESS
Development of chemical probes and drug leads
One of the focus areas of our group is the development of
chemical probes to be used as tools to study enzymes and development of drug leads to facilitate the development of therapeutics and to validate enzymes as drug targets. Our drug
discovery efforts have recently been focused on tankyrases
(TNKS1/ARTD5 and TNKS2/ARTD6), which are promising drug targets due to their involvement in the Wnt signalling pathway. In recent years several new inhibitors have
been described and they can be classified by their binding to
the nicotinamide subsite, to the adenosine subsite or to both
sites. We have studied different inhibitors anchoring either to
the nicotinamide or to the adenosine binding sites, which has
enabled us to develop several potent and selective tankyrase
inhibitors. We use protein X-ray crystallography to study
protein–ligand interactions and this molecular information
is combined with various techniques ranging from enzyme
inhibition measurements to calorimetry (Figure 1). Several
new inhibitor scaffolds to be used as drug leads have been
developed together with our collaborators. We have analysed
inhibitor selectivity with activity assays, which we have now
optimized for the majority of recombinantly produced human ARTDs. In addition to structure-based design we are also
screening random compound libraries and this way we have
been able to find new chemical probes, which are currently
under characterization and further development.
ABSTRACT
ADP-ribosylation is a covalent post-translational protein modification involved in
many cellular signalling events. We study the structures and mechanisms of the
enzymes involved and aim to develop potent and selective inhibitors to be used
as drug leads and as chemical tools to study the enzymes. We develop assays
suitable for high-throughput screening of inhibitors and use structural information for more rational design. Protein X-ray crystallography is our main tool used
to understand structure-activity relationships of compounds. We also use small
angle X-ray scattering and other biophysical techniques to study the functions of
these multidomain proteins.
Figure 1. Ligand-binding studies using different techniques.
A) Dose–response measurement of ARTD10 inhibitor with
a fluorescence-based activity
assay. B) Thermal shift assay of
different small molecules showing various degrees of protein
stabilization. C) Isothermal titration calorimetry of a ligand
to a macro domain. D) Crystal
structure of a tankyrase-inhibitor complex.
perform ADP-ribosylation, bind and recognize the modification or hydrolyse it. There are many unanswered questions
regarding the functions of the enzymes involved in ADP-ribosylation and we aim to understand the molecular details using
activity assays, biophysical methods and structural studies.
Project workers:
Vesa Hautaniemi, M. Sc.
(Sigrid Jusélius Foundation, Biocenter Oulu)
Syed Sahan, B. Sc. (Sigrid Jusélius Foundation)
Mikko Hynönen (Sigrid Jusélius Foundation)
Project Leader:
Lari Lehtiö, Ph.D., Docent
(Biocenter Oulu, Academy of Finland)
Teemu Haikarainen, Ph.D. (Academy of Finland)
Alexander Ignatev, Ph.D. (Jane and Aatos Erkko Foundation)
Mirko Maksimainen, Ph.D. (Academy of Finland)
Figure 2. Crystal structures of Trypanosoma macro domains. A) Snowman-like crystal and B) crystal structure of ADP-ribose bound to a macro domain.
Crystal structures of enzymes involved in ADP-ribosylation
We are using protein X-ray crystallography in order to study
structures of the enzymes involved in ADP-ribose signalling.
This technique allows us to look at the molecular details of
the enzymes and their interactions (Figure 2). In combination
with supporting biochemical and biophysical methods available at Biocenter Oulu, we can understand how these enzymes
control important cellular signalling events. These studies will
result in better understanding of the proteins involved and
will also reveal new possibilities to create therapeutic agents
against human diseases.
Structural and functional studies of multidomain ARTDs
ARTD enzymes contain a homologous transferase domain
usually located at the C-terminus, and in addition to that
many other domains which differ between enzymes. These
domains contribute to the function and localization of the
enzymes. The interplay between domains in interaction and
activation processes is largely unknown and we use both biophysical and structural methods in order to understand how
the enzymes function. We use X-ray crystallography to study
the structures of full-length enzymes, individual domains and
macromolecular complexes and combine this with low-resolution structural studies (Figure 3). Activity assays, which we use
also for screening of compound libraries, allow us to study the
effects of interaction partners on catalytic activity. We use calorimetry and fluorescence polarization to study affinities and
stoichiometry between macromolecules and we create various
truncated constructs to study the roles of individual domains.
We have particularly studied enzymes related to DNA repair,
both in humans and in the human parasites Trypanosoma cruzi and brucei. The studies reveal what type of DNA damage
these proteins recognize, which lesion models activate the enzymes, which domains are required for affinity and activation
and how the proteins are organized when binding to different
DNA models.
Margherita Miele, University of Perugia, Italy
Ph.D. Students:
Harikanth Venkannagari, M.Sc.
(Jane and Aatos Erkko Foundation, Sigrid Jusélius
Foundation, Academy of Finland)
Ezeogo Obaji, M.Sc. (Sigrid Jusélius Foundation,
Jane and Aatos Erkko Foundation)
Yves Nkizinkiko, M.Sc. (Biocenter Oulu)
Sudarshan Murthy, M.Sc. (Sigrid Jusélius Foundation)
Yashwanth Ashok, M. Sc. (Biocenter Oulu)
Kumpan K, Nathubhai A, Zhang C, Wood PJ,
Lloyd MD, Thompson AS, Haikarainen T, Lehtiö L, Threadgill MD. Structure-based design,
synthesis and evaluation in vitro of arylnaphthyridinones, arylpyridopyrimidinones and
their tetrahydro derivatives as inhibitors of
the tankyrases. Bioorg Med Chem 23:3013-32,
2015.
Nkizinkiko Y, Suneel Kumar BV, Jeankumar
VU, Haikarainen T, Koivunen J, Madhuri C,
Yogeeswari P, Venkannagari H, Obaji E, Pihlajaniemi T, Sriram D, Lehtiö L. Discovery of
potent and selective nonplanar tankyrase inhibiting nicotinamide mimics. Bioorg Med Chem
23:4139-4149, 2015.
Paine HA, Nathubhai A, Woon EC, Sunderland
PT, Wood PJ, Mahon MF, Lloyd MD, Thompson AS, Haikarainen T, Narwal M, Lehtiö L,
Threadgill MD. Exploration of the nicotinamide-binding site of the tankyrases, identifying
FUTURE GOALS
An important goal of the group is to develop effective and robust methods to screen inhibitors and especially to efficiently
test the inhibitors against all members of the human ARTD
family. Selective inhibitors will make it possible to study and
verify the biological functions of the enzymes. This would potentially also create new drug leads, as many of the enzymes
appear to have functions and interactions that are potentially
of pharmaceutical interest. We have already identified selective
inhibitors of some human ARTD enzymes and they are currently being evaluated using structural studies and cell-based
assays.
Figure 3. We use small angle X-ray scattering combined with crystal
structures and biophysical methods in order to understand how multidomain enzymes work. Rigid-body modelling of crystal structures of individual protein domains and DNA is shown, as fitted to the low-resolution
SAXS envelope.
We have also expanded the set of target proteins involved in
ADP-ribosylation and are currently carrying out structural
and functional studies as well as inhibitor development on
several related human and parasite protein families, which
3-arylisoquinolin-1-ones as potent and selective
inhibitors in vitro. Bioorg Med Chem 23:5891908, 2015.
Haikarainen T, Waaler J, Ignatev A, Nkizinkiko
Y, Venkannagari H, Obaji E, Krauss S, Lehtiö
L. Development and structural analysis of adenosine site binding tankyrase inhibitors. Bioorg
Med Chem Lett 26:(2):328-33, 2016.
The transcription factors (TFs) in a given genome can be
classified into distinct families by structurally conserved
DNA-binding domains (DBDs), often with similar DNA
recognition properties. However, the members of the family always display distinct functions and activities in various
biological processes in cancer and normal development. The
likelihood and nature are different in the network hubs of gene
transcriptional regulation, which is often composed of intertwined regulatory relationships between TF protein complexes
and chromatinized gene regulatory elements, such as promoters, insulators and enhancers (Wei et al. J Biochem 381:1-12,
2004 and Cell Res 15:292:292-300, 2005). Therefore, it is
of crucial importance to identify the genome-wide chromatin
locations of a given TF with biological significance. Meanwhile, we hypothesized that some cancer risk-associated single
nucleotide polymorphisms (SNPs) identified by genome-wide
association studies (GWASs) can disrupt TF–DNA binding at
key enhancers and initiate aberrant expression of SNP-linked
susceptibility genes. And this, in turn, may result in perturbation of TF regulatory networks that cause cancer initiation
and progression.
To address these questions, we carried out genome-wide
chromatin location analyses of several driver TFs such as
TMPRSS2-ERG, HOXB13, FOXA1 and androgen recep-
tor (AR) (Figure 1), which are often overexpressed and constitutively activated in many clinical prostate cancer tissues.
In combination with global ChIP-seq studies of enhancer
chromatin marks we have identified thousands of prostate
cancer cell-type-specific enhancers. We are using integrative and complementary genome-wide approaches including
ChIP-seq, RNA-seq, FAIRE-seq and 3C-derived methods in
combination with novel and classic molecular and biochemical assays, and also up-to-date computational and statistical
methods. Recently, we found that prostate cancer-associated
key TFs including HOXB13, FOXA1 and ERG extensively
cooperate with AR signaling to regulate target genes that are
implicated in prostate cancer cell growth and tumor progression (data not shown). In addition, we found that our integrative genomic data seems to be working well in functional
interpretation and mechanistic understanding of GWAS-discovered genetic variants in prostate cancer, including the SNP
rs339331, through enhancing HOXB13 chromatin binding
to drive up-regulation of the transcription factor gene RFX6,
which confers a risk of prostate cancer (Huang et al. Nat Genet 46:126-35, 2014).
RECENT PROGRESS
To map prostate cancer gene regulatory networks driven by
driver TFs, for the first time, we profiled genome-wide chromatin locations of HOXB13, which is known to be important
From left: Jihan Xia, Gonghong Wei, Yuehong Yang, Sufyan Suleman, Qin Zhang, Hang-Mao Lee, Nikolaos Giannareas, Ping Gao.
REGULATORY GENOMICS
AND FUNCTIONAL CANCER GENETICS
PROJECT LEADER
Gonghong Wei, Ph.D., Docent,
Academy Research Fellow
University of Oulu
ABSTRACT
Transcription factors (TFs) are sequence-specific DNA-binding proteins. The human genome contains approximately 1400 TFs contributing to complex gene regulation and accurate cell growth control. Aberrant expression of TFs is frequently
observed in cancer of the breast and prostate, Ewing’s sarcoma and leukemia.
Charting gene regulatory networks driven by the TFs in cancer cells is a systematic way to discover novel mechanisms and clinical markers for cancer risk
prediction and therapy. Here, we aim to uncover the cancerous roles of multiple
TFs including key members of the ETS, HOX and Forkhead classes of DNA-binding proteins. Together with data from genome-wide association studies, we aim
to see how cancer risk-associated single nucleotide polymorphisms (SNPs) alter
TF–DNA binding at sites of clinically important enhancers. We will validate key
findings including risk SNPs, enhancers and genes that are potentially prognostic
and diagnostic markers that could be used for cancer patient risk prediction and
stratification.
Figure 1. Genome-wide mapping of TF binding sites and enhancers in prostate cancer cells. Human chromosomes are shown around the outer ring. Other
tracks contain ChIP-seq data of the TFs and enhancer marks as indicated.
as regards prostate development and tumor progression. We
observed that prostate cancer GWAS SNPs are significantly
enriched at HOXB13 binding sites (Huang et al. 2014). Interestingly, the common genetic variant rs339331 at the prostate cancer susceptibility 6q22 locus lies within a functional
HOXB13 binding site, and is precisely located at a HOXB13
ChIP-seq peak summit. In a Japanese GWAS, the identification of rs339331 in RFX6 was reported as the SNP most associated with prostate cancer risk (P = 1.6 × 10-12; rs339331 T as
strongest risk allele). Interestingly, the significant association
between rs339331 and susceptibility to prostate cancer was
further observed in African Americans, men of European ancestry and the Chinese population, suggesting that rs339331
is a potential genetic marker to evaluate prostate cancer risk
across different ethnic groups.
We provided several lines of evidence to show that HOXB13
and AR favor binding to the risk T allele at rs339331 in vivo.
Linkage disequilibrium (LD) analysis based on the 1000 Genome Project indicates that GPRC6A and RFX6 are associated with rs339331 in a strong LD block (Huang et al. 2014).
However, the existing genomic data from multiple prostate
cancer cells and tissues suggested that only the RFX6 gene is
active at 6q22. In addition, we found that RFX6 is markedly
upregulated in prostate tumor samples. RFX6 upregulation
correlates with prostate cancer progression, suggesting that
the expression of RFX6 is a promoter for prostate cancer cell
growth and metastatic progression. Interestingly, we recently
observed that lower expression of RFX6 correlates with tumor
progression and metastasis in other solid tumors of breast,
colorectal and gastric cancer. Our study suggests that RFX6
is a plausible causative gene linked to rs339331 conferring a
risk of prostate cancer. Consistently, eQTL analysis revealed
that the rs339331 T risk allele was significantly correlated with higher RFX6 mRNA levels in a Swedish cohort of
prostate cancer samples. We therefore proposed a model in
which rs339331 is a DNA-binding motif disruptor for AR/
HOXB13 heterodimer, and enhanced chromatin binding
of HOXB13 and AR to the T risk allele at rs339331 results
in increased RFX6 expression, conferring predisposition to
prostate cancer (Figure 2). We are currently working on the
genes and pathways that are regulated by the TF RFX6, and
we are also investigating the functional roles of RFX6 in other
types of cancers (breast, colon and stomach). In addition, we
are working towards systems annotation of all prostate cancer risk-associated loci using integrative data sets of regulatory genomics, bioinformatics, statistics and high-throughput
eQTL analysis with prostate tumor samples (Whitington et
al., in press). Meanwhile, my lab is also actively involved in
some fruitful collaboration with other scientists from the University of Oulu, identifying a novel functional genetic variant
that predisposes people to primary hip and knee osteoarthritis
(Taipale et al., 2015), the University of Helsinki as regards
discovering HOXB7 target genes in breast cancer (Heinonen
et al., 2015), and Wake Forest University School of Medicine
in the USA as regards systematic enrichment analysis of all
prostate cancer risk-associated SNPs discovered in GWASs
(Chen et al., 2015).
FUTURE GOALS
The mechanisms by which the aberrant expression of TFs
contribute to cancer development is generally not understood,
even for the well-studied transcriptional regulators, such as
AR, ETS factors and HOX family members. GWASs have
identified thousands of SNPs associated with predisposition
to various diseases including cancer. However, the molecular
mechanisms underlying the causal actions and biological effects of these SNPs remain poorly understood. We will continue to address these questions and aim to see how these SNPs
affect TF binding to key enhancers, which in turn alter the
SNP-associated gene expression that confers cancer susceptibility. We will carry on systematic analysis of gene regulatory
networks downstream of the TFs using classical molecular and
biochemical methods, as well as state-of-the-art functional
genomics and systems biology approaches – the combined
strength of genomics, genetics and bioinformatics.
Project Leader:
Gonghong Wei, Ph.D. (Academy of Finland)
Gonghong Wei, Ph.D. (24 March – 15 April 2015) had a
short-term international research visit to professor Depei Liu’s
lab at Institute of Basic Medical Sciences, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing,
China
Chandan Thapa, B.Sc. (20 April – 20 May 2015), a master
student from International Master Program, University of
Oulu, Oulu, Finland
Hang-Mao Lee, Ph.D. (Academy of Finland)
Wei Song, Ph.D. (China Scholarship Council and Foundation)
Ph.D. Students:
Ping Gao, M.Sc.
(Academy of Finland and China Scholarship Council)
Qin Zhang, M.Sc. (Academy of Finland and Foundation)
Project Researcher:
Yuehong Yang, M.Sc. (University of Oulu)
Project Workers:
Shuai Ni, B.Sc. (Foundation)
Sufyan Suleman, B.Sc. (Foundation)
Ilaria Svezia, B.Sc. (Erasmus fellowship and Foundation)
Chen H, Yu H, Wang J, Zhang Z, Gao Z, Chen
Z, Lu Y, Liu W, Jiang D, Zheng SL, Wei GH,
Issacs WB, Feng J, Xu J. Systematic enrichment analysis of potentially functional regions
for 103 prostate cancer risk-associated loci.
Prostate. 75:1264-1276, 2015.
Taipale M, Jakkula E, Kämäräinen OP, Gao P,
Skarp S, Barral S, Kiviranta I, Kröger H, Ott
J, Wei GH, Ala-Kokko L, Männikkö M. Targeted
re-sequencing of linkage region on 2q21 identifies a novel functional variant for hip and knee
osteoarthritis. Osteoarthritis Cartilage. doi:
10.1016/j.joca.2015.10.019, 2015.
Heinonen H, Lepikhova T, Sahu B, Pehkonen
H, Pihlajamaa P, Louhimo R, Gao P, Wei GH,
Hautaniemi S, Jänne OA, Monni O. Identification of several potential chromatin binding sites
of HOXB7 and its downstream target genes in
breast cancer. Int J Cancer. 137:2374-2383,
2015.
Figure 2. Graphic representation of our model of the regulatory relationships between
HOXB13/AR, rs339331 and RFX6 with potential
clinical value for risk prediction and stratification in prostate cancer. HOXB13 and AR bind to
the rs339331 region within the RFX6 gene and
regulate the expression of RFX6. The prostate
cancer risk-associated T allele at rs339331 results in increased HOXB13 and AR chromatin
binding and thus upregulates RFX6 expression,
which consequently leads to an increased risk
of the development of prostate cancer.
Whitington T, Gao P, Song W, Ross-Adams H,
Lamb AD, Yang Y, Svezia I, Klevebring D, Mills
IG, Karlsson R, Halim S, Dunning MJ, Egevad
L, Warren AY, Neal DE, Grönberg H, Lindberg
J, Wei GH, Wiklund F. Gene regulatory mechanisms underpinning prostate cancer susceptibility. Nature Genetics, in press.
COORDINATORS’ PROJECTS AND SELECTED CORE FACILITIES
Secretory as well as absorptive organs, such as lung, salivary
gland, kidney and mammary gland have a specialized luminal
space within an epithelial tube where fluid is transported to its
destination. The luminal space is formed during embryogenesis and begins by segregation of transmembrane proteins such
as ion channels to distinct membrane domains. Cell junctions
are responsible for maintenance of these specialised domains
and differentiated phenotype of the epithelium. Tools to control cell adhesion in tissues range from gene expression to cell
proliferation, migration and apoptosis. In addition to cell–cell
contacts, integrin-mediated cell–ECM contacts are crucial for
the differentiation of epithelial cells.
In the kidney, water and electrolytes are secreted and reabsorbed in different segments of nephrons in a highly regulated
manner. In renal failure, glomerular filtration may be reduced,
leaving only tubular secretory mechanisms in nephrons as a
means to form urine. Hence, efficient secretory and absorptive
mechanisms are especially needed when the organism is facing drastic changes in the environment and kidney epithelium
must have the capacity to regulate transcellular transport as
well as lumen size in order to keep the epithelium functional.
Janne Capra and Sinikka Eskelinen.
DIFFERENTIATION AND MALIGNANT
TRANSFORMATION OF EPITHELIAL
CELLS: 3D CELL CULTURE MODELS
PROJECT LEADER
Docent Sinikka Eskelinen, Ph.D.
Biocenter Oulu and Unit of Cancer Research
and Translational Medicine, Faculty of Medicine,
University of Oulu
The differentiated architecture of an epithelial layer is disrupted in carcinogenesis and filling of the luminal spaces of
epithelial glands is a hallmark of early epithelial tumours.
Transformed tumour cells populate the lumen and this process
requires both enhanced proliferation induced by oncogenes
and inhibition of apoptosis by antiapoptotic signals. Three-dimensional cell culture systems can be of utmost importance
in testing the therapeutic responses of tumour cells to various
compounds or in evaluating the invasive capacity of transformed cells. We have developed methods to monitor epithelial–mesenchymal and mesenchymal–epithelial transformation
in a highly regulated manner and to analyse the sequence of
events with state-of-the-art spinning disc confocal and light
sheet microscopes.
RECENT PROGRESS
ABSTRACT
Factors regulating transepithelial water transport
and generation of hydrostatic pressure within the lumen
We have built up an MDCK cell culture model where we
can analyse the mechanics of lumen formation and enlargement within short periods of time, without interference by
alterations in cell number through proliferation or apoptosis,
by using high-resolution confocal microscopy and imaging
software. We have used the method to elucidate the capacity
of cells to respond to changes in the composition of the extracellular milieu or changes in membrane potential within
short time intervals. This gives valuable information on the
behaviour of kidney cells under extreme situations or under
the influence of drugs affecting ion pumps or channels.
We observed that when the cells were subjected to a hyperpolarizing environment, i.e. external medium with no sodium or
potassium, apical secretion of chloride ions and transepithelial
water transport is initiated. As a consequence the lumen is
rapidly expanded. In contrast, a depolarizing environment,
i.e. equalizing internal and external potassium levels, leads to
cell swelling and ultimately death. Hence, water influx to the
lumen could be induced solely by hyperpolarization, which
activates the cellular machinery, cystic fibrosis transmembrane
receptors (CFTRs) and aquaporins. An antioxidant molecule,
N-acetylcysteine (NAC) has been widely used in the treatment
of cystic fibrosis patients and in contrast-induced nephropathy. We wished to elucidate the mechanism of NAC action in
our experimental set-up. It completely prevented water flow
and luminal expansion in hyperpolarizing conditions and depolarized the cells analogously to ionophores to monovalent
cations. These findings open new perspectives for pharmacological treatment of increased or decreased water secretion.
Malignant transformation
of epithelial cells by the Src oncogene
Lumen formation by MDCK cells in a 3D environment
We aim to understand the mechanisms behind the maintenance of a differentiated phenotype and analyse the electrophysiological parameters which regulate
the transport capacity of kidney epithelial cells. We also aim to understand the
circumstances in which polarity programs are changed during malignant transformation to cancer cells. We use non-transformed and Src-transformed kidney
epithelial MDCK cells as models. On the basis of the results we will try to find
pharmacologically active compounds regulating secretion or acting as tumour
suppressors in cell culture environments, and draw conclusions concerning their
capacity to do the same in vivo.
vitro, the differentiation process is flexible and the cells have
the capacity to correct erroneous cell divisions and restructure
the multilumina into a well-organized epithelial structure with
one lumen surrounded by a single layer of cells having uniform apico-basal orientation.
Canine kidney epithelial MDCK cells can differentiate in a
3D environment into spherical cysts with a lumen inside and a
basal membrane facing the extracellular matrix. We have monitored cyst formation of MDCK cells stably transfected with
Venus-tagged CD59 protein (kind gift from Reika Watanabe
and Guillaume Castillon, Dept. Biochem., Univ. Geneva) as
a function of time using state-of-the-art spinning disc microscopy techniques. Since CD59 is a lipid-anchored, secreted
protein, it served as a marker for the apical membranes and
lumina. MDCK cells grown in matrigel for 2–3 days form
preapical patches (PAPs) between adjacent cells which develop into a large lumen 2–3 days later. Time lapse imaging
of Venus-CD59 MDCK cells grown within matrigel under
the microscope resulted in the conclusion that there is a third
mechanism of lumen formation besides hollowing and cavitation. We have called it “coalescence”. In this process, the cells
initially divide irregularly, but rapidly reorganize themselves
along the cyst periphery in a dynamic way. Hence, at least in
In order to study malignant transformation in cultured cells,
an inducible culture system is required. A good cell culture
model consists of ts-Src-transformed canine kidney MDCK
cells which, when cultivated at 40.5 oC, behave as normal epithelial cells, whereas after a shift to 35 oC, Src tyrosine kinase is activated and the transformation process begins. In a
2D environment, the cells become mesenchymal with poor
cell–cell contacts and a flattened shape. In a 3D environment
they form irregular clusters without lumina. This feature gives
us a tool to analyse the regulatory factors of the transformation process induced by the v-Src oncogene just by changing
the culture temperature. With this model we have monitored
changes in gene expression, protein phosphorylation, cadherin
internalization, mitochondrial activity and apoptosis. Recently we have focused on the physical mechanisms behind lumen
filling and epithelial–mesenchymal transition. Preliminary results show that Src activation leads to rapid luminal collapse
as a result of opening of tight junctions and release of intraluminal hydrostatic pressure, and subsequent lumen filling by
cell migration and proliferation. The spinning disc confocal
microscope enables visualization of the time course and mechanism of lumen filling once Src is activated.
Role of survivin in malignant transformation
So that cancer cells can grow freely and metastasize, they need
to overcome their own proliferation regulation and inhibit
apoptosis. A member of the inhibitor of apoptosis (IAP) protein family, survivin, is expressed in most tumour cells and is
considered to be a promising therapeutic target. We have observed downregulation of survivin in untransformed MDCK
cells and elevated expression in Src-transformed MDCK cells
grown in a 3D environment (Töyli et al. 2010). There are
controversial views on the mechanism of how survivin promotes the transformation process. In addition to inhibition of
apoptosis, it regulates proliferation and promotes migration.
We aim to discover whether there is a correlation and causal
relationship between expression of survivin and cell migration
or proliferation.
Project Leader:
Sinikka Eskelinen, Ph.D. (University of Oulu)
Ph.D. Students:
Janne Capra, M.Sc. (Biocenter Oulu)
FUTURE GOALS
We aim to understand the mechanisms behind the morphogenesis, functionality and malignant transformation of epithelial MDCK cysts in 3D model systems. To achieve these goals
we are trying to find answers to the following questions:
1. What determines the ultimate size of the lumen and cyst in
3D culture of MDCK cells?
2. What is the source of the lumen negative potential in kidney cells and how can it be regulated in vitro and in vivo?
3. What are the roles of survivin and matrix composition in
3D differentiation?
From left: Outi Lampela, Ioannis Beis, Andre Juffer, Leonardo Garma, Pierre Leprovost.
PROJECT LEADER
André H. Juffer, Ph.D.
University of Oulu
COMPUTER SIMULATION
OF BIOLOGICAL PROCESSES
4. What are the targets of Src tyrosine kinase in two-dimensional and three-dimensional cell cultures?
5. What are the mutual roles of reactive oxygen species (ROS)
and antioxidants in malignant transformation and its inhibition in Src-transformed MDCK cells in a 3D environment?
The main methods are visualization of cells grown in extracellular matrix (matrigel), using spinning disc confocal microscopy and light sheet confocal microscopy for living cells and
point scanning confocal microscopy for fixed cells.
ABSTRACT
This project is focused on the development and application of software tools
for simulating biological systems on a computer, with the aim of explaining the
behavior of a given process. The techniques range from detailed QM/MM (quantum mechanics/molecular mechanics) and dynamic models of proteins to multiple-scale approaches that combine, for instance, diffusion models with cellular
automata (e.g. brain tumor simulations). Recent applications have been concerned, among other things, with the investigation of the structure and function
of the T-cell receptor and the enzymes thiolase and chitinase.
Computer simulation techniques have become important
tools to study the properties of biomolecules and their interactions with other molecules. These techniques are employed
with the general objective of predicting and explaining physical quantities and phenomena, such as protein-ligand associations, reaction rate constants of enzyme-catalyzed reactions,
acid dissociation constants of proteins, protein domain motion, protein folding and stability, and the like. The underlying
computational model provides direct insight into the workings
of these molecules, such that statements about their functional
properties can be offered. Such knowledge is frequently relied
upon, for instance, in the drug design industry to speed up the
process of drug finding and drug target analysis.
Computer simulation and modeling are not limited to the
study of proteins, but can be and are being applied to many
different domains of science. This project is specifically concerned with the study of biological processes. Whereas bioinformatics is largely concerned with the generation and analysis
of biological data, computer simulation and modeling are applied with the objective of explaining the behavior of a given
biological process. Each simulation model typically deals with
different time- and length scales. Chemical reactions at the
active site of an enzyme occur in very short timescales (fs),
protein dynamics generally take place in the range of ps to
ns or even longer timescales, interfacial processes (e.g. protein
diffusion at membrane surfaces) cover the ms to ms timescale,
protein translation falls in the ms to s range, and the growth of
tumors occurs over months to years. Each model must therefore involve different simulation and modeling methods.
Simulation and modeling methods rely on the application of
techniques that are based in physics (theoretical biophysics),
mathematics and chemistry. This project is predominantly
concerned with the development and application of such techniques. These range from very detailed QM/MM (quantum
mechanics/molecular mechanics) and dynamic models of proteins to multiple-scale approaches that combine, for instance,
diffusion models with cellular automata (CA) in the case of
brain tumor simulation.
RECENT PROGRESS
Comparison of non-sequential sets of protein residues
A methodology for performing sequence-free comparison of
functional sites in protein structures is introduced. The method is based on a new notion of similarity among superimposed
groups of amino acid residues that evaluates both geometry
and physico-chemical properties. The method is specifically designed to handle disconnected and sparsely distributed
sets of residues. A genetic algorithm is employed to find the
superimposition of protein segments that maximizes their
similarity. The method was evaluated by performing all-toall comparison on two separate sets of ligand-binding sites,
comprising 47 protein-FAD (Flavin-Adenine Dinucleotide)
and 64 protein-NAD (Nicotinamide-Adenine Dinucleotide)
complexes, and comparing the results with those of an existing
sequence-based structural alignment tool (TM-Align). The
quality of the two methodologies is judged by the methods’
capacity, among other things, to correctly predict the similarities in the protein-ligand contact patterns of each pair of
binding sites. The results show that use of a sequence-free
method represents a significant improvement versus the sequence-based one, resulting in 23 significant binding-site homologies being detected by the new method but ignored by
the sequence-based one (Garma and Juffer, 2015). See also
Figure 1.
Figure 1. Superimposition of the NAD-binding sites of 1uxh and 1v8b. The
panels on the left show the results produced by G-GA (our method) and
the ones on the right illustrate the superimposition generated by TM-Align.
The backbone of the two proteins is shown using a cartoon representation,
with 1vb8 in blue and 1uxh in red. The Cα values of the binding sites are
highlighted in the top panels and represented by their Van der Waals radii.
In the lower panels the NAD ligands are shown using a licorice representation with the same color as their corresponding complex.
Wnt5a
Wnt proteins can be considered to be candidates involved in
CAKUT (Congenital Anomaly of the Kidney and Urinary
Tract) diseases, since they take part in the control of kidney
organogenesis. Of these proteins, Wnt5a is expressed in the
ureteric bud (UB) and its deficiency leads to a duplex collecting system, uni- or bilateral kidney agenesis (10/90), hypoplasia with an altered pattern of ureteric tree organization
(42/90) and lobularization defects with partly fused ureter
trunks (25/90), unlike control animals. In our studies the
UB also had notably fewer tips due to Wnt5a deficiency, being 306 at E15.5 and 765 at E16.5, versus 428 and 1022 in
controls (p < 0.02 and p < 0.03 respectively). These changes
due to Wnt5a knockout are associated with anomalies in the
ultrastructure of UB daughter epithelial cells. The basement
membrane (BM) was malformed, so that its thickness was increased from 46.3 nm to 71.2 nm (p < 0.01) at E16.5 in Wnt5a knockout animals when compared with controls. Expression of a panel of BM components such as laminin and type
IV collagen was also reduced as a result of Wnt5a knockout.
Expression of the P4ha1 gene, which encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I), and overall C-P4H enzyme activity were elevated by around 26% as a
result of impairment of Wnt5a function. Compound Wnt5a
+/- ;P4ha1 +/- embryos demonstrated Wnt5a -/- related defects, for example local hyperplasia in the UB tree. An R260H
WNT5A variant was identified from a renal human disease
cohort. Functional studies and simulation (Figure 2) of the
consequence of the mouse variant in comparison with normal
Wnt5a reduced signaling and enlarged the putative frizzled
receptor-binding pocket. Altogether, Wnt5a has a novel function in kidney organogenesis by contributing to the patterning
of UB-derived collecting duct development contributing putatively to congenital disease.
activity. Electrostatic interactions and pH control the degree
of binding of ligands to proteins, as may be judged from the
ionic strength and pH dependency of the affinity of the ligand
for the protein. The protein’s charge distribution may have
been optimized during evolution to perform specific functional tasks. Clearly, any theoretical molecular model for describing biological processes involving proteins should account for
possible variations in the protein’s charge distribution due to
proton (dis)association by ionizable groups in the protein. In
this project we propose a novel model for charge fluctuations
in coarse-grained molecular dynamics (CGMD), a simulation
methodology that has become one of the most important
techniques for the study of proteins and membranes at large
length and long time scales. Conventional CGMD is based
on Newtonian mechanics. Like standard atomistic molecular dynamics (MD), it computes forces on CG particles or
“beads” to generate a sequence of states (particle coordinates
and velocities) as a function of time, from which numerous
molecular, thermodynamic and time-dependent properties of
the system can be calculated. However, charge fluctuations in
CGMD are completely ignored. This subproject is aimed at
correcting this serious omission. See also Figure 3.
Figure 2. Modeling of the putative influence of c.779G>A on the in silico-simulated 3D structure of WNT5A protein suggested around 22%
expansion of the frizzled receptor-binding pocket, the value changing
from wild-type at 3.06 nm (SD 0.437 nm) to the variant at 3.74 nm (SD
0.53 nm) (in red/blue, respectively). The frizzled receptor binding loop
region of wild-type WNT5A protein oscillated in the range of 1.5–4.3 nm
while the corresponding value for the R260H variant was 2.5–5.18 nm.
Proton transfer events in coarse-grained
molecular dynamics
Molecular properties of proteins, such as stability, solubility,
enzyme activity and ligand interaction, are strongly affected
by the protein’s charge distribution, this typically biased by the
protein’s location in various cell compartments. Many membrane proteins carry more basic residues than cytosolic ones
to facilitate binding at negatively charged membrane surfaces.
The pH affects the charge distribution, as manifested, for example, by the pH dependency of protein stability, governed,
among other things, by differences in acid dissociation constants of ionizable groups in the folded and unfolded state.
Also, proteins near charged membranes may partially open up,
temporarily exposing ionizable groups, affecting the protein’s
charge distribution and consequently its ability to bind to
membranes. Enzyme and ribozyme (RNA-based catalyst) activity strongly depend on pH. They frequently display a maximum within a particular pH interval. Small deviations from
the optimal pH range, affecting protonation states of titrating
residues such as histidine, may result in complete abolition of
Figure 3. Illustration of a coarse-grained (CG) procedure in which a number of atoms (AT) are grouped into larger particles (left). As a result, the
details of the protons are obscured, so that the proton transfer process
at the atomistic level (right, black line, particle in a double well potential)
cannot be explicitly modeled. One can, however, realize the net effect of
the transfer in terms of mass and charge fluctuations, modeled as jumps
between particles (right, red and green line).
Figure 4. Illustration of a detected water trail in
one of the snapshots generated from an atomistic molecular dynamics simulation of thiolase
(1dm3). The target residues (in green) are located in subunit B (C89B, A316B, N327B). The network (in red) extends from the active site area
to the surface of the protein. Water molecules
are represented as blue crosses.
Thiolase
FUTURE GOALS
This subproject is concerned with the importance of hydrogen bond networks (HBNs) for the efficient catalysis of the
Zoogloea ramigera thiolase active site (Figure 4). This will be
investigated by in silico modeling and simulation and structural enzymological approaches. These studies will benefit very
much from the presence of a range of key structures of this
thiolase, complexed with various substrates, substrate analogs
and intermediates. Two HBNs will be studied, being related
to the properties of oxyanion hole 1 (OAH1, stabilizing an
enolate intermediate) and oxyanion hole 2 (OAH2, stabilizing a tetrahedral intermediate) of thiolase. The hydrogen bond
donors (HBDs) of these oxyanion holes are from main chain,
side chain or water molecules. In the HBN of OAH1 water
molecules play a key role, whereas in OAH2 a peptide-mediated HBN is involved. These HBNs will be studied by computational approaches. This study capitalizes on the enormous
amount of structural data already available on the thiolase
system. In addition, the computational studies will be complemented and validated by structural enzymological studies
(collaboration with Prof. Wierenga’s group).
1. Development of a novel method for the inclusion of proton
binding in coarse-grained simulation of proteins.
2. Deriving scientifically sound and probabilistic optimal
principles for coarse-grain molecular systems for simulating
protein adsorption at interfaces.
Project Leader:
André H. Juffer, Ph.D. (Biocenter Oulu)
• A method for handling electronic polarization effects was
completed (Lampela, PhD thesis defense in spring 2016).
PROJECT LEADER
Aki Manninen,
Adjunct Professor of Cell Biology, Ph.D.
University of Oulu
REGULATION OF EPITHELIAL
CELL FUNCTIONS BY INTEGRINS
AND THE EXTRACELLULAR MATRIX
Yi-Ling Chen
(UO strategic funding targeted for BF operations)
Ph.D. Students:
Outi Lampela, M.Sc.
Leonardo Garma, M.Sc. (Biocenter Oulu)
Pierre Leprovost (Biocenter Oulu)
Other work
From left: Satu Myllymäki, Ulla-Reetta Kämäräinen, Irina Raykhel, Riitta Jokela, Kai Zhang, Jaana Träskelin, Aki Manninen, Sandhanakrishnan Cattavarayan.
ABSTRACT
Polarized epithelial cells are crucial building blocks of most of our organs. Our
group studies the role of the extracellular matrix (ECM) and ECM-binding integrin
receptors in the regulation of epithelial cell differentiation and morphogenesis.
Deregulated epithelial cell functions contribute to common and severe diseases
such as fibrosis and cancer. We study epithelial cell differentiation using various
cell biological and biochemical methods in combination with reverse genetic tools
in advancedd in vitro cell/tissue models.
Garma L and Juffer AH. Comparison of non-sequential sets of protein residues. Comput Biol
Chem 61:23-38, 2015.
Pietilä I, Prunskaite-Hyyryläinen R, Kaisto S,
Nicolaou N , van Eerde AM , Salo AM, Garma
L, Miinalainen I, Feitz WF, Bongers EMHF,
Juffer AH, Knoers NVAM, Renkema KY, Myllyharju J, Vainio SJ. Wnt5a Deficiency Leads
to Anomalies in Ureteric Tree Development,
Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in
Kidney Collecting Duct Patterning. PloS ONE,
11(1):e0147171, 2016.
A polarized structure, and directional growth and migration
of epithelial cells are crucial for both organ development and
maintenance of their asymmetric functions, such as excretion
of waste by the kidney filtering system. The ECM, deposited
by the cells themselves, not only scaffolds tissues but is also
rich in regulatory signals that orchestrate cellular functions
and responses. A laminin-rich basement membrane (BM) is
of particular importance as it orients the polarity of epithelial cells by serving as a ligand for members of the integrin
family of ECM receptors. Twenty-four different integrin heterodimers have been reported in humans. How the different
integrins convey signals from the ECM into the cells is not
thoroughly understood. However, it is clear that even subtle
dysfunctions in this reciprocal communication can disrupt
organ functionality. Therefore, it is important to build up
comprehensive knowledge of the regulation of epithelial cell
function by cell–ECM interactions.
RECENT PROGRESS AND FUTURE GOALS
Dissecting the functional roles
of specific integrins in epithelial cells
mechanisms by which integrins transmit BM-derived signals
to regulate epithelial cell polarization and morphogenesis.
To better understand the molecular mechanisms underlining
these phenomena, our aim is to identify the components of
the integrin interaction complex by mass spectrometry.
αV-integrins regulate cellular mechanotransduction responses in epithelial cells (Sandhanakrishnan Cattavarayane)
As mentioned above, integrins are crucial sensors and effectors of cellular biomechanics. Our RNAi-based screening approach revealed a central role for αV-integrins as regulators
of the maturation of focal adhesions (FAs) in MDCK cells
(Teräväinen et al., PLoSONE, 2013). When aV-integrins were
depleted, MDCK cells became unable to correctly respond to
changes in ECM stiffness. We are currently focusing on dissecting the molecular machinery associated with αV-integrins
at maturing focal adhesions. Integrins lack enzymatic activity,
so they rely on associated proteins which modulate cellular
signal transduction pathways and thereby cellular functions.
By using mainly proteomics-based approaches we aim to identify novel molecular effectors that are functionally associated
with αV-integrins.
MDCK
KRas12V
Figure 1. 3D models of epithelial morphogenesis and invasion of normal and oncogene-transformed MDCK cells. We are studying transformed cells as
separate clusters (left panel) and as individual transformed cells surrounded by normal cells (right panel).
Satu-Marja Myllymäki: Specific roles of epithelial integrins in
chemical and physical sensing of the extracellular matrix to
regulate cell shape and polarity. Acta Universitatis Ouluensis.
D, Medica 1308.
The bi-directional function of integrins enables ECM binding
by the cell while simultaneously sensing its chemical composition and physical properties. Sites of integrin adhesion serve
as localized organizing centres for the cytoskeleton, regulating
changes in the cell architecture. Integrin adhesion sites are also
sensitive to mechanical stimuli and capable of imparting contractility across the cytoskeleton, thereby facilitating processes
such as cell spreading and migration. A clear link exists between
integrin function, cell polarity and epithelial morphogenesis.
Integrin functions are known to be central to development as
well as to epithelial pathologies, but more detailed studies addressing their specific roles at a cellular level are still necessary.
A number of different integrins share common ligands and are
thought to have partially redundant functions. However, it is
becoming evident that even integrins with similar ligands have
differing cellular functions governed via mechanisms beyond
their ligand-binding specificity. A complex network of accompanying effector molecules at the cell membrane fine-tunes
the cellular response. By utilizing various advanced epithelial
model systems we are studying the cellular functions of specific integrins and their potential functional crosstalk.
Integrin-mediated signals in the regulation of epithelial cell
shape and morphogenesis (Satu Myllymäki)
In three-dimensional (3D) culture environments MDCK cells
recapitulate epithelial morphogenesis by forming polarized lumen-enclosing spherical cysts. By analysing the behaviour and
phenotypes of integrin-knockdown (Itg-KD) cells in various
culture conditions we have observed that α2b1- and b4-integrins are required for ECM recognition and provide cues
for the establishment of laminin-based basal cues in collagen-I
gels (Myllymäki et al., PLoSONE, 2011). α3b1-integrins
were found to be essential for control of the cell division axis in
the maintenance of lumen integrity in laminin-rich basement
membrane extract (BME) gels (Myllymäki et al., PLoSONE,
2011). In our ongoing studies we are addressing the molecular
Specific integrin functions in epithelial cancers
(Kai Zhang)
In several tumour types, the regulation of integrin-mediated
signalling is perturbed, thereby contributing to abnormal migration, invasion, proliferation and survival of tumour cells.
Although integrins themselves are not thought to transform
cells, recent data demonstrate that several integrins contribute
to tumourigenesis by inhibiting or cooperating with oncogenes. The vast majority of diagnosed cancers are carcinomas
that originate from epithelium of skin, breast, prostate, colon
or kidneys. Epithelial polarity and/or polarized organization
of epithelial cells within tissues have emerged as critical regulators of oncogenesis and multiple polarity-related genes appear
to function as tumour suppressors. As mentioned above, we
have previously characterized functions of specific integrins in
the regulation of epithelial polarity of untransformed MDCK
cells. In this project we are addressing the possible functional
synergies or antagonisms between selected integrins (implicated in the regulation of epithelial polarity in our previous
studies) and oncogenes. As model oncogenes we are focusing
on a few central genes known to transform epithelial cells in
vitro and in vivo. We will look specifically into those integrins
which are critical for epithelial polarity in non-transformed
cells and integrins whose expression is induced by transformation (such as α5b1-integrin). The oncogenic properties
of selected oncogenes are being studied in combination with
specifically modulated integrin-mutant cell lines. Modified
3D culture setups will be used as functional readouts (Figure
1). Our aim is to facilitate the development of novel targeted
therapeutic approaches to inhibit malignant transformation
by modulating reciprocal signalling between cancer cells and
their tumour microenvironment.
eGFP
b-cat
BME
Project Leader:
Aki Manninen, Ph.D. (Academy of Finland, Biocenter Oulu,
UO strategic funding targeted for BF operations and
University of Oulu)
Ph.D. Students:
Satu Myllymäki, M.Sc. (Academy of Finland, Foundations)
Sandhanakrishnan Cattavarayane, M.Sc. (Biocenter Oulu)
Kai Zhang, M.Sc. (Academy of Finland)
Jaana Träskelin, part-time (Biocenter Oulu)
Riitta Jokela (University of Oulu)
Irina Raykhel, Ph.D. (Biocenter Oulu, UO strategic funding
targeted for BF operations and University of Oulu)
Carrera M, Bitu CC, de Oliveira CE, Cervigne
NK, Graner E, Manninen A, Salo T, Coletta RD.
HOXA10 controls proliferation, migration and
invasion in oral squamous cell carcinoma. Int J
Clin Exp Pathol 8(4):3613-23, 2015.
Junttila S, Saarela U, Halt K, Manninen A,
Pärssinen H, Lecca RM, Brandli AW, SimsLucas S, Skovorodkin I, Vainio SJ. Functional genetic targeting of the embryonic kidney
progenitor cells ex vivo. J Am Soc Nephrol
26(5):1126-37, 2015.
Cattavarayane S, Palovuori R, Jayendrakishore TR, Manninen A. a6b1- and aV-integrins are required for long-term self-renewal of
murine embryonic stem cells in the absence of
LIF. BMC Cell Biol 16:3, 2015.
Manninen A. Epithelial polarity - Generating and
integrating signals from the ECM with integrins.
Exp Cell Res 334(2): 337-349, 2015.
Osteoarthritis (OA) is the most common degenerative disease
worldwide. It is characterized by progressive degradation of articular cartilage that leads to joint space narrowing, subchondral sclerosis, osteophyte and cyst formation, and eventually
loss of joint function. In Finland the Health 2000 national
survey indicated a prevalence of 6.1% in men and 8.0% in
women for clinically diagnosed knee OA. Certain forms of
OA have long been known to have a genetic component.
Recent studies have confirmed these findings and indicated
a significant role for genetic factors in primary OA. Degeneration of the lumbar spine has a prevalence of about 5% in the
Finnish population. Early epidemiological studies suggested
that low back syndromes are mainly caused by environmental
and anthropometric factors such as occupational load, back
injuries, height and obesity. However, considerable familial
predisposition to early onset sciatica and disc herniation has
been shown, and more recent epidemiological studies indicate
that disc degeneration may be explained primarily by genetic
influences.
From left: Mari Taipale, Minna Kraatari, Sini Skarp, Maiju Welling, Eeva Slitz, Aira Erkkilä, Anu Myllymäki, Anthi Kelempisioti, Minna Männikkö.
GENETIC SUSCEPTIBILITY
TO COMMON MUSCULOSKELETAL
DISEASES
PROJECT LEADER
Docent Minna Männikkö, Ph.D.
Center for Life Course Health Research,
Faculty of Medicine and Oulu Center
for Cell-Matrix Research,
University of Oulu
Osteoporosis (OP) in adults can be regarded as a complex disease with high social and economic costs and its diagnosis is
focused on the assessment of bone mineral density (BMD).
Every year over 7500 hip fractures occur in Finland, resulting
in an annual cost of 45 million euros. There are numerous risk
factors described in OP, the most significant being low bone
mass, age and being female. In general, risk factor scores show
poor specificity and sensitivity as regards the prediction of OP
and fracture risk, and some risk factors vary significantly with
age. Twin studies indicate that genetic factors account for up
to 80% of peak bone mass. Studies of candidate genes for OP
have been focused on cytokines, growth factors that regulate
bone turnover, genes that encode components of bone matrix,
and genes encoding receptors for calciotropic hormones. The
common form of OP is considered to be multifactorial, arising from the interaction of common polymorphic alleles with
multiple environmental factors.
RECENT PROGRESS
ABSTRACT
Degenerative musculoskeletal diseases such as osteoarthritis (OA) and osteoporosis (OP) are among the greatest reasons for disabilities among all medical conditions, particularly in the aging population. They cause considerable individual
suffering and are a significant economic burden on public healthcare. Many of the
underlying mechanisms driving disease progression are not fully understood, and
yet this knowledge is essential for better diagnostics, prevention and treatment of
the disease states. While these conditions can be secondary to various factors, in
OA, for example, the majority of cases are considered primary and the proportion
of heritability in OA can be as high as 65%. This project is aimed at improving
the understanding of molecular pathways underlying common musculoskeletal
diseases by identifying disease-associated genetic/epigenetic variants. Large
population-based cohorts, the Northern Finland Birth Cohort (http://www.oulu.
fi/nfbc/) and the Health 2000 Survey (http://www.terveys2000.fi/), are used
to study the genetic, metabolic and environmental determinants of degenerative
musculoskeletal diseases.
There is strong evidence that inflammatory mediators perpetuate disease progression in OA. In our previous candidate
gene studies we have observed that a certain interleukin 6 (IL6) promoter haplotype is associated with the severity of the
symptoms in hand and lumbar spine OA (Kämäräinen et al.
2008; Karppinen et al. 2008a; Karppinen et al. 2008b). We
have also observed that genetic variations in the interleukin-1
(IL-1) cluster and the matrix metalloproteinase-3 (MMP-3)
gene together associate significantly with type II modic changes and specific bone marrow lesions visible in magnetic resonance imaging (MRI), and are strongly related to lower back
pain (Karppinen et al. 2008b). Degeneration of the lumbar
spine is a common condition that progresses with aging, but
it has also been shown to be already frequent among young
adults. We observed in a subpopulation of the NFBC1986
that the IL6 promoter variants also increased the risk of spinal
degeneration (Kelempisioti et al. 2011). We have also investigated the role of environmental factors, low back pain history
and sciatica symptoms in this subpopulation and showed that
sciatica symptoms were already common in young adulthood
(Karjalainen et al. 2013).
In a collaborative study we identified the gene for carbohydrate sulphotransferase 3 (CHST3), an enzyme that catalyzes
proteoglycan sulphation, to be a susceptibility gene as regards
lumbar disc degeneration (Song et al. 2013). The initial finding came through linkage studies in Southern Chinese families, and further association analyses in a multi-ethnic population of 32,642 subjects indicated a variant that lies within a
potential microRNA-513a-5p binding site. Functional studies
showed that interaction of the susceptibility allele with the
microRNA was enhanced, and that CHST3 mRNA was significantly reduced in the intervertebral disc cells of human
subjects carrying this allele (Song et al. 2013).
In our genome-wide linkage analysis of Finnish OA families we
identified a susceptibility locus on chromosome region 2q21,
with a multipoint LOD score of 3.91. Targeted re-sequencing
of the region revealed an insertion variant, rs11446594, located in a predicted strong enhancer element. Insertion creates a
recognition sequence for two transcription factors, ELF3 and
HMGA1, which are predicted to play a significant role in articular cartilage homeostasis. Their DNA-binding affinity is
highly increased in the presence of the A allele of rs11446594
compared with the wild-type null allele. This variant is a potential novel functional variant that predisposes people to hip
and knee OA.
FUTURE GOALS
Our main goal is to identify metabolic pathways defective
in musculoskeletal diseases, by using genetic approaches.
Although genome-wide association studies (GWASs) have
revealed several interesting predisposing candidate genes for
OA, they all indicate relatively low individual risk. These
studies are also complicated by clinical and genetic heterogeneity and the contribution of numerous rare alleles that are
not identified in GWASs. We will apply next-generation sequencing techniques to identify rare variants with high genetic
impact on these common musculoskeletal diseases, using well
defined phenotypes in both population and family cohorts.
We will use a combination of in silico, in vitro and in cellulo
methodologies to demonstrate causality of the genetic variants on the disease state and to study the underlying molecular
mechanisms.
Project Leader:
Minna Männikkö, Ph.D., Docent (University of Oulu)
Elina Hietikko, M.D., Ph.D.
(Biocenter Oulu and University of Oulu)
Ph.D. Students:
Anthi Kelempisioti, M.Sc. (University of Oulu)
Minna Kraatari, Med.Cand. (University of Oulu)
Marika Löija, M.D.
Eeva Slitz, M.Sc. (University of Oulu)
Sini Skarp, M.Sc. (Biocenter Oulu and University of Oulu)
Mari Taipale, M.Sc. (University of Oulu)
Maiju Welling, Med.Cand. (University of Oulu)
(Biocenter Oulu, University of Oulu)
Taipale M, Jakkula E, Kämäräinen OP, Gao P,
Skarp S, Barral S, Kiviranta I, Kröger H, Ott
J, Wei GH, Ala-Kokko L, Männikkö M. Targeted
re-sequencing of linkage region on 2q21 identifies a novel functional variant for hip and knee
osteoarthritis. Osteoarthritis Cartilage. doi:
10.1016/j.joca.2015.10.019, 2015, Epub ahead
of print.
From left: Raija Soininen, Hanna Seppälä, Reetta Vuolteenaho, Kirsi Säkkinen, Outi Kettunen, Jonna Ranta. In front: Sanna Kauppinen.
PROJECT LEADER
Docent Raija Soininen, Ph.D.
University of Oulu
ANALYSIS OF GENE FUNCTIONS
IN VIVO BY GENERATION AND
CHARACTERIZATION OF GENETICALLY
MODIFIED MICE
ABSTRACT
The BCO Transgenic core facility provides a wide repertoire of services connected to the generation, archiving, and, to some extent, analysis of genetically
modified mice. New and emerging technologies in the field are constantly evaluated and set up. National and international activities, including the Biocenter
Finland-linked FinnMouse consortium, the ESFRI-Infrafrontier project, the European Mouse Mutant Archive, and NorIMM, the Nordic Infrastructure for Mouse
Models, have become important parts of our function.
Genetically modified (GM) mice are important tools in analysis of genes and gene products in vivo. They provide valuable information about gene functions and serve as models of
human diseases. The BCO Transgenic core facility provides
services in generation, archiving, re-derivation, and analysis
of genetically modified mice (www.oulu.fi/biocenter/tgcore).
Consultation, education of researchers, and following and being involved in the development and set-up of new methods
are also activities of the core facility. The core facility promotes
and fulfils the 3R’s (reduction, replacement, refinement) in
animal experiments.
The European Mouse Mutant Archive EMMA is a repository
for the collection, archiving (via cryopreservation) and distribution of relevant mouse mutant strains essential for basic
biomedical research. EMMA is organized as a decentralized
archive of 14 partners in 11 European countries. The partners
have established common operating procedures and highest-quality standards. The Biocenter Oulu transgenic core facility serves as the Finnish EMMA node. Since 2013, EMMA
has been part of the Infrafrontier research infrastructure
(RI). (www.infrafrontier.eu). Infrafrontier supplies GM
mouse models to researchers economically and efficiently (archiving and distribution), and provides information about the
phenotypic features of mouse models (systemic phenotyping),
such as analysis of development, morphology, physiology,
metabolism and behaviour. The Infrafrontier Technical
Working Groups meet twice a year to discuss and plan development of new methods and improvements of the ones
in use. The Infrafrontier RI closely collaborates with and
contributes to the International Mouse Phenotyping Consortium IMPC (www.mousephenotype.org). The IPAD-MD
project (Research Infrastructure for Phenotyping, Archiving
and Distribution of Mouse Disease Models) addresses global
cooperation and coordination between Infrafrontier and
complementary research infrastructures worldwide, contributing to the global effort of the IMPC.
Funding to provide repository services, cost-free cryopreservation of germ cells and embryos, generation of GM mouse
strains from embryonic stem (ES) cells, and re-derivation and
distribution of mouse strains is provided by the EC Infrafrontier-I3 grant for 2013–2016. The Finnish unit is archiving 70 mouse strains in 2013–2016, largely from the Finnish
research community, providing cost-free generation of six
mouse strains from ES cells as a transnational access project,
and distributing mice according to orders.
Finnish participation in Infrafrontier was included in the
Finnish Research Infrastructure Roadmap in 2009 and 2013,
and the University of Oulu is representing Finland as a partner
in the legal entity, Infrafrontier GmbH.
RECENT PROGRESS
We have continued to provide up-to-date services concerning GM mouse generation, re-derivation and archiving to
customers in Oulu, as well as other universities in Finland
and abroad. Since 2000, more than 200 gene-targeted mouse
strains have been generated, in addition to a large number of
transgenic mouse strains. In 2015 we served 57 customers involved in a total of 254 projects. As a special project, cleaning
and re-derivation of 40 mouse strains at the newly disinfected
animal facility SPF barrier unit was performed in 2015, and
cleaning of 17 strains will be finished in 2016.
Sperm cryopreservation and in vitro fertilization (IVF) were
started as new services in 2010, and currently mouse strains
are cryopreserved as sperm and two-cell embryos generated via
IVF. In 2015, a total of 27 mouse lines were cryopreserved for
in-house use, and 22 strains archived in EMMA. Generation
of iPS cells (induced pluripotent stem cells) and production of
mice from these cells have been accomplished. However, the
more recently set up 2i method for efficient isolation of ES
cells from mouse embryos is the method of choice in most cases. Using this method, we have already generated more than
100 ES cell lines in seven projects. These cells will mainly be
used for in vitro studies, but we have also performed a second
round of gene targeting and generated a mutant mouse strain
using ES cells generated by this method.
The BCO Transgenic core facility serves as the Finnish Infrafrontier-EMMA node, a national archiving facility. Full
national and international services in archiving and distribution of mouse lines have been provided in Oulu since the start
of the operational phase of the Infrafrontier-I3 project in
2013. So far, a total of 37 mouse strains originating in Finland
have been archived in EMMA, and an additional 16 strains
were submitted to the EMMA archive to be archived in 2016.
Furthermore, six gene-targeted mouse strains have been generated as an Infrafrontier transnational access service.
Since July 2011 the BCO Transgenic core facility has served as
a distribution node of the Wellcome Trust Sanger Institute, located in Hinxton, Cambridgeshire (UK), which generates new
mutant mouse strains as part of the EUCOMM (European
Conditional Mouse Mutagenesis) project. In 2015, 16 mouse
strains were shipped to customers in Australia, China, Japan,
the U.K. and the U.S.A. as live mice or embryos.
Research
Currently our main focus is in establishment of new methods
in generation and cryopreservation of genetically modified
mice. Therefore, technological development related to gene
modification in mice belongs to our research activities. The
latest addition is generation of gene-edited mice via CRISPR/
Cas9 technology. A method for site-specific modification of
the genome in early mouse embryos using cell-permeable Cre
fusion protein was also set up, and we have also successfully
tested and added ovary transplantation to our methods repertoire, and started using laser-assisted morula injection for generation of chimeric mice, reducing the number of donor mice.
Genotyping of blastocyst-stage embryos was established as
part of quality control of EMMA services, and tests including
shipment of embryos and sperm economically and efficiently without liquid nitrogen were performed. Most recently, a
method for cryopreservation of oocytes via vitrification was
successfully set up.
interacting with important signalling molecules. They also
bind water, and several extracellular matrix components have
affinity to heparan sulphate (HS). Perlecan is a large HSPG
found in all basement membranes (BMs) and also in the tissue
stroma of liver and cartilage, where no BMs are present. It carries three or four HS side chains that have been shown to bind
growth factors. One of our goals is to elucidate the functions
of HS chains in BMs, perlecan being the primary model proteoglycan, and study if variations in HS content could have a
role in symptoms of BM-connected diseases. For this, we have
generated mice in which the perlecan gene is targeted so that
the attachment sites of HS side chains are deleted.
FUTURE GOALS
The transgenic core facility will continue to provide high-level
services to researchers and keep updated in new technology,
especially as regards novel genome-editing tools.
We will be actively involved in national and international activities related to GM mice. In the Infrafrontier-I3 project,
the goal is cryopreservation of 70 mutant mouse lines in Finland by the end of 2016.
Project Leader:
Raija Soininen, Docent, PhD
(University of Oulu, Academy of Finland)
Laboratory supervisor:
Jonna Ranta MSc (University of Oulu)
Technicians: 3,5
(University of Oulu and Biocenter Oulu, UO strategic funding targeted for BF operations, Infrafrontier-I3)
Biocenter Finland Model Organisms network FinnMouse
(chair)
NordForsk project
Researcher network project Nordic Infrastructure for Mouse
Models NorIMM (coordination) 2014–2016
EU project
EC FP7 project Infrafrontier-I3 (partner), 2013–2016
ESFRI project
University of Oulu is a founding member in Infrafrontier
GmbH, which was created to coordinate the transnational acitivities of the Infrafrontier RI. Other founding members
are the Helmholtz Zentrum München, Germany, the Centre
National de la Recherche Scientifique, France, the Institute of
Molecular Genetics of the Czech Academy of Sciences, Czech
Republic, and the Biological Research Center ‘Alexander
Fleming’, Greece.
Researcher:
Reetta Vuolteenaho, PhD (Biocenter Oulu, UO strategic
funding targeted for BF operations)
E, Myllyharju J. Severe extracellular matrix abnormalitiesand chondrodysplasia in mice lacking collagen prolyl 4-hydroxylase isoenzyme II
in combination with a reduced amount of isoenzyme I. J Biol Chem 290(27):16964-78, 2015.
Colombelli C, Palmisano M, Eshed-Eisenbach
Y, Zambroni D, Pavoni E, Ferri C, Saccucci S,
Nicole S, Soininen R, McKee KK, Yurchenco
PD, Peles E, Wrabetz L, Feltri ML. Perlecan is
recruited by dystroglycan to nodes of Ranvier
and binds the clustering molecule gliomedin. J
Cell Biol 208(3):313-29, 2015.
Hurskainen T, Kokkonen N, Sormunen R,
Jackow J, Löffek S, Soininen R, Franzke CW,
Bruckner-Tuderman L, Tasanen K. Deletion of
the major bullous pemphigoid epitope region of
collagen XVII induces blistering, autoimmunization, and itching in mice. J Invest Dermatol
135(5):1303-10, 2015.
INFRAFRONTIER Consortium. INFRAFRONTIER--providing mutant mouse resources as
research tools for the international scientific
community. Nucleic Acids Res 43 (Database
issue):D1171-5. 20, 2015.
Ronkainen J, Huusko TJ, Soininen R, Mondini
E, Cinti F, Mäkelä KA, Kovalainen M, Herzig
KH, Järvelin MR, Sebert S, Savolainen MJ,
Salonurmi T. Fat mass- and obesity-associated
gene Fto affects the dietary response in mouse
white adipose tissue. Sci Rep 18, 5:9233, 2015.
We are also studying the functions of heparan sulfate proteoglycans (HSPGs), which influence biological processes by
Scavizzi F, Ryder E, Newman S, Raspa M,
Gleeson D, Wardle-Jones H, Montoliu L,
Fernandez A, Dessain ML, Larrigaldie V,
Khorshidi Z, Vuolteenaho R, Soininen R,
André P, Jacquot S, Hong Y, de Angelis MH,
Ramirez-Solis R, Doe B. Blastocyst genotyping
for quality control of mouse mutant archives: an
ethical and economical approach. Transgenic
Res 24(5):921-927, 2015.
RECENT DEVELOPMENT
INSTRUMENTATION
The specimen preparation laboratory and the electron microscopes are located in the facilities of Biocenter Oulu, providing excellent opportunities for efficient and smooth operation.
Personnel representing both Biocenter Oulu and the Department of Pathology are involved in running the facility.
Scanning electron microscope
(Sigma HD VP, Carl Zeiss AB, Germany)
Transmission electron microscope
(Tecnai Spirit, 120 kV, FEI Company)
CCD cameras Quemesa and Veleta
(Olympus Soft Imaging Solutions, GMBH)
Tissue processor (Lynx)
High pressure freezing device
(EM Pact, Leica, Vienna, Austria)
Freeze substitution device (Leica EM AFS 2, Leica)
Block trimmer (Leica)
Ultramicrotome (Leica Ultracut UCT with EM FCS, Leica)
Leica EMKMR2 glass knife maker
Critical Point Dryer (K850, Quorum Technologies)
Sputter coater and carbon evaporator
(Q150T ES, Quorum Technologies)
Scanning electron microscopic analysis of biological samples
started in Biocenter Oulu premises at the beginning of 2015.
A new SEM instrument was installed in November 2014 with
Biocenter Finland FIRI2013 and University of Oulu strategic
funding. It enables versatile analysis of surface structures of
cells and tissues with multiple detectors. Because of its good
performance using low acceleration voltage and a variable
pressure mode, samples sensitive to beam damage and samples which are nonconductive can be readily analysed. Furthermore, the instrument is equipped with Shuttle & Find
software, facilitating its use in correlative light and electron
microscopy applications.
In front: Raija Sormunen, Ilkka Miinalainen. 2nd row from left: Päivi Tyni, Sirpa Kellokumpu, Tarja Piispanen.
ELECTRON MICROSCOPY
CORE FACILITY
DESCRIPTION OF THE FACILITY
The Biocenter Oulu electron microscopy core facility provides both instrumentation and experienced personnel to assist researchers in projects where ultrastructural imaging is needed. One main task is to teach users about the possibilities of the methods and how to use them efficiently. The specimen preparation
laboratory is jointly run by personnel from Biocenter Oulu and the Department
of Pathology. Techniques routinely used include plastic- embedding, sectioning
and staining. Additionally, expertise in the special techniques of cryosectioning,
immunolabelling, negative staining of single proteins and macromolecules, high
pressure freezing, SEM (scanning electron microscopy) and FIB-SEM (focused
ion beam scanning electron microscopy) is available. We also work on the development of new approaches and methods in electron microscopy. In addition to
Biocenter Oulu groups, we serve other groups at Oulu University and collaborate
with other universities in Finland as well as with research institutes abroad.
PROJECT LEADER
Docent Raija Sormunen, Ph.D.
Biocenter Oulu and Cancer and Translational
Medicine Research Unit, Faculty of Medicine,
University of Oulu
Focused ion beam scanning electron microscopy allows automated acquisition of data sets for 3D ultrastructural reconstruction of tissues or cells by sequentially imaging a freshly
cut, resin-embedded block face at a spatial resolution below
10 nm. Specimen preparation and the image contrast mechanism are based on resin-embedding of fixed or high-pressure
frozen specimens. FIB-SEM is essential for understanding
cellular networks which need to be reconstructed throughout
a large volume. Instrumentation for FIB-SEM is situated in
the Micro- and Nanotechnology (MNT) centre at Linnanmaa
campus and is operated together with MNT personnel.
Vascular corrosion casting is an excellent tool for morphological examination of organ and tissue microcirculation. In this
method, an animal is perfused with a low viscosity resin and
surrounding tissue is removed by alkaline treatment to reveal
a replica of the vasculature. Casts are normally imaged using
SEM but can be further processed for analysis using confocal microscopy (resin is autofluorescent), micro-computed
tomography (microCT), or optical projection tomography
(OPT), which allows the production of 3D reconstructions.
LABORATORY PERSONNEL
Coordinator:
Raija Sormunen, Ph.D. (Biocenter Oulu)
Post-doctoral Investigator:
Ilkka Miinalainen, Ph.D. (University of Oulu strategic
funding targeted for BF operations)
Laboratory Technicians: 3 technicians (Biocenter Oulu,
University of Oulu, University of Oulu strategic funding
targeted for BF operations and University of Oulu)
We have applied modified negative staining for the imaging of exosomes. In this method, an exosome preparation is
placed on a grid, immunostained and contrasted by covering
the preparation with a methylcellulose film containing uranyl
acetate.
E, Myllyharju J. Severe Extracellular Matrix
Abnormalities and Chondrodysplasia in Mice
Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount
of Isoenzyme I. J Biol Chem 290(27):16964-78,
2015.
Konzack A, Jakupovic M, Kubaichuk K, Görlach
A, Dombrowski F, Miinalainen I, Sormunen R,
Kietzmann T. Mitochondrial Dysfunction Due to
Lack of Manganese Superoxide Dismutase Promotes Hepatocarcinogenesis. Antioxid Redox
Signal 23(14):1059-75, 2015.
Kutchuk L, Laitala A, Soueid-Bomgarten S,
Shentzer P, Rosendahl AH, Eilot S, Grossman
M, Sagi I, Sormunen R, Myllyharju J, Mäki
JM, Hasson P. Muscle composition is regulated by a Lox-TGFb feedback loop. Development
142(5):983-93, 2015.
Lemma SA, Pasanen AK, Haapasaari KM,
Sippola A, Sormunen R, Soini Y, Jantunen E,
Koivunen P, Salokorpi N, Bloigu R, Turpeenniemi-Hujanen T, Kuittinen O. Similar chemokine receptor profiles in lymphomas with central
nervous systeminvolvement - possible biomark-
ers for patient selection for central nervoussystem prophylaxis, a retrospective study. Eur J
Haematol doi: 10.1111/ejh.12626, 2015.
Moilanen JM, Kokkonen N, Löffek S, Väyrynen JP, Syväniemi E, Hurskainen T,Mäkinen
M, Klintrup K, Mäkelä J, Sormunen R, Bruckner-Tuderman L, Autio-Harmainen H, Tasanen
K. Collagen XVII expression correlates with the
invasion and metastasis of colorectal cancer.
Hum Pathol 46(3):434-42, 2015.
Nätynki M, Kangas J, Miinalainen I, Sormunen
R, Pietilä R, Soblet J, Boon LM, Vikkula M,
Limaye N, Eklund L. Common and specific effects of TIE2 mutations causing venous malformations. Hum Mol Genet 24:6374-6389, 2015.
Sokka M, Rilla K, Miinalainen I, Pospiech
H, Syväoja JE. High levels of TopBP1 induce
ATR-dependent shut down of rRNA transcription and nucleolar segregation. Nucleic Acids
Res 43(10):4975-89, 2015.
Vered M, Lehtonen M, Hotakainen L, Pirilä E,
Teppo S, Nyberg P, Sormunen R, ZlotogorskiHurvitz A, Salo T, Dayan D. Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor
prognosis: an in-vivo and in-vitro study. BMC
Cancer 15:25, 2015.
Vähätalo LH, Ruohonen ST, Mäkelä S,
Kovalainen M, Huotari A, Mäkelä KA, Määttä
JA, Miinalainen I, Gilsbach R, Hein L, Ailanen
L, Mattila M, Eerola K, Röyttä M, Ruohonen S,
Herzig KH, Savontaus E. Neuropeptide Y in the
noradrenergic neurones induces obesity and
inhibits sympathetic tone in mice. Acta Physiol
(Oxf) 213(4):902-15, 2015.
Zlotogorski-Hurvitz A, Dayan D, Chaushu
G, Korvala J, Salo T, Sormunen R, Vered M.
Human saliva-derived exosomes: comparing
methods of isolation. J Histochem Cytochem
63(3):181-9, 2015.
Åkerfelt M, Bayramoglu N, Robinson S, Toriseva M, Schukov H-P, Härmä V, Virtanen J,
Sormunen R, Kaakinen M, Kannala J, Eklund
L, Heikkilä J, Nees M. Automated tracking of
tumor-stroma morphology in microtissues
identifies functional targets within the tumor
microenvironment for therapeutic intervention.
Oncotarget 6:30035-30056, 2015.
Karsikas S, Myllymäki M, Heikkilä M, Sormunen R, Kivirikko KI, Myllyharju J, Serpi R,
Koivunen P. HIF-P4H-2 deficiency protects
against skeletal muscle ischemia-reperfusion
injury. J Mol Med (Berl) Epub ahead of print,
2015.
First row from left: Shiv Sah-Teli, Tiila-Riikka Kiema, Rik Wierenga. Second row from left: Chandal Thapa, Shruthi Sridhar, Rajesh Harijan.
Third row from left: Abhinandan Murthy, Ville Ratas, Rajaram Venkatesan. Fourth row from left: Ed Daniel, Kristian Koski.
PROJECT LEADER
Prof. Rik K. Wierenga, Ph.D.
University of Oulu
STRUCTURAL ENZYMOLOGY:
A QUANTITATIVE APPROACH
ABSTRACT
Good progress has been made towards obtaining working expression and purification protocols for human and E. coli (anaerobic) trifunctional enzyme (TFE)
and human collagen prolyl 4-hydroxylase (C-P4H), providing sufficient yields for
crystallisation, biophysical characterisation and enzyme kinetics. The structure
of the CdsD protein of Chlamydia trachomatis suggests how this protein might
form a ring-like assembly in the basal body of its T3SS injectisome. The Mycobacterium smegmatis T1-like thiolase tetrameric structure shows intrinsic asymmetry that could be of functional relevance. The crystal structure of the ternary
complex of rat peroxisomal multifunctional enzyme, type-1 (MFE1) suggests a
mechanism for its substrate channelling properties.
Enzymes have the unique property of converting a bound
molecule, the reactant, into another molecule, the product.
The (very) high energy barrier between reactant and product
somehow (almost) disappears once the reactant is bound in
the active-site pocket of the enzyme. Understanding of this
phenomenon, known as biocatalysis, is one of the main aims
of much current enzyme research, pointing to the importance
of the electrostatic properties as well as the dynamics of the
active site. Protein crystallographic studies provide information on the three-dimensional structure of the active site, how
the atoms are arranged, as well as, to some extent, the dynamic properties of the active-site residues. Detailed analysis
of the structures, including bioinformatics and biocomputing
approaches, is an essential component of our structural studies. In addition, we complement our structural studies with
biophysical characterisation and enzyme kinetic experiments.
We particularly study enzymes of lipid metabolism that
are CoA-dependent and which catalyse reactions in which
thioester chemistry plays a key role. Some of these enzymes
are large complexes, such as the human CoA-dependent, tetrameric trifunctional enzyme (TFE, 260 kDa, a2b2), which is
membrane-associated. TFEs have three active sites, catalysing
three subsequent reactions of the b-oxidation pathway. For
mammalian TFE, substrate channelling between the active
sites has been established experimentally using 2-trans-hexadecanoyl-CoA as the substrate (Yao and Schulz, 1996).
Monofunctional CoA-dependent enzymes that we study are
isovaleryl-CoA dehydrogenase, enoyl-CoA isomerase and thiolase. We also study human and rat MFE1 (multifunctional
enzyme, type 1), which has two active sites that catalyse the
hydratase and the dehydrogenase reactions of the b-oxidation
pathway, respectively.
Another set of enzymes that we study in depth are the collagen prolyl 4-hydroxylases (Myllyharju, 2006), in particular
tetrameric collagen prolyl 4-hydroxylase (C-P4H, 240 kDa,
a2b2). The latter enzyme is equipped with a peptide substrate-binding domain (the PSB domain), separate from the
catalytic domain. The substrate of C-P4H is procollagen and
it is believed that the function of the PSB domain is related
to capturing this polymeric substrate. The interplay between
the PSB domain and the catalytic domain is an intriguing
mystery, most likely providing the enzyme with processivity
properties. We aim to understand the mechanistic properties
of C-P4H by way of structural enzymology approaches. In
humans there are three isoenzymes, C-P4H-I, -II and -III, and
the PSB domain may also provide these isoenzymes effectively with different in vivo substrate-specificity properties and
therefore different functions.
RECENT PROGRESS
Structural studies depend on high-quality protein samples. A
major effort in protein structural studies therefore concerns
the development of reliable protein expression and purification protocols. This is more difficult to achieve if the studied
protein is a complex of two different polypeptide chains, as
in our projects on the trifunctional enzyme and the collagen
prolyl 4-hydroxylases. We study three different TFEs: Myco-
bacterium tuberculosis TFE (MtTFE), human (mitochondrial)
TFE (HsTFE) and (anaerobic) Escherichia coli TFE (anEcTFE). MtTFE is a soluble enzyme for which a suitable expression and purification protocol exists. HsTFE and anEcTFE
are actually membrane-associated enzymes, which means that
a suitable detergent has to be found for solubilising these complexes. In the TFE and C-P4H projects, two chains, a and b,
need to be expressed simultaneously. Using codon-optimised
synthetic genes and selecting appropriate plasmids and E. coli
host strains, it is now possible to express about 0.5 mg of HsTFE and 0.4 mg of anEcTFE per litre of culture medium, using
the best possible detergent. CryoEM studies with the Butcher
group (University of Helsinki) have been initiated. A particular complexity of the C-P4H project concerns the presence of
SS bridges. According to current knowledge there are possibly
eight SS bridges in the full-length tetramer. Another problem
concerns aspecific (?) proteolytic cleavage, via which truncated
complexes are purified. In this project, in collaboration with
the Myllyharju group, we work on C-P4Hs of human (types I,
II, and III) as well as Caenorhabditis elegans and Brugia malayi
C-P4H. With help from the Ruddock group (BCO, FBMM)
we have developed a more efficient expression protocol, which
currently works best for human C-P4H-II, of which we can
now purify 2 mg of full-length tetramer per litre of culture
medium. Together with Bergmann (BCO, FBMM) and Janis
(University of Eastern Finland, Joensuu) we are trying to determine the exact nature of the SS bridges of C-P4H by way
of mass spectrometric approaches.
Several structures of a Mycobacterium smegmatis thiolase
(MSM-13 thiolase) were determined by the Murthy research
group (Bangalore, India). This thiolase is a tetrameric thiolase
of unknown function. Three different crystal forms have been
obtained and in each of the crystal forms the tetramer has
been crystallised as an asymmetric tetramer, unexpectedly (Janardan et al., 2015). The shape of these tetrameric thiolases
has unique features, such that the active site is located at the
end of a bulk solvent-accessible cleft that is formed by loops
of each of the four subunits. The asymmetry of the tetramer
generates a “narrow” cleft and a “wide” cleft, as schematically visualised in Figure 1. In the structure of the CoA-bound
complex the highest occupancy of bound CoA is found in
the active sites facing the “narrow” cleft. MSM-13 thiolase has
been classified as a thiolase with unknown function (Anbazhagan et al., 2013), but pairwise sequence comparisons show
that it has much sequence similarity with the mitochondrial
T1 thiolase, which is a tetrameric, degradative thiolase. Study
of the active site geometry of MSM-13 thiolase and human
T1 thiolase nevertheless shows significant structural differences and enzyme kinetic assays also suggest different kinetic
properties for MSM-13 and T1 thiolase. The substrate specificity, and therefore the function of the MSM-13 thiolase remains unknown, despite the large amount of available data.
In another project, two structures of the periplasmic part of
CdsD of the human pathogen Chlamydia trachomatis (CdsD,
Figure 2) were completely refined and deposited (Meriläinen
et al., manuscript submitted for publication). CdsD is a building block of the basal body of the Type-III Secretion System
(T3SS) of Chlamydia trachomatis. This system translocates
proteins from within the bacterium to the eukaryotic host cell.
The basal body of the T3SS crosses the inner membrane (IM)
Figure 1. Schematic drawing of the tetrameric thiolase, consisting of subunits A (green), B (blue), C (yellow) and D (magenta). The tetramer is assembled
as a dimer of two tight dimers, being the AB dimer and the CD dimer. The two dimers interact with each other via the four tetramerisation loops, labelled
“t”. Each active site (marked *) is located at the end of a bulk solvent-accessible cleft, which is shaped by loops protruding out of each of the other three
subunits: “di” is a dimer interface loop. “t” is a loop that is part of the tetramerisation motif, “c” is the cationic loop pointing towards the active site of the
opposing dimer. In this way the active site of subunit B is completed by the “di” loop of subunit A, the “t” loop of subunit C and the “c” loop of subunit D.
In the MSM-13 thiolase crystal form that was obtained in the presence of CoA, the active sites of subunits B and C, facing the narrow cleft, have a bound
CoA molecule (PDB entry code 5BZ4) (Janardan et al., 2015).
and the outer membrane (OM) of this gram-negative bacterium. The basal body is built from three structural proteins,
CdsD, CdsJ and CdsC. CdsD and CdsJ form two concentric
rings of 24 proteins which cross the IM. CdsD forms the outer
ring, CdsJ forms the inner ring. The CdsC protein forms a
15-mer ring structure that crosses the OM. Determination of
the crystal structure of chlamydial CdsD has been challenging,
as the routine molecular replacement procedures for obtaining initial-phase information failed. Therefore, experimental
phases had to be achieved by making a suitable heavy-atom
derivative, which in the end was successful after generating a
new crystal form in a mother liquor that was suitable for soaking experiments with heavy-atom compounds. The structure
shows an extended shape with three domains, each having an
abbab fold (Figure 2), which are assembled in a linear fashion, generating a molecule of about 90Å in length. Two crystal
forms have been obtained, showing the same (extensive) crystal packing interactions, which might reflect packing of the
CdsD molecule in the outer ring assembly of the basal body of
the chlamydial T3SS injectisome.
The recently determined crystal structures of MFE1 suggest a
hypothesis for the channelling mechanism of this multifunctional enzyme with two active sites. The N-terminal domain
harbours the hydratase active site and the C-terminal region
harbours the second, dehydrogenase active site. In one of these
structures, the second active site is complexed with a 3-ketodecanoyl-CoA molecule as well as with NAD+ (or NADH).
crystallographic binding studies and computational studies
(in collaboration with the Mroginsky group, TU-Berlin) to
elucidate its substrate specificity. For MFE1 and TFE it has
been observed that the product of the first active site is ‘channelled’ to the second active site without being released in bulk
solvent. Our research on MFE1 will focus on understanding
the mechanism of this substrate channelling. In addition,
in the long term, our structural enzymology studies provide
insight that can facilitate the use of natural enzymes for the
synthesis of desirable chemicals using more sustainable production methods. Also, we will put considerable effort into
understanding the reaction mechanisms of these enzymes by
extending our studies towards the discovery of high-affinity
transition-state analogues (with the Schramm group, Albert
Einstein College, New York). These compounds capture key
structural features of the transition state of enzyme catalysed
reactions (Schramm, 2015) and consequently can have picomolar affinity and are important for understanding the respective reaction mechanisms. Such compounds can also be
exploited in drug discovery research projects.
OTHER REFERENCES
Anbazhagan, P., Harijan, R.K., Kiema, T.R., Janardan, N.,
Murthy. M.R., Michels, P.A, Juffer, A.H., Wierenga, R.K.
(2014) Phylogenetic relationships and classification of thiolases and thiolase-like proteins of Mycobacterium tuberculosis and
Mycobacterium smegmatis. Tuberculosis (Edinb). 94:405-412.
Figure 2. The abbab fold of the PD1, PD2 and PD3 domains of the periplasmic part of CdsD of the Chlamydia trachomatis Type III Secretion System (T3SS). The N-terminus and C-terminus are at opposing ends. The
three subsequent PD domains of CdsD generate an elongated molecule
(PDB entry 4QO6, 4QQ0). This fold was originally recognised as the BON
domain (Yeats and Bateman, 2003) and is characterised by conserved
hydrophobic residues and glycines. DALI searches in the PDB show that
this module also occurs in other proteins with a totally different function.
The crystal form of this ternary complex has been obtained
by a complicated protocol in which the crystal is equilibrated
with 2-trans-decenoyl-CoA, provided by Dr Schmitz, University of Wurzberg, Germany. This compound is hydrated in the
crystal by the first active site, then diffuses to the second active
site and, in the presence of NAD+, is then converted to 3-ketodecanoyl-CoA. Preliminary analysis of this ternary complex
suggests that the linker helix between the two domains might
be involved in allosteric communication between the two active sites, which then could explain the substrate channelling,
as observed for the 2-trans,4-trans-decadienoyl-CoA substrate
(Yang et al., 1986).
FUTURE GOALS
Our immediate goal in the human and anEc TFE and C-P4H
projects is to crystallize these enzymes and determine their
crystal structures. The structural information will then be used
to understand their function. For MtTFE, we will carry out
Myllyharju, J. (2008) Prolyl 4-hydroxylases, key enzymes in
the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets. Ann Med. 40:402417.
Schramm, V.L. (2015) Transition States and transition state
analogue interactions with enzymes. Acc Chem Res.48:10321039.
Yao, K.W. and Schulz, H. (1996) Intermediate channeling on
the trifunctional beta-oxidation complex from pig heart mitochondria. J Biol Chem. 271:17816-20.
Yang, S.Y., Cuebas, D., Schulz, H. (1986) Channeling of
3-hydroxy-4-trans-decenoyl coenzyme A on the bifunctional
beta-oxidation enzyme from rat liver peroxisomes and on the
large subunit of the fatty acid oxidation complex from Escherichia coli. J Biol Chem.261:15390-15395.
Undergraduate Students:
Chandan Thapa (thiolases)
Paurnima Patil (MCE)
Subam Kathet (ECI2)
Project Leader:
Rik Wierenga (University of Oulu)
Rajaram Venkatesan (Academy of Finland)
Tiila Kiema (senior postdoc, thiolases, IVDH, MFE1,
maintenance of the diffraction unit, Biocenter Oulu)
Kristian Koski (senior postdoc, C-P4H, University of Oulu
strategic funding targeted for BF operations)
Prasad Kasaragod (postdoc, MFE1, myomaker,
Sigrid Jusélius Foundation)
Rajesh Harijan (postdoc, thiolases, PNP, Albert Einstein
College of Medecine, New York)
PhD. Students:
Goodluck Onwukwe (PhD student, yeast and human ECI1,
ECI2, University of Oulu, Ehrnrooth Foundation)
Abhinandan Murthy (PhD student, human C-P4H,
University of Oulu)
Shiv Kumar Sah-Teli (PhD student, Escherichia coli and
human TFE, University of Oulu, Academy of Finland)
Ville Ratas
(technician, crystallisation of proteins, Biocenter Oulu)
Ed Daniel (programmer, development of xtalPiMS,
Diamond Light Source, UK)
Biocenter Finland Structural Biology network, chair person
Biostruct-X, EU, coordinator of the Oulu TID-centre
iNEXT, EU, coordinator of the Oulu TID-centre
BESSY, Berlin, Germany, member of the macromolecular
crystallography beamline proposal evaluation panel
(EU-Biostruct-X, TID-center)
(EU-iNEXT, TID-center)
Daniel E, Onwukwe GU, Wierenga RK, Quaggin
S E, Vainio S, Krause M. ATGme: Open-source
web application for rare codon identification and
custom DNA sequence optimization. BMC Bioinformatics 16:303, 2015.
Janardan N, Harijan RK, Kiema TR, Wierenga R
K, Murthy MR. Structural characterization of a
mitochondrial 3-ketoacyl-CoA (T1)-like thiolase
from Mycobacterium smegmatis. Acta Crystallogr D Biol Crystallogr 71:2479-2493, 2015.
Krause M, Neubauer P, Wierenga RK. Structure based directed evolution of a monomeric
triosephosphate isomerase: towards a pentose
sugar isomerase. Protein Eng Des Sel 28:187197, 2015.
Onwukwe GU, Koski MK, Pihko P, Schmitz W,
Wierenga RK. Structures of yeast peroxisomal
Δ3,Δ2- enoyl-CoA isomerase complexed with
acyl-CoA substrate analogues: the importance
of hydrogen bond networks for the reactivity of
the catalytic base and the oxyanion hole. Acta
Crystallogr D Biol Crystallogr 71:2178-2191,
2015.
Onwukwe GU, Kursula P, Koski MK, Schmitz
W, Wierenga RK. Human Δ3, Δ2-enoyl-CoA
isomerase, type-2: a structural enzymology
study on the catalytic role of its ACBP-domain
and helix-10. FEBS J 282:746-768, 2015.
Yeats, C. and Bateman, A. (2003) The BON domain: a putative membrane-binding domain. Trends
Biochem Sci. 28:352-355.
Rajesh K. Harijan: Structural enzymological studies of
SCP2-thiolase and SCP2-thiolase like proteins of Trypanosomatidae.
Goodluck U. Onwukwe: Structural and enzymological characterization of human and yeast D3,D2-enoyl-CoA isomerases
(ECIs).
Krause M, Wierenga RK. Towards new non-natural TIM-barrel enzymes using computational
design and directed evolution approaches. In
“Understanding enzymes; Function, Design, Engineering and Analysis”, Editor: Svendsen, A.S.,
Pan Stanford Publishing Pte. Ltd, Singapore, in
press.
BIOCENTER OULU PROJECTS 2016–2019
BIOCENTER OULU PROJECTS 2016–2019
PRINCIPAL GROUPS
PROJECT LEADERS
Mika Ala-Korpela, Ph.D. and
Johannes Kettunen, Ph.D.
POPULATION-BASED STRATIFICATION OF INDIVIDUAL
CARDIOVASCULAR RISK — A MULTI-OMICS APPROACH
PROJECT LEADER
Inari Kursula, Ph.D.
UNDERSTANDING THE MECHANISM AND EVOLUTION
OF APICOMPLEXAN GLIDING MOTILITY – ON A ROAD
TO DRUG AND VACCINE DISCOVERY
PROJECT LEADERS
Johanna Myllyharju, Ph.D. and
Peppi Karppinen, M.D., Ph.D.
HYPOXIA RESPONSE PATHWAY AS A TARGET
FOR NOVEL THERAPEUTICS
PROJECT LEADERS
Outi Savolainen, PhD.,
Mikko Sillanpää Ph.D. and
Tanja Pyhäjärvi, Ph.D.
GENOMIC AND STATISTICAL ANALYSIS
OF GENOTYPE-PHENOTYPE RELATIONSHIPS
PROJECT LEADERS
Johanna Uusimaa, M.D., Ph.D. and
Reetta Hinttala, Ph.D.
DYSFUNCTION OF CELLULAR METABOLIC
AND SIGNALLING PATHWAYS BEHIND SEVERE
NEUROLOGICAL AND MULTIORGAN DISEASES
IN CHILDREN
PROJECT LEADER
Seppo Vainio, Ph.D.
MECHANISMS OF ORGANOGENESIS
PROJECT LEADERS
Taina Pihlajaniemi, M.D., Ph.D. and
Lauri Eklund, Ph.D.
PROJECT LEADER
Gonghong Wei, Ph.D.
CELL-EXTRACELLULAR MATRIX INTERACTIONS IN
STEM CELL NICHES AND VASCULAR STRUCTURES
REGULATORY GENOMICS AND FUNCTIONAL
CANCER GENETICS
PROJECT LEADER
Lloyd Ruddock, Ph.D.
PROJECT LEADER
Rikkert Wierenga, Ph.D.
MECHANISMS AND APPLICATIONS
OF DISULFIDE BOND FORMATION
MULTIFUNCTIONAL b-OXIDATION ENZYMES:
STRUCTURES, SUBSTRATE CHANNELING AND
CELLULAR FUNCTION
PROJECT LEADERS
Markku Savolainen, M.D., Ph.D.,
Karl-Heinz Herzig, M.D., Ph.D. and
Marjo-Riitta Järvelin, M.D., Ph.D.
PROJECT LEADER
Robert Winqvist, Ph.D.
MOLECULAR MECHANISMS OF AND PATHWAYS
TO METABOLIC SYNDROME
FACTORS AND MECHANISMS IN HEREDITARY
BREAST CANCER PREDISPOSITION
www.oulu.fi/biocenter/groups/principal
EVENTS
EVENTS
EVENTS
PHD SATELLITE SYMPOSIUM OF BIOCENTER DAY, MARCH 18
Karim Ullah, Minna Kihlström,
Thomas Kietzmann and Zarin Zainul
Minna Kraatari and Sini Skarp
Sini Skarp, Minna Kraatari,
Anna Tervasmäki, Niina Laurila and
Tuomo Mantere
Speakers from left to right: Kenneth Dawson (University College Dublin, Ireland), Tarja Laitinen (Turku University Hospital, Finland), Carol Shoulders
(Queen Mary University of London, UK), Paul Winyard (University College London, UK) and Hans Lehrach (Max Planck Institute for Molecular Genetics,
Berlin, Germany)
Poster exhibition
BIOCENTER DAY, MARCH 19
Johanna Myllyharju, Ari-Pekka Kvist, Ritva Saastamoinen, and Meri Rova
Organizing Committee from left to right:
Tuomas Hämälä, Karim Ullah, Eeva Slitz, Ulla
Saarela, Minna Kihlström, Anja Konzack, Zarin
Zainul, Ezeogo Obaji, Chi Zhang and Abhinandan Murthy
Tiila Kiema and Ulrich Bergmann
Commercial product exhibition
EVENTS
EVENTS
BIOCENTER OULU EVALUATION SITE VISIT, MAY 4–5
Evaluation Committee from left to right: Professor Kathryn Cheah (University of Hong Kong, Hong Kong), Professor Lars Klareskog (Karolinska Insitutet,
Sweden), Professor Janos Hajdu (Uppsala University, Sweden) and Professor Rita Horwath (Newcastle University, UK)
Johanna Myllyharju, Sinikka Eskelinen and Jouko Uusitalo (Business Development Manager, Technopolis Plc)
Raija Soininen and Reetta Vuolteenaho
Mikko Sillanpää, Mirko Maksimainen and Lari Lehtiö
Rajaram Venkatesan and Rik Wierenga
Ritva Saastamoinen, Johanna Myllyharju, Andre Juffer and Seppo Vainio
Ilkka Miinalainen, Sinikka Eskelinen and Aki Manninen
BIOCENTER OULU BRAINSTORM IN LASARETTI, AUGUST 20–21
EVENTS
EVENTS
BIOCENTER OULU WORKSHOPS FOR SCHOOL CHILDREN
DURING THE SCIENCE DAYS OF THE UNIVERSITY, SEPTEMBER 1
MINERVA FOUNDATION RESEARCH COUNCIL AWARDED
THE MEDIX PRIZE TO GONGHONG WEI, SEPTEMBER 21
Gonghong Wei received the Medix Prize for the study revealing a novel
mechanism by which men with a specific genetic variant have increased
susceptibility to prostate cancer (Nature Genetics, 2014. 46:126-135).
The Medix Prize was given for the best scientific article from Finland
published in 2014 in the field of biomedicine and clinical research.
Learning to pipet
Sini Skarp and DNA analysis
OULU-ULM JOINT PHD RETREAT IN OULU, NOVEMBER 18–19
Ulrich Bergmann introducing mass spectrometry
Ilkka Miinalainen introducing scanning electron microscopy
VISIT OF PROFESSOR ANDY MCMAHON, SEPTEMBER 1
Seppo Vainio and Andy McMahon
Professor Andy McMahon (University of Southern California) gave the
keynote lecture in the Opening of the Science Days of the University.
From left to right: Johanna Myllyharju, Taina Pihlajaniemi, Kalervo Hiltunen, Andy McMahon, Sakari Jussi-Pekka and Sinikka Eskelinen
EVENTS
EVENTS
KUPCZYK GUEST PROFESSORSHIP OF ULM UNIVERSITY
TO JOHANNA MYLLYHARJU, NOVEMBER 30
CAREER SEMINAR AND 20 YEARS OF DOCTORAL TRAINING,
DECEMBER 15
Johanna Myllyharju was awarded Kupczyk Guest Professorship of
Ulm University, Germany. This Guest Professorship is awarded to a
high-ranking scientist from abroad to strengthen cooperations with Ulm
University. In the photo from left to right: Prof. Dr. Michael Kühl, Johanna
Myllyharju and the President of Ulm University, Prof. Dr. Michael Weber.
Photo Elvira Eberhardt/Ulm University
GENES AND SOCIETY, ARGUMENTA SEMINAR, DECEMBER 8
From left to right former graduate school coordinators: Pekka Kilpeläinen (CEMIS-Oulu/Kajaani) , Sinikka Eskelinen (Strategic research services,
OU), Anthony Heape (University of Oulu Graduate School, OU), Erja Heikkinen (Ministry of Education and Culture) with Ritva Saastamoinen (BCO-DP
Coordinator). Photos by Kulmakuvaamo.
DISCOVERY OF THE YEAR EVENT, DECEMBER 15
From left to right: Professor Seppo Vainio,
professor Helena Kääriäinen (National Institute
for Health and Welfare, Helsinki), professor
Hannes Lohi (University of Helsinki), professor
Jaakko Kaprio (Institute for molecular medicine,
FIMM) and Coordinator Meri Rova (Genes and
Society, Argumenta Project).
The Winners of the Discovery of the year
award with a shared first authorship: Marjut
Nätynki and Jaakko Kangas
Speakers from left to right: Kateryna Kubaichuk, Jaakko Kangas, Marjut Nätynki, Antti Salo, Justiina
Ronkainen and Sandhanakrishnan Cattavarayane
Taina Pihlajaniemi, Kari Kivirikko and Johanna
Myllyharju
Professor Helena Kääriäinen and the audience in the Saalasti Hall. Photos by Kulmakuvaamo.
EVENTS
BIOCENTER OULU DOCTORAL PROGRAMME EVENTS 2015
ORAL PRESENTATIONS
AT THE BCO DISCOVERY OF THE YEAR 2015
PRESENTING AUTHOR ANTTI SALO:
Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice
Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with
a Reduced Amount of Isoenzyme I. *Aro E, *Salo AM, Khatri R, Finnilä M,
Miinalainen I, Sormunen R, Pakkanen O, Holster T, Soininen R, Prein C,
Clausen-Schaumann H, Aszódi A, Tuukkanen J, Kivirikko KI, Schipani E,
Myllyharju J. J Biol Chem. 290(27):16964-78, 2015
PRESENTING AUTHOR KATERYNA KUBAICHUK:
Mitochondrial dysfunction due to lack of manganese superoxide dismutase
promotes hepatocarcinogenesis. Konzack A, Jakupovic M, Kubaichuk K,
Görlach A, Dombrowski F, Miinalainen I, Sormunen R, Kietzmann T. Antioxid Redox Signal Sep 30 PMID: 26422659, 2015
PRESENTING AUTHOR SANDHANAKRISHNAN
CATTAVARAYANE:
a6b1- and aV-integrins are required for long-term self-renewal of murine
embryonic stem cells in the absence of LIF. Cattavarayane S, Palovuori R,
Tanjore Ramanathan J, Manninen A. BMC Cell Biol.16:3., 2015
PRESENTING AUTHOR JUSTIINA RONKANEN:
PRESENTING AUTHOR MAARIT NÄTYNKI AND
JAAKKO KANGAS:
Common and specific effects of TIE2 mutations causing venous malformations. *Nätynki M, *Kangas J, Miinalainen I, Sormunen R, Pietilä R, Soblet
J, Boon LM, Vikkula M, Limaye N, Eklund L. Hum Mol Genet. 24(22):6374-89,
2015
THE WINNERS OF THE DISCOVERY OF THE YEAR 2015
Marjut Nätynki and Jaakko Kangas with a shared first authorship.
THE BEST POSTER AWARD
Heli Härönen: Both transmembrane and shed collagen XIII are indispensable for maintenance and plasticity of the neuromuscular junction.
Härönen H, Heikkinen A, Zainul Z, Tu H, Sormunen R, Miinalainen I,
Oikarainen T, Santoleri S, Pihlajaniemi T.
SPECIAL ACTIVITY AWARD
EM Coordinator Raija Sormunen
Fat mass- and obesity-associated gene Fto affects the dietary response in
mouse white adipose tissue. Ronkainen J, Huusko TJ, Soininen R, Sebert
S, Mondini E, Cinti F, Mäkelä K, Kilpeläinen M, Herzig KH, Järvelin MR,
Savolainen MJ, Salonurmi T. Sci Rep. 5: 9233, 2015
SPRING 2015
All term
BCO Seminar Series
February 17
Kontinkangas Campus Science Day
February 24
Scanning Electron Microscopy Open House
February 25
BCO Info Session
March 18
BCO-DP Satellite Symposium
March 19
Biocenter Day 2015: Personalized Medicine: Hype or Hope?
March 24
Kontinkangas Seminar for Senior Researchers
March 25–27
Spring Meeting in cooperation with the International Graduate School in Molecular Medicine Ulm, Ulm, Germany
April 7–23
Biomedical Imaging Methods Course (in cooperation with MRC)
April 16
Oulu BioImaging Network OBI Day
May 6–7
Expression Data Analysis with Chipster Course
May 18–22
Practical X-ray Course on Methods in Protein Crystallography: Data Collection, Data Processing, and Phasing
May 27
Research Funding Seminar: Do’s and Don’ts of Funding Applications
(in cooperation with MRC and Faculty of Medicine)
May 27–28
Advanced Course on Light and Electron Microscopy
SUMMER 2015
June 1–5
EMBO Practical Course: Modern Biophysical Methods for Protein-Ligand Interactions
June 2
Argumenta Seminar: Genomitieto käyttöön – Genomitiedon merkitys Suomelle ja suomalaisten hyvinvoinnille /
Genome Data is at our hands: Significance of the new information to the Finnish society and people’s welfare
AUTUMN 2015
Heli Härönen, the Winner of the Best Poster Award, and Ritva Saastamoinen
Minna Männikkö, Sini Skarp and Mari Taipale were thanked for their
contribution in running DNA analysis core facility services. In the photo:
Minna Männikkö, Johanna Myllyharju, Pirkko Huhtala and Sini Skarp
Sinikka Eskelinen was thanked for her long term contribution as the BCO
light microscopy coordinator. In the photo: Sinikka Eskelinen, Johanna
Myllyharju and Pirkko Huhtala
Anne Vainionpää (on the right) congratulates Raija Sormunen, who
received the Special activity award, for her unique contribution in Biocenter Oulu EM core facility over the years.
All term
BCO Seminar Series
August 10–14
Logical Reasoning in Human Genetics Course (in cooperation with MRC)
September 2
Argumenta Lecture: “The past hidden in our genes”
September 14
BIG DATA Seminar (in collaboration with Infotech Oulu and Genes & Society Argumenta Project)
September 22–23
Method Course on Immunohistochemistry and Digital Microscopy
October 5
Nobel Prize in Physiology or Medicine: Kontinkangas Campus Event
October 6
Argumenta Lecture: “Active information in quantum physics, biology and beyond”
October 7–9
Fall Meeting in cooperation with the International Graduate School in Molecular Medicine Ulm, Ulm, Germany
October 20
Argumenta Seminar: Omat ja parhaan ystävämme koiran geenit
(in collaboration with the University of the Third Age), in Imatra
October 22
Minisymposium: Synthetic Biology in Finland
November 3
Argumenta Lecture: “How prenatal and early life events influence later psychological and physical well-being”
November 3
Yksilöllistetty lääketiede – Turun lääketiedepäivien tieteellinen symposiumi (in collaboration with Argumenta Project)
November 11
Argumenta Lecture: ”Understanding the impact by geospace processes on the atmosphere of Earth”
November 12
Workshop on Biomedical and Life Sciences Application of Synchrotron Radiation
November 13
Biocenter Oulu – Biocenter Kuopio Day
November 18–19
TissueHome Oulu-Ulm Joint PhD Retreat, hosted by Oulu
November 20
Oulu Bioscience Networking Event
December 8
Argumenta Seminar: Geneettinen tulevaisuutemme – Millaisia mahdollisuuksia geenitieto avaa yhteiskunnassa
December 10
Enterprise Forum: Academic Entrepreneurship – Doctors Creating New Business
December 15
Career Seminar & 20 Years of Doctoral Training in Biocenter Oulu Doctoral Programme
December 15
BCO Discovery of the Year 2015
BIOCENTER OULU SEMINAR SERIES 2015
BIOCENTER OULU SEMINAR SERIES 2015
SPRING 2015
SPRING 2016
January 12
Host: Aki Manninen
Markku Varjosalo, Institute of Biotechnology, University of Helsinki, Helsinki
Dissecting cellular signaling pathways using mass spectrometry based proteomics
All term
Biocenter Oulu Seminar Series
January 27
Genes and Society Argumenta project event: Document film “Genetic Me” and associated discussion
February 19
Host: Seppo Vainio
Michael Kühl, Institute for Biochemistry and Molecular Biology, Ulm University, Ulm, Germany
Molecular mechanisms of cardiogenesis in vertebrates
February 1
Workshop: Kidney manifestations in metabolic diseases
February 17
Biocenter Oulu Info Session
February 26
Seppo Vainio, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu
Morphogenetic guidance cues in emergence of structure & function during organogenesis
February 23
Kontinkangas Science Day
Biocenter Day 2015
Personalized Medicine: Hype or Hope?
March 3
Minisymposium: Electron microscopy in science
March 19
March 15
Joel Sussman, Structural Proteomics Center, Dept. of Structural Biology, Weizmann Institute of Science,
Rehovot, Israel
What is in common between Alzheimer’s drugs & nerve agents? Acetylcholinesterase
March 16
BCO Day 2016: Microbiome: The Good, the Bad, and the Ugly
March 30
Host: Rik Wierenga
April 6–8
Spring Meeting of the International Graduate School in Molecular Medicine Ulm, Ulm, Germany
April 12
Genes and Society Argumenta project seminar: Genomi- ja hyvinvointitiedon hyödyntäminen – sääntelystä sovelluksiin
April 9
Host: Raija Soininen
Lluis Montoliu, Dept. of Molecular and Cellular Biology, National Centre of Biotechnology, Madrid, Spain
Functional analysis of non-coding DNA genomic sequences with CRISPR-Cas9 approaches
April 5–29
Course: Biomedical Imaging Methods, (in cooperation with Medical Research Center)
April 20
Oulu BioImaging Network OBI Day
May 21
Host: Gonghong Wei
Johanna Schleutker, Dept. of Medical Biochemistry and Genetics, Institute of Biomedicine,
University of Turku, Turku
Prostate cancer genetics – old challenges and new developments
May 18–20
Course on Next Generation Sequencing and Population Genetics
May
Research mobility funding seminar for doctoral students and postdocs
SUMMER 2016
AUTUMN 2015
August 27
Host: Johanna Myllyharju
Dmitri Papkovsky, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland
In situ control and imaging of oxygenation in tissue models with cell-penetrating phosphorescent probes
September 1
Host: Seppo Vainio
Andy McMahon, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of
Southern California, Los Angeles, USA
Repair of the adult kidney
October 1
Host: Aki Manninen
October 8
Host: Rik Wierenga
Charles Streuli, Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK
Cell-matrix interactions in breast biology: Controlling gene expression, circadian clocks, and the beginnings
of cancer
Yorgo Modis, Dept. of Medicine, Cambridge Immunology Network, University of Cambridge, Cambridge, UK
Multiscale imaging of cell entry and innate immune recognition of enveloped RNA viruses
June 1
BCO 30 Years – Event for staff and researchers
June 8–10
Basic Course on Light and Electron Microscopy
June 12–15
EMBO conference: The biochemistry and chemistry of biocatalysis: From understanding to design
AUTUMN 2016
All term
Biocenter Oulu Seminar Series
August
Workshop: Early Stage Drug Discovery in Finland and in Oulu
October 3
Nobel Prize in Physiology or Medicine: Kontinkangas Campus Event
October 12–14
Fall Meeting of the International Graduate School in Molecular Medicine Ulm, Ulm, Germany
October
Theme-specific Minisymposium: Omics in Biomedicine
October 29
Host: Johanna Myllyharju
Volker Haase, Division of Nephrology & Hypertension, Nashville, USA and Karolinska Institutet, Sweden
Oxygen metabolism in the kidney: Insights from genetic models
November 13
BCO-BCK Minisymposium in Kuopio
Recent highlights of protein and metabolite research
October
Virus core facility course on microRNA and viral vectors
November
Oulu-Ulm Joint PhD Programme Annual Retreat, Ulm, Germany
BCO Discovery of the Year 2015
November
Biocenter Oulu – Biocenter Kuopio Day
November
Oulu Bioscience Networking Event
November
Introduction to biocomputing
December
Discovery of the Year 2015
TBA
Genes and Society Argumenta Project Seminars
December 15
DOCTORAL PROGRAMME CURRICULUM OUTLINE
BIOCENTER OULU DOCTORAL PROGRAMME
CURRICULUM OUTLINE 2017–2018
THEME-SPECIFIC
MINI-SYMPOSIA
CORE FACILITY
COURSES
2017
2018
Biological Rhythms and Oscillations
Cell Communication & Vesicle Transport
BioImaging
BioImaging
Mouse Models
Mouse Models
Microarrays & Sequencing
Microarrays & Sequencing
Protein Crystallography, Proteomics &
Protein analysis
Protein Crystallography, Proteomics &
Protein analysis
Biocomputing & Bioinformatics
Biocomputing & Bioinformatics
MicroRNA & Viral Vectors
BCO
RESEARCH-DRIVEN
COURSES, WORKSHOPS
AND SYMPOSIA
OTHER NATIONAL
AND INTERNATIONAL
COURSES, SYMPOSIA
& CONFERENCES;
LOCAL, NATIONAL
AND INTERNATIONAL
COOPERATION EVENTS
REGULAR GENERAL
BCO EVENTS
GENERAL AND
CROSS-SECTORAL
EDUCATION
TBA;
Contributed by each BCO group
at least once per 4-year term
TBA;
Contributed by each BCO group
at least once per 4-year term
Oulu-Ulm Joint PhD Programme
Annual Retreat, Ulm
Oulu-Ulm Joint PhD Programme
Annual Retreat, Ulm
Emerging Approaches and Technologies
Across Disciplines
Emerging Approaches and Technologies
Across Disciplines
Biocenter Oulu – Kuopio Day
Biocenter Oulu – Kuopio Day
Bioscience Networking Event
Bioscience Networking Event
Multidisciplinary events
organized in cooperation
with other Doctoral Programmes
Multidisciplinary events
organized in cooperation
with other Doctoral Programmes
BCO Seminars (All term)
BCO Seminars (All term)
BCO Day
BCO Day
BCO Discovery of the Year
BCO Discovery of the Year
Transferable Skills, Working Life
and Business Skills,
and General Education by UniOGS
Genes and Society Argumenta Project
Closing Seminar
Transferable Skills, Working Life
and Business Skills,
and General Education by UniOGS

BIOCENTER OULU

Transcription

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