Students Meeting - Faculdade de Medicina da Universidade de Lisboa

Transcription

doctoral program of the lisbon academic medical centre
PhD
X
Students Meeting
www.medicina.ulisboa.pt/phdmeeting
MAR /ABR’16
- EEM
anfit.58
-
Book of
30|31| 01 Abstracts
10h-18h30
9h-18h30
9h-18h30
Dear colleagues,
CENTRO HOSPITALAR
LISBOA NORTE, EPE
Welcome to the X Edition of the Instituto de Medicina Molecular (IMM)/Lisbon
Academic Medical Centre (LAMC) PhD Students’ meeting!
PhD Students Committee
CAML PhD Meeting
Tiago Amado
João Vieira
Mafalda Matos
Alexandra Vítor
Pedro Pappotto
Marie Bordone
Pedro Barbacena
Ana Margarida Vaz
Pizza Seminar
Ana Portêlo
Bethania Cassani
André Faustino
Ana Filipa Guedes
Idálio Viegas
IMM Retreat
Cátia Janota
Bárbara Gomes
Ana Martins
Marcelo Augusto
Rita Vaz
Juliana Perazzo
Mário Felício
Tiago Figueira
PhD Students’ Representatives 2016
Maria Rebelo
Sílvia Arroz Madeira
Antónia Ferreira, Instituto de Formação Avançada, FMUL
[email protected]
Sponsor
Support
It has been almost a decade since a bright group of students organized the first
IMM PhD Students Meeting. Since then several things changed yet, the will and
determination of IMM new generation of students remained the same.
The main goal of this meeting, fully organized by members of the PhD Students
committee, is to create the opportunity for students to present and discuss their
work with the overall IMM community. However, it is also a social gathering that
allows students to gain further insight and inspiration for their PhD work.
This year’s edition has been extended in order to accommodate all students. Oral
presentations will be given by students from the 1st and 4th year, while students
from the 2nd and 3rd will be presenting a poster.
Moreover, there will be two appealing workshops in Carreer Development held
by Filipa Moraes (PhD), Consultant in STEM (Science, Technology, Engineering &
Math) Higher Education and Mélanie Héroult (PhD), Innovation Director at Bayer.
Finally, we will have four excellent keynote speakers that will certainly inspire us all:
Paulo Oliveira (PhD) and Carlos Pereira (PhD) from the Center for Neuroscience
and Cell Biology - Universidade de Coimbra, Nuno Figueiredo (MD, PhD) from
Champalimaud Foundation and Kirk Deitsch (PhD), from Weill Cornell Medical
College, New York.
We would like to acknowledge everyone involved in the organization of this meeting,
specially the “Meeting gang” of the current PhD Student Committee who spent
a lot of time in order to plan and build such an interesting and successful conference. We would also like to appreciate all the IMM/CAML community for engaging
in their own PhD students’ activities with, not only your presence, but also your
discussions, making all our effort in helping to grow an active, enthusiastic and
critical mass of students worthwhile.
URPC
Unidade de
Relações Públicas
e Comunicação
We truly wish you enjoy this meeting.
Thank you all!
Maria Rebelo and Sílvia Arroz Madeira
PhD Students’ Representatives
PROGRAMME
MAR
30
MAR
31
09h00 Keynote Speaker – Filipe Pereira, PhD
10h00 Coffee break + Poster Session I
11h00 Oral Session
11h00
4
11h20
1
13h30 Opening Session
11h25
1
14h00 Keynote Speaker – Paulo Oliveira, PhD
11h30
1
15h00 Oral Session
11h35
4
O1 Rita Pinto
11h55
1
O2 Diana Chapela
12h00
1
O3 Marcelo Dias
12h05
1
O4 Inês Leal
12h10
4
15h45 Coffee break
12h30
1
16h15 Oral Session
12h35
1
10h00 Registration
15h00
4
15h20
1
15h25
1
15h30
1
O12 Sara Coelho
O13 Amilcar Santos
O14 João Gomes
O15 Rita Belo
O16 Marta Figueiredo
O17 Daniel Martins
O18 Pedro Gouveia
O19 Ângelo Dias
O20 Maria Rebelo
O21 Inês Albuquerque
O22 Sara Baptista
O5 Armando Cruz
13h00 Lunch
O6 Ana Armada-Moreira
14h00 Oral Session
16h15
4
16h35
1
16h40
1
16h45
1
16h50
4
17h10
1
17h15
1
O23 Ana Maceiras
14h00
4
14h20
1
14h25
4
O10 Carolina Constant
14h45
1
O11 Catarina Laborinho
14h50
4
15h10
1
15h15
4
O7 Catarina Lourenço
O8 Miguel Andrade
O9 Alda Silva
17h30 Workshop – Filipa Moraes, PhD
O24 Maria Helena Brigas
O25 Rita Vaz
O26 Andreia Pereira
O27 Francisco Mouro
O28 Filipe Figueiredo
O29 Sreerama C Sridhara
16h00 Coffee break + Poster Session II
17h00 Keynote Speaker – Nuno Figueiredo, MD, PhD
APR
01
09h00 Keynote Speaker – Kirk Deitsch, PhD
PhD Students Abstracts
10h00 Coffee break + Poster Session III
Oral Sessions
11h00 Oral Session
Pág
O30 Ana Rodrigues
4
O31 Elsa Sousa
1
O32 Ana Ferro
1
O33 Miguel Nobre
1
O34 André Faustino
4
O35 Patrícia Carvalho
1
O36 Susana Dias
1
O37 Julia Skalska
1
O38 Rita Domingues
4
O39 Ana Cardoso
1
O40 Mariana Oliveira
1
11h00
4
11h20
1
11h25
1
11h30
1
11h35
4
11h55
1
12h00
1
12h05
1
12h10
4
12h30
1
12h35
1
13h00 Lunch
14h30 Workshop – Mélanie Héroult, PhD
15h30 Oral Session
O41 Cláudia Lima
15h30
4
15h50
1
15h55
1
O1
| Rita Pinto
17
O2
| Diana Chapela
18
4
O3
| Marcelo Dias
18
1
O4
| Inês Leal
18
4
O5
| Armando Cruz
19
1
O6
| Ana Armada-Moreira
20
4
O7
| Catarina Lourenço
20
4
O8
| Miguel Andrade
21
1
O9
| Alda Silva
22
1
O10 | Carolina Constant
22
1
O11 | Catarina Laborinho
23
4
O12 | Sara Coelho
24
1
O13 | Amilcar Santos
24
1
O14 | João Gomes
24
1
O15 | Rita Belo
25
4
O16 | Marta Figueiredo
26
1
O17 | Daniel Martins
26
1
O18 | Pedro Gouveia
26
4
O19 | Ângelo Dias
27
1
O20 | Maria Rebelo
28
1
O21 | Inês Albuquerque
28
4
29
1
30
4
4
1
1
1
4
O42 António Ascenso
1
O43 Mara Alves
1
16h00 Coffee break + Poster Session IV
1
15h30 Oral Session
4
O44 Sérgio Ribeiro
1
O45 Sofia Mensurado
1
17h00
4
17h20
1
17h25
4
O46 Sara Fernandes
Pág
1
O22 | Sara Baptista
4
O23 | Ana Maceiras
O24 | Maria Helena Brigas
18h00 Awards Ceremony & Closing Session
20h30 Celebration dinner
7
30
O25 | Rita Vaz
31
O26 | Andreia Pereira
32
O27 | Francisco Mouro
33
O28 | Filipe Figueiredo
33
O29 | Sreerama C Sridhara
33
O30 | Ana Rodrigues
34
O31 | Elsa Sousa
35
O32 | Ana Ferro
36
O33 | Miguel Nobre
36
O34 | André Faustino
37
O35 | Patrícia Carvalho
38
O36 | Susana Dias
38
O37 | Julia Skalska
38
O38 | Rita Domingues
39
O39 | Ana Cardoso
40
O40 | Mariana Oliveira
40
O41 | Cláudia Lima
41
O42 | António Ascenso
42
O43 | Mara Alves
42
O44 | Sérgio Ribeiro
43
O45 | Sofia Mensurado
44
O46 | Sara Fernandes
44
Poster Sessions
MAR 31st
16:00 – 17:00
10:00 – 11:00
Poster Session I
Pág
Poster Session II
P1
Fernando M Martins
47
2
P3
Válter R Fonseca
48
3
P5
Tiago N Figueira
49
3
P7
Rui Traquete
50
3
3
2
3
3
3
P31
Bruno F Gonçalves
75
2
P33
Ana Catarina Policiano
76
2
P35
Viriato M'bana
77
3
P37
Sofia R Fernandes
78
2
P39 Ana Paula Barbosa
79
2
Tiago Amado
62
P8
Sílvia Arroz Madeira
63
2
P10
Gabriela Ribeiro
64
2
P12
David Angelo
65
3
P14
Mafalda R Pimentel
66
Oth
P16
Rita Vaz-Drago
67
2
P18
Ana Filipa Guedes
68
2
P20 Bethania García Cassani
69
3
Alexandra C Vitor
53
P15
Padma Akkapeddi
54
3
55
3
P19
Ana Portêlo
56
Oth
P21
Debanjan Murkherjee
57
2
P23
Mariana T Ferreira
58
3
P25
Mário R Felício
59
2
2
74
P6
P13
2
Vasco Conceição
2
3
3
P29
2
2
P17 Maria Inês Chendo
3
61
51
3
73
João Gama Marques
52
Oth
Vânia Cardoso
P4
Raquel Freitas
Poster Session IV
Pág
P27
2
Margarida Sanches-Vaz
3
Poster Session III
60
P9
3
Pág
Margherita Fabbri
P11
3
16:00 – 17:00
10:00 – 11:00
P2
2
2
APR1st
P28
Ângela Raquel A Leal
88
P30
Joana B Santos
89
P32
Nádia R Henriques Rei
90
P34
Emanuel F Fernandes
91
P36
Carlos Noronha Ferreira
92
P38
Ana de Souto Martins
93
P40
Idálio Viegas
94
P42
Filipa Gil Marques
95
P44
Cátia Janota
96
P41
Pedro H Papotto
80
3
P43
Pedro Laires
81
2
P45
Joana Ministro
82
Oth
P47
Marie Bordone
84
3
P49
Pedro Barbacena
85
3
P51
Mafalda R. Matos
86
P22
Jessica G Bevan
70
2
P24
João Mello-Vieira
71
2
P26
Ana Catarina Farinha
72
3
P46 Isanete Alonso
9
Pág
97
P48
Joana C Reino Carvalho
98
P50
Nataniel G Rosa
99
Keynote Speaker
Keynote Speaker
Paulo J. Oliveira
Carlos Filipe Pereira
Group Leader, Mitochondrial Toxicology
and Experimental Therapeutics
Center for Neuroscience and Cell Biology,
Universidade de Coimbra, Portugal
Group Leader, Cell Reprogramming and
Developmental Hematopoiesis
Center for Neuroscience and Cell Biology,
Universidade de Coimbra, Portugal
In 1999, Paulo J. Oliveira received his Bachelor of Science from Universidade de
Coimbra in Biochemistry. In 2003, he completed his PhD in Cellular Biology from
the same University. After completing his doctorate, Paulo Oliveira spent more
than three years working at the Medical School of the University of Minnesota,
Duluth, USA, where he collaborated with several researchers and contributed
to the publication of several peer-reviewed manuscripts. Paulo Oliveira current
research interests include the alteration of cardiac mitochondrial function by physical activity and diet, cardiac mitochondrial dysfunction and cell death caused by
anti-neoplastic agents, mitochondrial alterations during cancer stem cell differentiation and carcinogenesis or rational designing of mitochondrial-directed agents.
He is currently one of the most well-known Portuguese researchers in this area,
collaborating with several collaborations in Europe, Africa and in the USA. He has
also received several prizes from the Portuguese Cardiology Society for his work on
cardiac mitochondrial research.
Besides research, Paulo Oliveira is often involved in teaching at the Department of
Life Sciences, University of Coimbra, as well as in other Universities in Portugal and
elsewhere, besides in numerous science communication activities.
Of importance, he has maintained a consistent primary role in the organization of
national and international scientific meetings, including the International Courses
in Toxicology (2005-2010 in Coimbra), 2013 Annual Meeting of the European
Society for Clinical Investigation (Albufeira, Portugal) and the 2014 Meeting of the
Portuguese Biochemical Society. Since April 2013, Paulo Oliveira is also Councilor
for the European Society for Clinical Investigation, having an important role in the
management of the Society and of the Annual Meetings.
Filipe Pereira is currently the group leader of the Cell Reprograming and Developmental Hematopoiesis Laboratory at the Center for Neuroscience and Cell Biology,
University of Coimbra, Portugal. The laboratory is focused on cell fate engineering
and hematopoiesis. Filipe Pereira has long held an interest in understanding how
cell identity is acquired, maintained and ultimately modified or reversed. He has
completed his Biology Degree from the University of Porto, Portugal and the Ph.D.
from Imperial College of London, U.K. He has made several contributions to the
field of Cellular Reprogramming during his graduate and short post-doctoral studies in Prof. Amanda Fisher’s laboratory at the MRC Clinical Sciences Centre, by
addressing the molecular mechanisms underlying reprogramming. Filipe Pereira
then joined the group led by Prof. Ihor Lemischka, Icahn School of Medicine at
Mount Sinai, New York, USA, as a postdoctoral fellow. In New York he identified
the combination of transcription factors Gata2, Gfi1b, cFos, and Etv6 that efficiently induce endothelial-like precursor cells with the subsequent appearance of
hematopoietic progenitor cells. Filipe Pereira has published 27 papers in international peer-reviewed journals with over 1600 citations and h-index of 16, including
papers in high impact factor journals (Cell Stem Cell, Nature Cell Biology, Plos
Genetics, Genes & Development, Molecular Cell, etc…). He is also a co-inventor
of a patent held by Mount Sinai that describes a method to program hematopoietic stem cells from fibroblasts.
He has received a PhD fellowship from FCT (GABBA program), postdoctoral fellowships from EMBO, Revson foundation and Marie Curie, 4 independent travels
awards and the Rotary club merit award. Filipe Pereira has recently obtained funding, as principal investigator, from FCT. He is an associate Faculty Member of
the Faculty of 1000 and an editorial board member of the International Journal of
Stem Cell Research and Transplantation.
11
Keynote Speaker
Keynote Speaker
Kirk W. Deitsch
Nuno F. L. Figueiredo
Professor of Microbiology and Immunology
Weill Cornell Medical College, New York, USA
Digestive Surgeon and Investigator,
Champalimaud, Lisbon, Portugal
Kirk W. Deitsch received his Bachelor of Science from Central Michigan University
in 1989. He earned his PhD in genetics investigating the molecular biology of mosquito reproduction at Michigan State University in 1994. After completing his doctorate, Deitsch spent five years as a postdoctoral fellow at the National Institutes
of Health, where he began working with the parasites that cause severe malaria,
Plasmodium falciparum, under the direction of Thomas Wellems. Kirk Deitsch has
devoted his career studying how P. falciparum evades the host immune response.
The focus of the research being conducted at Weill Cornell Medical College, where
he arrived in 2001, is the var gene family that controls the process of antigenic variation. Currently, his lab studies the molecular basis for antigenic variation and epigenetic regulation of gene expression in P. falciparum, as well as other cellular processes associated with this parasite, namely DNA repair and nuclear organization.
Specifically he is addressing how malaria parasites intentionally switch expression
of PfEMP1, the protein encoded by the var gene family, on the surface of an infected
red blood cell to avoid destruction by the immune system and maintain infection.
Moreover, in collaboration with other research colleagues, the Deitsch lab also
investigates mechanisms of drug resistance and aims to develop new antimalarials.
Kirk Deitsch is a recipient of a New Scholar Award in Global Infectious Diseases
from the Ellison Medical Foundation as well as a 2002 Presidential Early Career
Award in Science and Engineering. In addition to his research interests, he is the
co-director of the Molecular and Cell Biology PhD Program at Weill Cornell. He also
serves on several editorial boards and NIH study sections.
Nuno Figueiredo graduated from Faculdade de Medicina, Universidade de Lisboa in
2001. His general internship was done in Centro Hospitalar de Cascais (2002 -2003)
and he then made is residency in General Surgery at Hospital Santa Maria, Lisbon
(2004-2010). He earned his PhD in 2013, investigating the protective role of Epirubicin in severe sepsis. After completing his doctorate, Nuno Figueiredo integrated
the International Laparoscopic Colorectal Surgery Masterclass in Queen Alexandra
Hospital, Portsmouth. From 2013 to the present day he is invited Professor of Surgery at Faculdade de Medicina, Universidade de Lisboa.
Nuno Figueiredo’s group is interested in different aspects of the oncology field, focusing mainly in colorectal cancer (CRC), tumour response to neo-adjuvant treatment
and robotic and laparoscopic surgery. His group is part of the consortium of International Watch & Wait Database, and is also involved in the ENVISAGE (Enhanced
Near-infrared VISualisation Antibody-guided imaGEbased surgery) project, which
aims to develop innovative and robust tumour-specific molecular imaging techniques
that will aid surgical procedures, improve clinical decision making and improve treatment strategies for patients with rectal cancer, through the creation of innovative
fluorescent small antibody-derived probes. Additionally Nuno Figueiredo’s group has
been trying to establish a personalized colorectal cancer therapy platform using novel
patient-derived primary and metastatic colorectal cancer xenografts in zebrafish. Zebrafish “xenopatients” can then be tested with the anti-cancer agents currently available in the clinic, thus helping to achieve a personalized, tailor-made treatment.
Apart from being surgery consultant in different portuguese and international hospitals, Nuno Figueiredo is a member of several societies such as: Sociedade de Ciências Médicas de Lisboa, Sociedade Portuguesa de Cirurgia, Sociedade Portuguesa
de Cuidados Intensivos and Sociedade Portuguesa de Coloproctologia VIII.
He earned several awards for best oral presentations and in 2013 he received the
Pfizer award for Fundamental Research.
13
Workshop Speaker
Workshop Speaker
Filipa Moraes
Mélanie Héroult
Consultant in STEM (Science, Technology,Engineering & Math)
Higher Education; Mentoring and Career Development
Innovation director, Bayer,
Leverkusen, Germany
Since 2015 Filipa Moraes has been a consultant in STEM Education, Mentoring
and Career Development. She studied Applied Chemistry/Biotechnology at the
Faculdade de Ciências e Tecnologia -Universidade Nova de Lisboa. During her PhD,
she worked at the Instituto Gulbenkian de Ciência with Dr. Moisés Mallo and in
2010 she received her doctorate degree in Biology from ITQB-UNL. Subsequently,
in 2010 she began her postdoctoral studies at Yale University, USA, in Dr. Michael
Simons’ lab, where she studied signaling pathways in arteriogenesis. In 2014, she
held a Teaching Scholar Appointment in Biology at the Faculty of Arts and Sciences
at Yale University. As a qualified Scientific Teaching Instructor (Howard Hughes
Medical Institute (HHMI)/ Yale Teaching Fellows Program), Filipa has proven experience in teaching at Yale University (Undergraduate and Graduate level) in different areas such as Genes & Development, Evolution, Mentoring, Research Conduct,
Scientific Teaching, Research Proposal & Paper Writing.
From October 2015 to March 2016, Filipa was the Advanced Training Manager at
Instituto de Medicina Molecular (iMM), where she managed the advanced training programs and provided mentoring and career development support to PhD
students and Postdoctoral trainees.
Mélanie Héroult studied Biology, Biochemistry and Molecular Biology at the University of PARIS XII, graduated as a ph.D. in Biochemistry . She subsequently
spent several years in the Maine Medical Research Institute (MMCRI - Portland,
US) and at the Clinic for tumor biology (KTB - Freiburg, Germany) where she
worked as postdoctoral researcher in the fields of Vascular Embryology Research
and Oncology Research. Mélanie Héroult then held a position as junior group
leader at the German Cancer Research Center (DKFZ - Heideilberg, Germany) in
the field of Oncology and Metastasis Research.
Mélanie Héroult joined Bayer in 2008 as Lab leader in Oncology Research in Wuppertal, (Germany) and in Berlin (Germany) research facilities. She led early drug
discovery programs and took part to the overarching research program between
Pharma and Cropsciences scientists. Mélanie Héroult joined the corporate Innovation Strategy team (Leverkusen, Germany) in 2014 where she led the group-wide
Innovation Culture project. Mélanie Héroult was newly appointed in 2016 to lead
the corporate R&D team to support the member of the board of Management at
BAYER for Innovation.
In 2015, Filipa moved to Portugal and has since been developing her course portfolio specializing in mentoring, career strategy and scientific teaching training.
15
4
Abstracts
Oral Sessions
O1
Asymmetry versus Symmetry: the role of Dmrt2a
in the formation of the vertebrate body plan
R.A. Pinto1 2, J. Almeida-Santos1 2, L. Saúde1 2
Instituto de Medicina Molecular e Instituto de Histologia e Biologia do Desenvolvimento, Faculdade
de Medicina da Universidade de Lisboa, Lisboa, Portugal;
2
Instituto Gulbenkian de Ciência, Oeiras,Portugal
1
[email protected]
Dmrt2a, a zinc finger-like transcription factor, has been identified as a coordinator
of two key processes that set up the vertebrate body plan: left-right asymmetry
in the lateral plate mesoderm (LPM) and bilateral symmetry in the presomitic
mesoderm (PSM). In order to understand Dmrt2a dual function, we will uncover
its mechanism of action. To achieve this goal, we built two heat-shock inducible
transgenic lines (Hsp70:Venus-dmrt2a and Hsp70:HA-dmrt2a) to overexpress
Dmrt2a in a controlled temporal manner and two mutant lines resorting to the
TALEN technology.
Our results show that in embryos where Dmrt2a is overexpressed at the bud stage:
(1) symmetric genes, such as deltaC and her1, become asymmetrically expressed
in the PSM, (2) left-sided genes, such as spaw and pitx2, become expressed in the
right LPM or bilaterally, (3) an asymmetrically located organ (heart), is affected in
both its key development times, (4) somite markers (myoD, mespb, papc) show
the absence of proper somitic borders and also somite fusions, (5) wavefront
markers (raldh2, fgf8, cyp26a1) are disturbed.
Also, we produced mutations within the first exon of dmrt2a and dmrt2b that will
be validated soon. Dmrt2b might cooperate with Dmrt2a in its biological functions, thus we will characterize its phenotype both per se and in double mutants.
According with our goal, we will use these lines in a microarray approach to
uncover the transcriptional regulatory network where Dmrt2a operates.
17
O2
1
Innovative Therapies for Spinal Cord Injury: a
zebrafish approach
D. Chapela1 2, L. Saúde1, S. Sousa2
1
2
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
Technophage
4
O5
The role of endocannabinoids upon hippocampal
ltp in the absence of adenosine a1 receptor-mediated signalling
A. Cruz1 2, M. Carlstrom3, A.M. Sebastião1 2
1
[email protected]
Institute of Pharmacology and Neurosciences, Faculdade de Medicina;
Unit of Neurosciences, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa;
3
Department of Physiology and Pharmacology, Karolinska Institutet, Sweden
2
[email protected]
O3
1
Time coding in contextual memories
M. Dias1, M. Remondes1
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
[email protected]
O4
1
The role of certolizumab in non-infectious uveitis
I. Leal, J.E. Fonseca1 2, C.M. Neves3 4 5
Serviço de Reumatologia, Centro Hospitalar Lisboa Norte;
Unidade de Investigação em Reumatologia, Instituto de Medicina Molecular, Faculdade de
Medicina, Universidade de Lisboa, Lisboa, Portugal;
3
Serviço de Oftalmologia, Centro Hospitalar Lisboa Norte;
4
Clínica Universitária de Oftalmologia, Faculdade de Medicina Universidade de Lisboa, Lisboa, Portugal;
5
Centro de Estudos das Ciências da Visão
1
2
[email protected]
Adenosine and endocannabinoids are important modulators of neuronal activity. Cannabinoid CB1 receptor (CB1R) activation inhibits GABA and glutamate release in the hippocampus, these effects being partially reduced by co-activation
of adenosine A1 receptor (A1R), suggesting an interaction between these modulatory pathways (1,2). We now studied the impact of CB1R on hippocampal (CA1)
long-term potentiation (LTP) and the A1Rs involvement on this modulation.
Extracellular electrophysiological recordings of field-excitatory post-synaptic potentials (fEPSPs) were performed at the CA1 area of hippocampal slices taken
from adult (8-18 weeks-old) wild type (WT) or A1R KO mice (C57Bl/6). LTP was
induced by electrical stimulation of the afferents with a weak- �-burst (five 100Hz
bursts, 4 stimuli, separated by 200 ms) or a strong- �-burst (ten 100Hz bursts, 4
stimuli, separated by 200 ms). The magnitude of LTP was quantified at 50-60 minutes after �-burst stimulation.
Both in WT and A1R KO mice, LTP magnitude induced by the weak- �-burst in the
presence of the CB1R antagonist, AM251 (1µM), was higher (P<0.05) than in its
absence. With strong- �-burst stimulation, LTP magnitude in both conditions was
of similar magnitude (P>0.05). Unexpectedly, AM 251 (1 µM) blocked LTP induced
by the strong- �-burst in A1R KO mice but not in WT mice. LTP induced by a strong�-burst was also decreased (P<0.5) by AM251 (1 µM) in slices from WT mice with
A1R blocked by the selective antagonist DPCPX (50nM).
These results suggest that under conditions of hampered A1R signalling, CB1R
activation by endogenous cannabinoids is crucial for maintenance of LTP induced
by intense neuronal firing.
1- Sousa VC et al (2011) Neuropsychopharmacology, 36, 472.
2- Hoffman AF et al (2010) J Neurosci. 30:545
19
O6
1
Astrosomes: a novel approach for the regulation
of neuronal communication
A. Armada-Moreira1, S.H. Vaz2 3, B. Städler4
4
The In / Del genotype of angiotensin converting
enzyme and blood pressure level are independent
factors of longevity
1
A.Silva1 2 3, A. Matos1 2, A. Gil1 2, J. Gorjão-Clara3 4, M. Bicho1 2
2
1
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
3
Institute of Pharmacology and Neurosciences, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
4
Interdisciplinary Nanoscience Centre (iNANO), Aarhus University, Aarhus C 8000, Denmark
[email protected]
O7
1
Regulation of adenosine levels as a new therapeutic strategy for Rett Syndrome
C. Miranda-Lourenço1 2, M.J. Diógenes1 2, A.M. Sebastião1 2
1
2
Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
[email protected]
O8
1
The development of episodic memory and
cognitive control: Behavioral and neural insights
from adolescence
M.A. Andrade1, A.L. Raposo2
1
2
Faculdade de Psicologia, Universidade de Lisboa, Lisboa, Portugal
[email protected]
O9
Genetics Laboratory and Environmental Institute of Health, Faculdade de Medicina da Universidade de Lisboa;
Instituto Rocha Cabral, Lisbon;
3
Universitary Geriatric Unit of Faculdade de Medicina, Universidade de Lisboa, Portugal;
4
Lisbon Academic Medical Centre - North of Lisbon Hospital Center
2
[email protected]
Objective: Assess the distribution of genotypes associated with the renin-angiotensin-aldosterone axis, in a sample of Portuguese centenarian population and its
association with hypertension and compare with a sample of lower age group of
individuals either hypertensive or normotensive.
Design and method: The study comprised a Caucasian population of 253 centenarians (77.9% women), with a mean age of 100.3±2.0 years, from different regions of
Portugal, and a control group of 268 individuals with mean age of 67.52±3.271, both
genders. It were hypertension criteria those of ESH/ESC 2013 and or under anti hypertensive medication. Controlled BP values were considered according to JNC8. For the
blood pressure measurements a Colson MAM BP3AA1-2 device was used. Genotyping was done for ACE, angiotensinogen, and angiotensin receptor 2 by DNA Microchip
platform using iPlex MassAssay system (Sequenom), Malditof mass spectrometry.
Results: According to hypertensive criteria, 62.8% (N=159) of the centenarian’s individuals were hypertensive and 79.5% (N=213) of control group were hypertensive.
We found to be normotensive is a protective factor in achieving longevity OR=0.252
CI 95%=0.138-0.461, p<0.00001. Furthermore the ACE II genotype, adjusted to
the level of blood pressure, was a risk factor in either homozygous (II) OR=7.510
CI95%=3.397-16.605, p<0.00001 both in heterozygous (ID), in relation to the referent (DD) OR=2.072 CI 95%=1.223-3.508, p=0.007. The same was verified considering as a criterion for normotensive, controlled BP values according to JNC8:
Genotype II, OR=8.263 CI95%=3.7-18.453, p<0.00001, genotype ID, OR=2.146
CI95%=1.262-3.648, p=0.005. There were no significant differences in the distribution of frequencies of other genotypes among subgroups of the sample.
Conclusion: The deletion mutation, latest in the ACE gene may be the result of
an adaptive mechanism for the human being acquires greater ability to longevity.
Since we cannot interfere with genes, we can, however, interfere with modifiable
factors, such as blood pressure, thus contributing to greater longevity.
21
O10
O12
1
4
Novel lung function parameters as pathophysiologic markers for the diagnosis and prognosis in
four different chronic obstructive lung diseases
in childhood – time for paradigm shift?
Lost in Time. A neurophilosophical quest to
understand the perception of time in MCI patients
S. Coelho1, M. Guerreiro1, C. Chester1, D. Silva1, J. Maroco2, M. Coelho1, F. Paglieri3,
A. de Mendonça1
Institute of Molecular Medicine and Faculdade de Medicina, Universidade d Lisboa, Lisboa Portugal;
William James Center for Research, ISPA-IU, Rua Jardim do Tabaco, 34, 1149-041 Lisboa;
3
Institute for Cognitive Sciences and Technologies of the CNR, Via S.Martino della Battaglia 44,
00185 Rome, Italy
1
C. A. Constant , T.I.C.C. Bandeira , A.O.C.A. Falcão
1
2
3
Centro de Estudos da Função Respiratória, do Sono e da Ventilação. Departamento de Pediatria,
Hospital de Santa Maria, Centro Hospitalar Lisboa Norte;
2
3
Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal
1
2
[email protected]
[email protected]
O11
1
Molecular Biomarkers Associated with Respiratory
Insufficiency in Amyotrophic Lateral Sclerosis
A.C.P. Laborinho1 2, M. Carvalho1 2 3
Instituto de Fisiologia, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
3
Department of Neurosciences, Hospital de Santa Maria ,Centro Hospitalar Lisboa Norte, Lisboa, Portugal
1
2
Patients with mild cognitive impairment (MCI) may have difficulties in time perception. Objective: We aimed to test whether the internal clock is changed in
MCI patients as compared to healthy controls of the same age. Additional, we
wanted to see if this change can influence the subjective passage of time judgments and intertemporal choices. Method: Fifty-five MCI patients and fifty-seven
healthy controls underwent an experimental protocol for time perception on interval length, a questionnaire for the subjective passage of time, an intertemporal
choices questionnaire and a neuropsychological evaluation. Results: MCI patients
presented no changes in the perception of interval length. However, they reported
the time passing slower than controls. This experience was significantly correlated with memory deficits and memory complaints, but not with performance
in executive tests, depressive or anxiety complaints. Finally, MCI patients have no
alterations in temporal preferences in comparison with the control group Conclusions: Memory deficits do not affect neither the perception of interval length nor
temporal preferences, but are associated with alterations in the subjective experience of time.
[email protected]
23
O13
O16
1
Spinal Cord Stimulation as a new therapy for Unipolar
and Bipolar Depression – a translational project.
A. Silva-dos-Santos1 2, A. Sebastião2 3
Psychiatry Unit, Hospital Vila Franca de Xira, Vila Franca de Xira, Portugal; 2 Unit of Neurosciences,
3
Institute of Pharmacology and Neurosciences, Faculdade de Medicina, Universidade de Lisboa,
Lisboa, Portugal
1
[email protected]
O14
4
Notch and Hedgehog in thymus/parathyroid
common primordium: a crosstalk to organ formation
M. Figueiredo1 2 *, J. Silva1 *, S. Santos1, V. Proa1, I. Alcobia1 2, S. Dias2, R. Zilhão3,
A. Cidadão1, H. Neves1 2
* co-authors
1
Instituto de Histologia e Biologia do Desenvolvimento, Edifício Egas Moniz, Faculdade de Medicina da
Universidade de Lisboa, Lisboa, Portugal;
2
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal;
3
Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
[email protected]
1
BDNF receptor cleavage: relevance for AD
pathophysiology
J. Fonseca-Gomes1 2, A. Jerónimo-Santos1 2, A.M. Sebastião1 2, M.J. Diógenes1 2
Institute of Pharmacology and Neurosciences, Faculdade de Medicina, Universidade de Lisboa,
Lisboa, Portugal; 2 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,
Lisboa, Portugal
1
[email protected]
O15
1
Kyotorphin as new pharmacological therapeutic
strategy for Alzheimer’s Disease
Avian thymus and parathyroids (T/PT) common primordium derives from the endoderm of the third and fourth pharyngeal pouches (3/4PP). The molecular mechanisms that govern its development are not fully understood. Herein we studied
the effects of Notch and Hedgehog (Hh) signalling modulation during common
primordium development using in vitro and in ovo approaches. The impairment
of Notch activity reduced Foxn1/thymus-fated and Gcm2/Pth/parathyroid-fated
domains in the 3/4PP and further compromised the development of parathyroids.
When Hh signalling was abolished, we observed a reduction in Gata3/Gcm2 and
Lfng expression-domains at the median/anterior and median/posterior territories
of the pouches, respectively. In contrast, Foxn1 expression-domain at the dorsal
tip of the pouches was ventrally expanded into the Lfng expression-domain. Hh
signals were also required for the survival of pharyngeal tissues and organs formation. This study offers new evidence to the role of Notch signalling in the T/PT
common primordium development, in an Hh-dependent manner.
R.A.F. Belo1, M.J. Diógenes1 2, V.L.S. Neves1, A.M.F. Sebastião1 2
1
2
[email protected]
25
O17
O20
1
Oxytocin - Dopamine interplay in Human Social
Decision-Making: a pharmaco-genetics-fMRI
approach
4
Antimalarial Drug-Resistance: Development of a
Novel Drug Assay for Plasmodium falciparum
M.S. Rebelo1, T. Hanscheid1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, 1649-028
Lisboa, Portugal
1
D. Martins1 2, M. Mehta2 , D. Prata1 2
1
2
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal ;
Centre for Neuroimaging Sciences, Institute of Psychiatry (IoP), King’s College London
[email protected]
O18
1
Breast volumes for breast cancer surgery planning
P. Gouveia1, J.P. Monteiro2, H.P. Oliveira2, M.J. Cardoso1, J.S. Cardoso2
1
Breast Unit, Champalimaud Clinical Center, Lisboa, Portugal; 2 INESCTEC, CTM, Porto, Portugal
[email protected]
O19
1
Reinforcement learning and Pavlovian bias in
children with Tourette syndrome
Â. Dias1, L. Ernst1, T. Maia1
1
[email protected]
[email protected]
Antimalarial-drug resistance is a threat for malaria control. In vitro antimalarial
sensitivity testing is crucial to detect and monitor drug resistance. Current assays
have been successfully used to detect drug effects on parasites. However, they
have some limitations, such as the use of radioactive or expensive reagents or
long incubation times. During my PhD I have developed a novel assay to detect
antimalarial drug effects, which might overcome some of the limitations of existing assays. This assay is based on the flow cytometric detection of hemozoin (Hz).
Hz is produced by the parasite itself during its erythrocytic cycle and its content
increases as the parasite matures, thus constituting an optimal growth indicator. The usefulness of this assay was investigated both in vitro, using laboratoryadapted strains, and ex vivo, using blood samples collected from malaria patients
in a malaria-endemic country.
Detection of Hz was accomplished by modifying a common flow cytometer, which
allowed the detection of light depolarization. Synchronized in vitro cultures of P.
falciparum were incubated with several antimalarial drugs for 48h. While, blood
from malaria patients was incubated for 72h with increasing concentrations of
chloroquine, artesunate and artemisinin. The percentage of depolarizing red
blood cells (RBC) was used as the maturation indicator and measured every 24
hours during the incubation period to determine parasite growth and drug effects.
Analysis of depolarizing events, corresponding to parasitized RBCs containing Hz,
allowed the detection of parasite maturation and antimalarial-drug effects already
after 24h of incubation, both in vitro and ex vivo.
These results suggest that this new approach could be developed into a simple (no
added reagents), rapid (24-hour) and objective drug assay. This would be a useful
tool not only in malaria endemic countries but also for rapid resistance testing in
returning travellers.
27
O21
O23
1
A modular approach to homogenous synthetic
glycoprotein vaccines
M.I.S. Albuquerque1, G.J.L. Bernardes1 2, R. Adamo3
1
2
University of Cambridge, UK; 3 GSK Vaccines, Siena, Italy
[email protected]
4
Different TCR-specificity requirements in T
follicular helper and T follicular regulatory cells
from the same germinal centers
A.R. Maceiras1 2, S.C.P. Almeida1 2, E. Mariotti-Ferrandiz3 4, W. Chaara4 5, F. Jebbawi4,
A. Six3 4, D. Klatzmann3 4 5, J. Faro2 6 7, L. Graça1 2
Instituto Gulbenkian de Ciência, Oeiras, Portugal;
3
Sorbonne Universités, UPMC Univ Paris 06, UMRS 959, Immunology-Immunopathology-Immunotherapy
(i3), F-75005, Paris, France;
4
INSERM, UMRS 959, Immunology-Immunopathology-Immunotherapy (i3), F-75005, Paris, France;
5
AP-HP, Hôpital Pitié-Salpêtrière, Biotherapy and Département Hospitalo-Universitaire
Inflammation-Immunopathology-Biotherapy (i2B), F-75651, Paris, France;
6
Área de Immunoloxía, Facultade de Bioloxía, and Centro de Investigacións Biomédicas (CINBIO),
Universidade de Vigo, Vigo, 36310 Vigo, Spain;
7
Instituto Biomédico de Vigo, 36310 Vigo, Spain
1
2
O22
1
Identification and characterization of novel
malaria liver stage antigens
S. Baptista1, M.M. Mota1, V. Zuzarte-Luis1
1
[email protected]
[email protected]
Immunization with an antigen leads to formation of germinal centers (GC) containing T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Tfr cells can
regulate GC responses and have been implicated in the prevention of autoimmunity. Therefore, given the preferential differentiation of Tfr cells from thymic
Tregs, we investigated the hypothesis that Tfr cells are predominantly autoreactive
differing from the Tfh TCR repertoire. By transferring defined TCR-transgenic T
cells we demonstrated preferential GC recruitment of Tfh cells specific to the immunizing antigen, while this preferential recruitment was not observed among Tfr
cells. In parallel, we compared the TCR diversity of polyclonal populations of Tfh
and Tfr cells in the peak of the GC response, from the same draining LN. While
Tfh cells show oligoclonal expansion of clones with particular TCR usage, Tfr cells
from the same GCs retain a broad TCR usage similar to the TCR diversity observed
in the precursor thymic-derived regulatory T cell population. Taken together, our
data show a difference in TCR usage by Tfh and Tfr populations in line with a different function of the two populations: Tfh cells promote antigen-specific B cell
responses and Tfr cells prevent autoimmunity.
29
O24
1
Searching for innate and adaptive immune cell
fingerprints in Alzheimer’s disease progression
M.H.C. Brigas1, L. Lopes1, J. Ribot1
1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
[email protected]
enables the identification of compounds with locomotor rescuing properties. This
assay also gives a hint about the dose of the selected compounds, which then was
translated to a rodent model. A rodent model that mimics the non-responsive
periods of L-Dopa was characterized, so that the selected compounds in the zebrafish screening were tested in a pre-clinical model. This mouse model exhibited
an aggravation of gait impairments after L-Dopa effectiveness peak, when chronically treated with L-Dopa. The compounds selected for rescuing gait impairments
in this mouse model are potential candidates to be new therapeutic alternatives
for late-stage PD and with the study of their mechanism of action new doors may
be opened to better understanding PD etiology.
O25
4
O26
1
Selection of a new therapeutic compound for
Parkinson’s disease: screening approach
R. Vaz1, D. Chapela1, J.E. Coelho2, L.V. Lopes2, N.D. Afonso1, T.F. Outeiro3 , S. Sousa1
TechnoPhage, SA, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
3
Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
1
2
[email protected]
Unravelling the specific properties of neuronal
mitochondria
A.S.F. Pereira1, V. Morais1
1
[email protected]
Parkinson’s disease (PD) is the second most common neurodegenerative disease.
The cardinal symptoms of the disease are bradykinesia, resting tremor, muscular
rigidity and postural instability. Levodopa (L-dopa) is a powerful treatment for motor symptoms in PD. However, its effectiveness decreases over time and the nonresponsive periods to this therapeutic approach tend to increase in late-stages
of the disease. The lack of alternative therapeutic compounds for these periods,
together with an aggravation of the symptoms are major concerns in the disease.
With the increase of prevalence of late-stage PD, new therapeutic options for this
stage of the disease are needed, along with new animal models. We here propose
a new selection process of alternative therapeutic options for late-stage PD. The
compounds were selected in an in vivo screening assay with zebrafish model that
31
O27
O28
4
1
Adenosine A2a receptors: hand-in-hand with cb1rs
mediating memory consolidation deficits
Unraveling The Nutritional Determinants In
Multiple Sclerosis
F.M. Mouro1 2, V.L. Batalha2, D.G. Ferreira2, C.E. Muller3, L.V. Lopes2, J.A. Ribeiro1 2,
A.M. Sebastião1 2
1
Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa;
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa.
3
Pharmazeutisches Institut, University of Bonn, Germany
1
2
[email protected]
Adenosine and endocannabinoids modulate neuronal activity at the hippocampus,
a brain area targeted by cannabinoids to disrupt memory. Evidence shows that
CB1R agonist WIN 55,212-2 is able to disrupt memory consolidation in Novel Object Recognition Test (NOR). Also, WIN disrupts hippocampal long-term potentiation (LTP). We wanted to evaluate whether chronic/acute blockade of adenosine
A2A receptors (A2ARs) could affect both CB1R-mediated memory deficits in a
NOR paradigm and CB1R-mediated decrease in hippocampal LTP.
Concerning NOR paradigm, A2ARs were inactivated either chronically (30 days), using KW-6002 (3mg/kg/day), or acutely, through SCH 58261 (0.5 mg/kg,i.p). Animals
(Black-Six mice aged between 8 and 12 weeks) were exposed to two identical objects
(familiar) in the training stage, and 24 hours later, to teste long-term memory, to two
dissimilar objects (one familiar and a novelty). As expected, control mice reacted to
novelty, spending more time exploring the novel object. On the other hand, mice
that received WIN chronically (1mg/kg,i.p during 30 days) or acutely (1mg/kg,i.p),
did not discriminate between familiar and novel objects, indicating pronounced
memory impairment. This effect was prevented in the presence of AM 251 (3mg/
kg,i.p), a CB1R selective antagonist, indicating a CB1R-mediated effect. Interestingly,
mice chronically treated with KW or acutely with SCH, behaved like control mice,
spending more time exploring novelty. Regarding extracellular recording, LTP potentiation was significantly lower whenever WIN was present., Whenever A2ARs were
simultaneously blocked, WIN effect was significantly attenuated.
These results show that chronic/acute blockade of A2ARs activity prevents the
CB1R-mediated disruption of memory consolidation in NOR and attenuates CB1Rmediated inhibition in hippocampal LTP, linking A2ARs activity to these effects.
F. Figueiredo1, P. Friedemann2, J. Sanches1
2
Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal;
Charité–Universitätsmedizin Berlin
[email protected]
4
O29
Transcription dynamics prevents RNA-mediated
genomic instability through SRPK2- dependent
DDX23 phosphorylation
S.C. Sridhara1, S. Carvalho1, A.R. Grosso1, M.Carmo-Fonseca1, S. F.de Almeida1 *
* corresponding author
1
[email protected]
Genomic instability is a hallmark of cancer, aging and many other diseases.
Genomic instability is frequently caused by R-loops, nucleic acid structures
formed by an RNA:DNA hybrid and a displaced single-stranded DNA. These structures arise during transcription when the nascent RNA molecule hybridizes with
the template DNA strand. In addition to an important regulatory role in diverse
cellular processes such as gene expression and DNA repair, R-loops also pose
great threats to genome stability and should therefore be kept under physiological levels. Nevertheless, the mechanisms that discriminate between physiological and deleterious R-loops are not known. Here we show that changes in transcription dynamics signal the location of aberrant R-loops and trigger a molecular
pathway towards their suppression. We found that RNA polymerase II (RNA Pol
Key words: Cannabinoids; Adenosine; Memory; Novel Object Recognition
33
II) pausing drives serine/arginine protein kinase 2 (SRPK2)-dependent phosphorylation of the DDX23 helicase. We show that in the absence of either SRPK2 or
DDX23, accumulation of R-loops leads to massive genomic instability as revealed
by high levels of DNA double strand breaks (DSBs) throughout the cell nucleus.
Importantly, the DDX23 mutations found in cancer samples occur frequently in
the domain that is used for SRPK2 recognition and phosphorylation. Moreover,
homozygous deletions of the entire DDX23 locus were detected in 10 (17%)
adenoid cystic carcinoma samples. Our results unravel the molecular details of a
novel link between transcription dynamics and RNA-mediated genomic instability
that may play important roles in cancer development and progression.
O30
4
Differences of bone Wnt regulators expression
between fragility fractures and osteoarthritis patients
A.M.F. Rodrigues1
1
[email protected]
Introduction: Recently, some studies pointed to the importance of Wnt signaling,
a crucial pathway for osteoblast differentiation and a master bone mass regulator.
With this study, we aim to compare bone expression of Wnt regulators between
fragility fracture and osteoarthritis patients.
Results: 118 patients submitted to total hip replacement surgery, 55 due to fragility fracture were evaluated. Fragility fracture patients had more women (84% vs
49%, p<0.001) were significantly older (80±7 vs 68± 12, p<0.001), had significantly
more previous fragility fractures (33% vs 5%, p<0.001) than osteoarthritis group.
Univariate analysis showed that RANKl/OPG ratio a marker of osteoclastogenisis was significantly higher in the fragility fracture group (p<0.001). On the other
hand osteocalcin expression, a marker of osteoblast maturation was significantly
lower in fragility fracture group (p<0.001). Regarding Wnt regulators, WNT 10b
(β=-0,398, p<0.001) and DKK1 (β=-0,387, p<0.001) expression was significantly
higher in osteoarthritis group even after adjusting for age and gender. In multivariate analysis SOST (β= 0.236, p= 0.0411) was significantly lower in the fragility
fracture group. Semi quantitative analysis of Immunohistochemistry results for
DKK1 revealed a significantly high staining (p= 0.04) in osteoarthritis group.
Conclusion: in fragility fracture patients, bone SOST expression is upregulated. In
osteoarthritis patients, DKK1 gene expression and protein staining is significantly
higher than in fragility fractures. These results improve the knowledge of WNT disturbances in these two diseases and can help us to identify new treatment targets.
O31
1
Linking biomechanics, inflammation and bone
formation in psoriatic arthritis
E.C.V. Sousa1 2, S. Silva1, I. Perpétuo1, G. Cruz3, M. Castro4, P.A. Ribeiro2, P. Filipe5,
M.T. Silva4, P. Alves6, H. Canhão1, J.E. Fonseca1
Serviço de Reumatologia, Hospital de Santa Maria, Lisboa, Portugal;
3
4
Unidade de Biomecatrónica, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal;
5
Serviço de Dermatologia, Hospital de Santa Maria, Lisboa, Portugal;
6
Serviço de Imagiologia, Centro Hospitalar Lisboa Central
1
Methods: Patients submitted to total hip replacement surgery either due to a fragility fracture or to osteoarthritis at the Orthopedics department of Hospital de
Santa Maria, were recruited. Clinical risk factors (CRFS) for osteoporotic fractures
were collected. RNA was extracted from a small trabecular bone piece and analyzed by quantitative real-time RT-PCR. Immunohistochemistry analysis for DKK1
and SOST was done.
2
[email protected]
35
O32
O34
1
4
Contribution for the evaluation of dental
implants with immediate load performed in
periodontal patients
Conformational changes governing dengue virus
capsid protein disordered N-terminal region and
its inhibition by pep14-23
A. Ferro1 2, F. Salvado1 2
A.F. Faustino1, G.M. Guerra1, R.G. Huber2, A. Hollmann1, M.M. Domingues1,
G.M. Barbosa3, F.J. Enguita1, P.J. Bond2, M.A.R.B. Castanho1, A.T. Da Poian3,
F.C.L. Almeida3, I.C. Martins1, N.C. Santos1
2
Malo Clinic
1
Bioinformatics Institute, A*STAR, Singapore;
3
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, RJ, Brazil
1
[email protected]
2
[email protected]
O33
1
Contribution for the prognostic evaluation of
dental implants in patients with peri-implant
pathology.
M. Nobre1 2, F. Salvado e Silva1 2
1
2
Malo Clinic
[email protected]
Dengue virus (DENV) infection affects millions of people and is becoming a major
global disease for which there is no specific available treatment. pep14-23 is a recently designed peptide, based on a conserved segment of DENV capsid (C) protein
[1-3]. This peptide inhibits the interaction of DENV C with host intracellular lipid
droplets (LDs) [2, 3], essential for viral replication [1, 4]. Moreover, pep14-23 also
inhibits DENV C interaction with very low-density lipoproteins [5, 6], which may prevent lipoviroparticle formation. Here [7], combining bioinformatics and biophysics,
we analyzed pep14-23 structure and ability to bind different phospholipids, relating that information with the full-length DENV C protein. We show that pep14-23
acquires α-helical conformation upon binding to anionic phospholipid membranes,
displaying an asymmetric charge distribution structural arrangement. Structure
prediction for the N-terminal segment reveals four viable homodimer orientations
that alternatively shield or expose the DENV C hydrophobic pocket. Taken together,
these findings suggest a new biological role for the disordered N-terminal region,
which may function as an autoinhibitory domain mediating DENV C interaction with
its biological targets. The results fit with our current understanding of DENV C and
pep14-23 structure and function, paving the way for similar approaches to understanding disordered proteins and improved peptidomimetics drug development
strategies against DENV and similar Flavivirus infections.
REFERENCES:
[1] Carvalho FA et al., 2012, J Virol 86, 2096-108; [2] Martins IC et al., 2012, Biochem J 444, 405-15;
[3] Martins IC et al., 2012, International Patent Office Nr WO2012/159187; [4] Samsa MM et al.,
2009, PLoS Pathog 5, e1000632; [5] Faustino AF et al., 2014, Nanomedicine: NBM 10, 247-55;
[6] Faustino AF et al., 2015, Sci Rep 5, 10592; [7] Faustino AF et al., 2015, ACS Chem Biol 10, 517-26.
37
O35
O38
1
Maximizing biomolecules signal detection for
study of single protein ligand interaction events
4
Transcriptional Regulation of Innate Lymphoid Cells
R.G. Domingues1, D. Fonseca-Pereira1, C. Godinho-Silva1, H. Veiga-Fernandes1
1
P.I.D.M. Carvalho1, I. Matins1, N. Santos1
1
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
[email protected]
[email protected]
O36
1
Towards the development of antimicrobial
peptides active against bacterial biofilms
S. Dias1 2, A.S.R.N. Veiga1 2
1
2
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
[email protected]
O37
1
Biophysical studies to unravel the mechanism of
action of anticancer peptides
J. Skalska1, D.M.D. Gaspar1
1
Innate lymphoid cells (ILCs) are a family of innate effectors that play key roles
in lymphoid organ development, tissue homeostasis and immunity to infection.
ILCs originate from a common innate lymphoid cell progenitor (CHILP) and based
on lineage-specific transcription factors and discrete cytokine profiles are classified into three distinct groups, ILC 1, 2 and 3.
Additionally, recent evidence indicates that ILCs are also regulated by dietary signals. Nevertheless, how ILCs perceive, integrate and respond to environmental
signals remains mostly unknown.
In this study we shed light into the transcriptional program that regulate ILC development and reveal a novel molecular link between innate immunity and environmental sensing. We show that the transcription factor NFIL3 is a key regulator
of all ILC subsets, controlling the commitment of CHILP as they emerge from
the common lymphoid progenitor. Conditional gene targeting, cell-autonomous
differentiation assays and bone-marrow mixed chimeras allowed us to restrict
the role of Nfil3 prior to ILC commitment. We further demonstrate that Nfil3 expression was strongly up-regulated by cytokines in CHILP and that NFIL3 binds
directly to the locus of the master transcription factor of CHILP, Id2, altering its
chromatin configuration and expression. Importantly, ectopic Id2 expression in
Nfil3 null precursors rescued defective ILC lineage development in vivo. Together
our data establishes NFIL3 as a key regulator of CHILP as they emerge during
early lymphopoiesis. Interestingly, NFIL3 is also a transcriptional regulator that
can integrate circadian clock signals. Most strikingly, preliminary data indicate
cell-autonomous molecular clocks in ILC progenitors and oscillations in their genetic signature expression. We now seek to investigate novel sensing mechanisms
by which ILCs integrate environmental cues impacting on mucosal homeostasis
and defence.
[email protected]
39
O39
O41
1
4
Regulation of type 2 innate lymphoid cells at
barrier sites
Communication and Language acquisition in
autism spectrum disorders: early markers of
neurodevelopment
A.F.R. Cardoso1, H. Veiga-Fernandes1
1
C.B. de Lima1 2, G. Oliveira3, M. Baptista1, M. Vigário2
Centro Hospitalar Lisboa Norte – Hospital de Santa Maria;2 Universidade de Lisboa - Faculdade de
Letras;3 Centro Hospitalar e Universitário de Coimbra – Hospital Pediátrico
1
[email protected]
[email protected]
O40
1
Studying the impact of IL7R on T-cell leukemia
using zebrafish models: from basic biology to
therapeutics.
M.L. Oliveira , D.M. Langenau , J.T. Barata
1
1
2
2
Harvard Stem Cell Institute, Boston, MA, USA
[email protected]
1
Autism Spectrum Disorder (PEA) is a neurodevelopmental disorder with early
manifestations, but often the diagnosis is delayed. For an early diagnosis is essential to identify the warning signs. The absence or delay in language development
is the principal motive for referral; But the problem arises long before the appearance of the first words. Before speaking, babies learn to communicate through
nonverbal means such as gesture, facial expression, eye contact and joint attention. To these they associate vocalizations. In children with ASD the intention to
communicate is compromised, as well as their pre-verbal communication skills.
What are the indicators of pre-verbal communication that are related to the acquisition and development of language?
Objective: To investigate communication and language development of children
with ASD, in order to define early criteria for diagnosis and prognosis for this
disorder.
Methodology: longitudinal prospective study of a sample of 19 children diagnosed
with ASD and 13 children with typical development conducted by collecting: i)
clinical data; ii) speech production in periodic recordings of parent-child interactions for a period of two years; iii) speech perception data using the eye tracking
methodology.
Results: It is expected to obtain statistically significant correlations between the
severity ASD traits, the communication skills, the language and cognitive level.
The aim is also specifically identify the pre-linguistic factors that most strongly
correlate with the language acquisition. At this stage we present preliminary data.
Conclusion: The aim is to get pre-linguistic markers of neurodevelopmental for the
language acquisition in children with ASD, that can have a diagnostic and prognostic value not only for clinical terms, but also for intervention and family knowledge.
41
O42
O44
1
Physical activity consultation in the treatment of
pediatric obesity
4
Casein Kinase 2 controls the survival of normal
thymic and leukemic γδ T-cells via modulation of
AKT signalling
A. Ascenso1, H. Fonseca2
1
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 2 Pediatric Obesity Clinic,
Department of Pediatrics, Hospital de Santa Maria, Lisboa, Portugal
[email protected]
O43
1
More than the sum of its parts: exploring the neural
and cognitive processes of semantic integration
M. Alves1, A. Raposo2, P. Figueiredo3
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 2 Faculdade de Psicologia,
Universidade de Lisboa, Lisboa, Portugal; 3 Instituto Superior Técnico, Universidade de Lisboa, Lisboa,
Portugal
1
[email protected] S.T. Ribeiro1, J.C. Ribot1, J.T. Barata1, B. Silva-Santos1
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa,Portugal
[email protected]
The thymus is the major site for normal and leukemic T-cell development. In
T-cell acute lymphoblastic leukemia (T-ALL), the serine-threonine protein kinase
Casein Kinase 2 (CK2) has been shown to support tumor cell survival and
expansion. Here we disclose an unanticipated role of CK2 in healthy human
thymocytes that is selective to the (less abundant and poorly-studied) γδ T-cell
lineage. By studying pediatric thymic samples and a γδ T-cell acute leukemia
(T-ALL) line, and combining specific chemical inhibition and immunoblotting, we
show that CK2 regulates the PTEN/AKT signaling pathway towards promoting
survival of normal and leukemic γδ T-cells. These cells displayed higher CK2
activity than their γδ counterparts, and were strikingly susceptible to death
upon specific CK2 inhibition. Mechanistically, we demonstrate that TCR/CD3
stimulation positively regulates CK2 activity in normal γδ thymocytes, while γδ
T-ALL cells further enhance CK2 activity upon CD27 costimulation. The highly
specific and clinical-grade CK2 inhibitor, CX-4945, induced G2/M cell cycle arrest
and apoptosis in γδ T-ALL cells, which were markedly more sensitive than their
γδ counterparts. These data identify CK2 as a novel determinant of normal and
leukemic γδ T-cell survival, and may thus impact their manipulation in both
hematology and immunotherapy.
43
O45
1
Regulation of T cell fitness in the tumour
microenvironment: from myeloid cells to dietary
factors
S. Mensurado1, M. Rei1, T. Lança1, N.G. Sousa1, H. Kubo1, K. Serre1*; B. Silva-Santos1*
*these authors contributed equally to this work
1
[email protected]
O46
4
Post-transcriptional regulation events that impact
embryonic development: a focus on Qkia and Fgf8a
regulation of mRNA stability. However, the post-transcriptional mechanisms that
operate during somite formation have not yet been uncovered.
In order to address this issue, we adopted two approaches. First we focused our
attention on a candidate post-transcriptional regulator of somite formation - the
RNA binding protein Quaking A (QkA). Secondly we focused on the 3’UTR sequence of the wavefront gene Fgf8a.
We characterised the QkA loss-of-function phenotype, using a morpholino approach, which revealed not only alterations in somite size but also in the left-right
positioning of the internal organs and in the development of the inner ear. A candidate target of QkA – Cadherin11 – was identified as a potential mediator of one
or more of the QkA loss-of-function phenotypes.
Regarding Fgf8a, previous studies in mouse and chick have shown that FGF8
mRNA has different stabilities in different embryonic tissues. In addition, two
independent RNAseq studies revealed the existence of alternative Fgf8a 3’UTRs
formed through alternative polyadenylation events. Using fluorescent reporters
we have shown that these alternative 3’UTRs can have different impacts on gene
expression levels.
S.F. Fernandes1, M. Gama-Carvalho2 3 , L. Saúde1
Instituto de Medicina Molecular e Instituto de Histologia e Biologia do Desenvolvimento, Faculdade
de Medicina da Universidade de Lisboa;
2
Gene Expression and Regulation Unit, BioISI - Biosystems & Integrative Sciences Institute, Faculdade
de Ciências da Universidade de Lisboa;
3
Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa
1
We are currently using qPCR and microdissection to determine the relative abundance of these UTRs in different embryonic tissues. In addition, mutant lines and
a morpholino approach are being used to determine the impact of these alternative polyadenylation events on embryonic development.
[email protected]
Embryonic development is highly controlled, both spatially and temporally, and
in many developmental processes this control relies on mechanisms of post-transcriptional regulation.
This study is focused on the formation of transient mesodermal structures,
termed somites, which contain the precursors of the vertebrae, skeletal muscles
and dermis of the back. Somite formation is governed by two mechanisms, a molecular clock and a wavefront of differentiation, both of which heavily rely on a tight
45
P1
3
Abstracts
Poster Sessions
Rheuma-mir.org - microRNAs in rheumatic diseases
F. Martins1, F. Enguita1, H. Canhão1
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa,Portugal
[email protected]
Background: The effectiveness of many drugs depends on the correct expression of
specific genes. The overexpression of specific miRNAs could decrease the expression levels of genes that promote drug’s effectiveness. Conversely, if some miRNAs
are under expressed may increase the expression levels of genes that inhibit the
effectiveness of the drug treatment.
Objectives: To collect from several databases the data related with the role of the
microRNAs in rheumatic diseases, allowing future investigations. We are mainly
interested in prediction tools than can support laboratorial findings.
Methods: We are using databases such as the PharmGKB - Pharmacogenomics
Knowledge Base, Pharmaco miR - The miRNA Pharmacogenomics Database,
DrugBank – Open Data Drug & drug Target Database, public databases predicting
the genes that bind to the promoter sequences of miRNAs (Mapper 2, Match 1.0,
Patch 1.0, P-Match 1.0, PROMO 3.0.2, TFBind, TFSEARCH 1.3. and PROmiRNA)
and several databases predicting the targets of miRNAs (miRWalk, mirTarBase,
Diana micro-T v4.0, miRanda, MicroCosm and TargetScan).
For the in silico analysis of the of the transcription factor binding sites we used two
databases of microRNA promoters: Marson et al., 2008, Cell, 134, 521-533 (PMID
18692474) and Marsico et al., 2013, Genome Biol., 14(8):R84 (PMID 23958307) and
we obtained the sequences of microRNA promoters that are needed for the prediction tools of transcription factor binding sites.
Results: So far, this tool is focused on Rheumatoid Arthritis (RA), and 155 genes
were identified as directly or indirectly associated to RA, after analysis of research
articles and databases.
Twelve genes associated to RA are predicted as regulating the transcription of 1110
microRNAs, and the RA genes are target protein-coding genes of 1776 different
microRNAs Data and the developed tools are available at http://rheuma-mir.org.
Conclusions: Very few RA genes are predicted as regulating the expression of miRNAs. However, all identified RA genes are target protein-coding genes of many different miRNAs.
47
P2
P3
2
Do non-motor symptoms respond to levodopa in
late-stage Parkinson’s disease patients: reports from
a levodopa challenge test
2
Follicular helper T cell subsets in the peripheral
blood and salivary gland biopsies in Sjögren´s syndrome patients
M. Fabbri1, M. Coelho1 2, D. Abreu1, L.C. Guedes1 2, M.M. Rosa1 2 3, N. Costa1, J.J. Ferreira1 2
V.R.Fonseca1, A. Água-Doce1, L. Graça1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,Lisboa, Portugal;
2
Department of Neurosciences, Serviço de Neurologia, Hospital de Santa Maria, Centro Hospitalar
Lisboa Norte, Lisboa, Portugal; 3 Laboratory of Clinical Pharmacology and Therapeutics, Faculdade
de Medicina, Universidade de Lisboa, Portugal
1
1
[email protected]
[email protected]
Background: Non-motor symptoms (NMS) are extremely common among latestage Parkinon’s disease (LSPD) patients owing a huge impact on patients’ quality
of life. Levodopa (L-dopa) responsiveness seems to decrease with disease progression but very few study has investigated this response among LSPD patients and its
effect on NMS during this latest disease phase needs still to be investigated.
Objective: Our aim was to study the NMS response to L-dopa in a LSPD population.
Methods: 20 LSPD patients, defined as Schwab and England ADL Scale < 50 or
Hoehn Yahr Stage >3 (MED ON), underwent a L-dopa challenge test. Blood pressure assessment, VAS for pain and fatigue and Strait Trait Anxiety Scale were evaluated before and after L-dopa intake. The same protocol was applied to with 22 PD
patients treated with subthalamic deep brain stimulation (DBS) to better inform the
interpretation of the results in the late stage population. NMS symptoms have been
investigated by means of the MDS-UPDRS-I, Non-Motor Symptoms assessment
scale for PD, Neuropsychiatric inventory scale, Mini Mental State Examination and
Pill questionnaire.
Results: 15%, 40% and 65% of LSPD patients were not able to fill out respectively
the Geriatric Depression Scale, the VAS and the STAIS, while the measure of blood
pressure during orthostatic position was not possible in four LSPD patients. L-dopa
intake did not show acute effect on NMS among LSPD patients, while it had a positive effect on anxiety and pain among DBS patients. L-dopa related adverse effects
(AEs), namely symptomatic orthostatic hypotension and sleepiness, were more
common among LSPD patients.
Conclusions: Self-reported scales applicability for NMS evaluation is very limited
among LSPD patients. Even considering the limits of questionnaires applicability
our exploratory study suggests the decrease of L-dopa effect on NMS in the latest
disease phase and its association whit frequent AEs.
Self-reactive antibodies have been implicated in human autoimmunity. These
antibodies are thought to be generated by B cells that undergo class-switching in
ectopic germinal centers (GC) frequently present in target tissues from autoimmune patients. It has been suggested that regulation of GC responses is maintained
by the balance between follicular helper and regulatory T cells (Tfh and Tfr). While
Tfh cells support the production of high-affinity antibodies, Tfr cells restrict GC
responses. Although the GC is a key location for the pathogenesis of autoimmunity, so far, most studies on the role of Tfh cells in human autoimmune disease have
focused on circulating cells.
To clarify the relationship between blood and tissue Tfh and Tfr cells, we studied
those populations in the peripheral blood and salivary glands from Sjögren’s syndrome (SS) patients. We found an increased percentage of circulating PD-1+CXCR5+
Tfh cells compared to age-matched healthy donors, although the percentage of
total circulating CXCR5+ Tfh were the same in the two groups. The circulating
PD-1+CXCR5+ Tfh correlated positively with the percentage of CD4 T cells infiltrating the salivary glands, as measured by flow cytometry. We also found an increased
percentage of circulating CXCR5+FoxP3+CD25+ T cells in SS patients which also
correlated positively with infiltrating CD4+ T cells in salivary glands.
Our study shows a relationship between circulating PD-1+CXCR5+ Tfh cells (but not
CXCR5+ T cells) and the emergence of ectopic lymphoid tissue with infiltrating CD4
T cells in the target organ of SS patients. In addition, patients with greater CD4 T cell
infiltrates have increased numbers of circulating cells with a Tfr phenotype.
49
P4
P5
3
3
Unconjugated bilirubin in schizophrenia,
schizoaffective and bipolar disorders: a
retrospective observational and controlled study
Using surface plasmon resonance to quantify
molecular partition towards lipid membranes:
equilibrium and kinetic data revisited
J.G. Marques1 2, I. Tinoco2, I. Pedro2, F. Leote3,R. Silva4, S. Ouakinin1
T.N. Figueira1, J.M. Freire1, A.S. Veiga1, M.A.R.B. Castanho1
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 2 Centro Hospitalar Psiquiátrico
de Lisboa; 3 Instituto Português de Línguas; 4 Faculdade de Farmácia, Universidade de Lisboa,
Lisboa, Portugal
1
1
[email protected]
[email protected]
High Unconjugated Bilirubin (UCB) levels have been found in psychiatric populations when compared with general population. These higher UCB levels have been
already correlated with acute psychotic states, with both positive and negative symptoms and also with a poorer outcome in patients with schizophrenia. It seems that
UCB may have some neurotoxic effect on brain development and may play a role in
brain tissue connectivity changes found in patients with schizophrenia.
In this observational transversal study we evaluated a sample composed of four subgroups including 50 schizophrenic, 69 schizoaffective and 85 bipolar patients versus
55 healthy controls, comparing the UCB mean levels and searching for a possible
correlation with duration of acute admission in the psychiatric ward.
We found a statistically significant difference between UCB mean levels of bipolar
(0,29mg/dL) versus both schizoaffective (0,36 mg/dL, p≤0,01), and schizophrenic
patients (0,39 mg/dL, p≤0,0001). We also found statistically significant difference
between mean duration of admission of schizophrenic patients (31 days) versus
both schizoaffective (22 days, p≤0,05), and bipolar patients (19 days, p≤0,001).
Our results suggest that UCB may contribute an important role in the complex
physiopathology of psychotic conditions, especially in the schizophrenia spectrum.
In future studies we will try to find correlation between these findings and other
psychopathological, neuropsychological and psychosocial variables.
Molecular interactions with lipid membranes are ubiquitous in nature and have
been increasingly studied for their potential biomedical and biotechnological applications. These range from drug delivery vectorization to antimicrobial development,
through the use of membrane active molecules such as peptides. Quantifying
molecular partition towards the lipid phase with adequate parameterization, is thus
fundamental when studying these processes.
Surface Plasmon Resonance (SPR) is a powerful technique to study molecular association, but is limited in its quantitative interpretation of molecular membrane partition data. We have developed and applied a novel analytical method for SPR data
treatment which enables the experimental determination of equilibrium and kinetic
membrane partition parameters. This methodology makes use of two complementary mathematical fitting models for SPR sensorgrams association and dissociation
data. The partition constants (Kp) determined for the single-domain antibody F63,
the anti-HIV peptide enfuvirtide, and the endogenous neuropeptide kyotorphin
towards POPC membranes were comparable to literature data obtained with other
techniques. Both models were further applied to the interaction of HRC4, a novel
Measles Virus fusion-inhibitor peptide dimer, revealing its increased partition and
retention in cholesterol-rich membranes.
Our work offers an alternative data treatment approach for SPR membrane interaction data and expands the applicability of the technique to the quantitative analysis
of lipid phase partition phenomena.
51
P6
P7
3
MicroRNA regulation of T cell function and
cytokine production
3
Small Molecule Drug Conjugates for Selective
Cancer Therapy
T. Amado1, N. Schmolka1, A.Q. Gomes1 2, B. Silva-Santos1
R. Traquete1, J. Seixas1, W. Heggie2, A. Koehler3, G. Bernardes1
2
Escola Superior de Tecnologia da Saúde de Lisboa, Instituto Politécnico de Lisboa, Lisboa, Portugal
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa,
Portugal; 2 Hovione FarmaCiencia SA, Lisboa, Portugal; 3 David H. Koch Institute for Integrative
Cancer Research, Department of Biological Engineering, Massachusetts Institute of Technology,
Cambridge, MA, USA
1
[email protected]
1
[email protected]
MicroRNAs are small non-coding RNA molecules that shape gene expression at
the post-transcription level. They have emerged as important players in T cell physiology, from development and survival to activation, proliferation, and differentiation. One of the major classes of proteins involved in these processes are cytokines,
which are both key input signals and major products of T cells. Cytokine production
by T cells defines their identity and determines their effector function.
In this study we aim at understanding how microRNAs regulate the ability of T
cells to produce some of their hallmark cytokines, namely IFNγ and IL-17. In order
to assess the microRNA landscape in functionally distinct cells we make use
of cytokine reporter mouse strains that allow us to isolate T cells based on the
cytokines being produced. Using next-generation sequencing we can determine the
differences in microRNA expression of the isolated cells. By manipulating the differentially expressed microRNAs we plan on getting new insights on how microRNAs
regulate the functional status and cytokine production on gamma delta and CD8+
T cells thus highlighting the relevance of these post-transcription regulators in T cell
differentiation.
Standard of care cancer therapies still rely heavily on non-specific radiation- and
chemotherapy, which are commonly associated with severe toxicity and in many
cases only offer limited benefit for the patient. Targeted delivery of therapeutic
agents into malignant tissue can markedly improve the therapeutic index and overall
efficacy of such substances. Antibodies against markers of disease have been considered the ligands of choice as drug-delivery vehicles. To date two antibody-drug
conjugates with unprecedented efficacy and safety profiles have been approved,
while more than 30 ADCs are currently in clinical development. More recently, the
use of low-molecular-weight ligands has emerged as a novel class of targeted cancer
therapeutics with improved properties. The miniaturization of the targeting vehicle,
coupled to a cytotoxic agent with a suitable linker, promises to dramatically improve
kinetics, selectivity and tumor-homing properties of current tumor-targeting agents.
We therefore propose to (1) develop a general chemical approach for building safe
small ligand-drug conjugates (LDCs), (2) demonstrate that a small organic molecule
targeting a specific intracellular target can selectively accumulate in cancer cells, (3)
develop a novel linker technology that is effective for drug delivery and (4) establish
that when chemically conjugated to a cytotoxic drug this novel LDC is effective for
the treatment of a certain cancer indication
53
P8
P9
3
Role of Retinoic Acid in Lymphocyte Development
S. Arroz-Madeira1, B. Raposo1, M. Ferreira1, D. Fonseca-Pereira2, H.Veiga-Fernandes1
Portugal; 2 Department of Immunology and Infectious Diseases and Department of Genetics and
Complex Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA
1
2
What can Trypanosomes teach us about new
anti-malarial strategies?
M. Sanches-Vaz, A.M. Mendes, L.M. Figueiredo*, M. Prudêncio*
*
1
[email protected]
[email protected]
In the last years a lot of attention has been given to dietary compounds and nutrition
in the immune system. Vitamin A, or its metabolite Retinoic acid (RA), signalling
has been shown to modulate different arms of the haematopoietic tree in different
ways: it enhances T helper cell type II responses in vivo; it increases B cell development in vitro; and it drives innate lymphoid cell (ILC) development in utero in a cellautonomous manner, setting the capacity of the individual to respond to infection
later in life. Vitamin A high dose diet also increased ILC2 numbers in mice at the
expenses of ILC3 making these animals more protected against nematode infection.
Consequently, lymphocytes cells can sense dietary cues and respond to it. In this
context, we wondered if RA modulates the whole immunity response impacting in
the commitment to a given lymphocyte lineage developmental path and not the
other.
In mice bearing a dominant negative form of the Retinoic Acid Receptor α (RARα) in
cells expressing the hCD2, there is an increase of early B cell progenitors in the bone
marrow (BM) and a reduction of the immature B cells in the BM or mature B cells at
periphery. Also in an in vitro approach for the development of B cells in the presence
or absence of RA signaling, the CLPLy6D+ (Common Lymphoid Progenitors that
mostly give rise to B cells) RA signaling impaired gave rise to less B cell colonies than
the controls. Moreover, CLPLy6D- (Common Lymphoid Progenitors that mostly rise
to T and ILC cells) RA signaling impaired, when forced to become a B cell, give rise
mainly to early B cell colonies, but not to mature ones.
These authors contributed equally to the present work
The parasites responsible for malaria (Plasmodium spp.) and sleeping sickness
(Trypanosoma brucei) have shared overlapping geographical distributions in subSaharan Africa for around 30 million years. Although the two parasite species share
humans as a common host, the mutual impact of these infections remains largely
understudied. In this project we aim to study whether and how an ongoing T. brucei
infection affects a subsequent infection by P. berghei. Employing a newly developed
co-infection mouse model, we observed that an ongoing infection by T. brucei leads
to a greater than 90% impairment of a subsequent liver infection by P. berghei. We
further employed qRT-PCR and quantitative analysis of immunofluorescence assay
of infected liver slices to show that P. berghei impairment can be observed as early
as 30 min post-infection, suggesting that most Plasmodium sporozoites do not succeed in invading hepatocytes when the mouse was previously infected by T. brucei.
Importantly, the Trypanosome inhibitory phenotype can be phenocopied by replacing live T. brucei infection by the injection of an equivalent amount of a T. brucei
lysate. When these lysates are treated with Proteinase K, the inhibitory effect is lost,
indicating that (a) T. brucei protein(s) has(ve) anti-Plasmodium properties.
The present study suggests that T. brucei elicits a mechanism that protects its host
from a Plasmodium infection. The underlying mechanism might contribute to our
understanding of host-pathogen interactions and may reveal novel strategies of
malaria control.
Our preliminary findings support that RA signalling does impact B cell development.
55
P10
P11
2
Flavor-nutrient conditioning from maltodextrin is
disrupted by weight-loss surgery
G. Ribeiro1 2, A. Fernandes3 , M.M. Oliveira4 , J.S. Duarte4 , R.M. Costa3 ,
A.J. Oliveira-Maia1 3 5
Neuropsychiatry Unit, Champalimaud Foundation;
2
Lisbon Academic Medical Centre;
3
Champalimaud Neuroscience Program, Champalimaud Foundation;
4
Department of Endocrinology, Diabetes and Metabolism, Centro Hospitalar de Lisboa Ocidental,
Lisboa, Portugal;
5
Department of Psychiatry and Mental Health, Centro Hospitalar de Lisboa Ocidental, Lisboa, Portugal
1
[email protected]
Flavor-nutrient conditioning (FNC) is the process whereby the preference for a particular flavor is altered by multiple pairings of that flavor with the postingestive consequences of a nutrient. FNC has been extensively demonstrated in rodents but
there is significant variability regarding methods and findings of FNC in humans.
Importantly, its relevance in the context of obesity and weight-loss has not been
explored. We hypothesized that weightloss surgery reduces postingestive food
reward and, to test this hypothesis, developed a novel method for controlled FNC
in humans. Our FNC protocol was developed and optimized in a sample of healthy
and non-obese volunteers (n=56). In a pre-conditioning day, 2 yoghurt flavors with
similar novelty and pleasantness were selected for each individual, and the induced
intake and pleasantness for each flavor was assessed. In the following days, subjects
drank one of flavors, enriched with maltodextrin, in the first and third day, and the
alternate, control flavor, in the second and fourth day. Subjects then returned to
the laboratory to repeat intake and pleasantness measurements. Following optimization, this protocol was also applied in a cohort of 32 patients from a hospitalbased weight-loss clinic, either prior to or after surgery, specifically, gastric bypass
or sleeve gastrectomy. We did not find, in either sample, significant conditioninginduced differences of flavor pleasantness, for both the conditioned and the control
flavor. However, with regards to flavor intake, in the non-clinical sample conditioning induced increased consumption of the flavor paired with maltodextrin. In the
clinical sample, however, only the pre-surgery obese group, but not the post-surgery
patients, had a significant increase in the intake of the flavor paired with maltodextrin.
Our findings suggest that flavor-nutrient conditioning contributes towards feeding
actions in humans, and that such mechanisms are altered after weight-loss surgery. 3
The effect of anti-CD6 (T1) in T cell function and
Immunopathology (EAE)
R.F. Freitas1 2, S.C.P. Almeida1 2, L. Graça1 2
1
2
Instituto Gulbenkian de Ciência, Oeiras, Portugal
[email protected]
CD6 glycoprotein is a cell surface receptor from the scavenger receptor cysteine
rich protein superfamily, expressed in mature T cells, thymocytes, B1a cells and
subsets of natural killer cells. One of its three ligands is the activated leukocyte
adhesion molecule (ALCAM) that binds to domain 3, thereby affecting T cell activation, proliferation, immune synapse formation and transmigration across the
blood brain barrier (BBB).
Genome wide association studies identified CD6 as a susceptibility locus for
Multiple Sclerosis (MS) and in clinical trials for rheumatoid arthritis and psoriasis
a humanized anti-CD6 Mab (T1h) has already been tested with promising results.
Investigation of CD6-targeting in MS has been hampered by lack of adequate
murine reagents. We will now test a “murinized” variant of anti-CD6 in a mouse
model of MS, in order to establish the therapeutic value of CD6-targeting for prevention of autoimmune neuroinflammation.
In addition we will investigate the mechanisms underlying the therapeutic action
of CD6-targeting on T cells. Our preliminary data show TCR-transgenic cells activated in vitro in different polarizing conditions can alter their functional specialization in presence of anti-CD6 antibodies.
We will establish the relationship between the impact of CD6-targeting on T cell
activation and acquisition of effector functions with the in vivo outcome of antiCD6 in neuroinflammation.
Finally we will validate our results using human cells. These experiments will allow
us to conclude whether targeting of human CD6 has similar impact on T cell activation as what we are observing with murine cells.
Our studies have the potential to provide proof of concept for subsequent clinical
development of CD6-targeting strategies for MS.
57
P12
P13
2
3
Preclinical development program for bioengineered
disc implants in Temporomandibular Joint (TMJ)
disc diseases: protocol of two exploratory Black
Merino Sheep randomized controlled trials
Reciprocal regulatory links between transcription
and DNA double strand break repair
A.C. Vítor1, R.M. Martin1, S. Carvalho1, S.F. Almeida1
1
D. Ângelo , J. Ferreira , R. Fernandes , N. Gonçalves , R. Gonzalez , F. Monje , F. Salvado
1
2
3
4
5
6
7
Stomatology Department, Hospital Santa Maria; 2 Clinical Pharmacology Unit, Instituto de
Medicina Molecular and Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 3 Clinical
Pharmacology Unit, Instituto de Medicina Molecular and Faculdade de Medicine, Universidade
de Lisboa, Lisboa, Portugal; 4 Clinical Pharmacology Unit, Instituto de Medicina Molecular and
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 5 Hospital Infanta Cristina,
Faculdade de Medicina, Universidade de Extremadura; 6 Hospital Infanta Cristina, Faculdade de
Medicina, Universidade de Extremadura; 7 Stomatology Department, Hospital Santa Maria
1
[email protected]
Background: There is no consensus concerning the therapeutic management of TMJ
disc diseases. The appraisal of TMJ trials is hampered by differences in patients’
characteristics and studied surgical procedures. No standard or predictive animal
models for therapeutic studies in TMJ are available. Disc implants can be an efficacious complement in bioengineered joint reconstruction and animal models may
offer the possibility to conduct informative preclinical studies. The main goal of
these trials is the evaluation of the feasibility of Black Merino Sheep animal model
for TMJ disc implant studies and to evaluate the histological and biomechanical
articular changes induced by TMJ implant surgery. Methods/Design: The authors
propose two sequential RCT in the Black Merino Sheep with blind outcome basement. In the first trial, 9 sheep will be randomized to three surgical bilateral procedures: discectomy group, discopexy group and sham surgery. In the second trial,
9 sheep will be randomized to three different TMJ discs implants. The primary
outcome is the histological gradation of TMJ degeneration. Secondary outcomes
are absolute masticatory time, rate masticatory time/masticatory cycle, masticatory kinetics and sheep weight. Conclusions: These will be the first trials using the
Black Merino Sheep as the animal model to study the efficacy of disc implants in
TMJ. Study design is innovative and will include a sham surgery group. Exploratory
outcomes include histological analysis and new functional masticatory parameters.
The proposed study methodology and the validation of such a large animal model
for TMJ disorders may add important information for the development program of
new therapeutic interventions for TMJ disc diseases.
[email protected]
How does ongoing transcription influence DNA repair in a coding region? How does
DNA damage within a gene impact on transcription of that same gene? Answering
to these questions is an outstanding task that will help us understand how genomic
instability shapes gene expression programs. Histone modifications are important
mediators of the crosstalk between the DNA damage response and transcription.
Typically, actively transcribed genes are “marked” by a characteristic set of histone
modifications. One of the chromatin marks of transcription is the trimethylation of
histone H3 (H3K36me3) by the SETD2 methyltransferase. We have recently shown
the presence of H3K36me3 in coding regions promotes the repair of DNA double
strand breaks (DSBs) by the error-free homologous recombination pathway. We suggest that transcription favors the error-free DSBs repair pathway in a coding region
to safeguard the integrity of genetic information. Now the question that follows is:
How does DNA repair impact on transcription? Our preliminary data suggest that
after induction of a DSB within a gene, transcription proceeds for a short period of
time before complete abrogation. However, we still miss a full picture of the molecular events that take place immediately after the RNA Polymerase II transcription
machinery encounters a DSB. This is mainly due to the lack of experimental model
systems to simultaneously follow transcription and DNA repair at single-molecule
resolution in live cells. To gain detailed insights with unprecedented resolution on
such events, we are currently establishing a new in vitro model system that makes
use of an endonuclease-triggered DSB within an inducible gene to follow, in realtime, the transcription and the DNA repair by high-resolution microscopy. With this
tool, we expect to identify the molecular details of the interplay between transcription and DNA repair. This knowledge will be instrumental to fully comprehend the
mechanisms that maintain genome integrity.
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The role of nuclear positioning in muscle function
M. Pimentel1, E.Gomes1
1
3
Generation and characterization of a fully human
antibody against the interleukin-7 receptor, a
potential target for T-cell Acute Lymphoblastic
Leukemia
[email protected]
P. Akkapeddi1 2, A. Gloger1, M. Matasci3, J.T. Barata2 #, G.J.L. Bernardes2 #, D. Neri1 * #
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss
Federal Institute of Technology Zürich, Zürich, Switzerland;
2
3
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK;
4
Philochem AG, Otelfingen, Switzerland
#
Co-senior authors
*
Corresponding author
1
Skeletal muscle is formed by multinucleated myofibers, the biggest cells in the
human body. Two decades ago it was proposed that each myofiber nucleus influences the region surrounding it, creating a virtual compartmentalization within
the cell. This was achieved by the localization of mRNA and respective proteins in
the vicinity of the nucleus of origin (Pavlath et al., 1989). These nuclear domains
are clearly observed at the neuromuscular and myotendinous junctions where
nuclei express specific mRNAs encoding proteins important for the function of
these subcellular regions. It is unknown if peripheral nuclei along mature myofibers form nuclear domains. We found nuclear positioning to be critical for skeletal muscle function. However, why nuclear positioning is important for muscle
function is unknown. We hypothesize that abnormal nuclear positioning leads to
nuclear domain coverage disruption resulting in muscle dysfunction. We developed an in vitro system capable of differentiating highly matured mouse myofibers. The system was further adapted for the formation of hybrid myofibers (heterokaryon) containing one human nucleus among several mouse nuclei. We
are using smFISH to determine the subcellular localization of essential mRNAs
throughout development and of specific human mRNAs at later developmental
stages. We are disrupting nuclear positioning and determining its effect in mRNA
localization. Protein localization is also being accessed using human specific antibodies in the heterokaryon system. Finally, we are developing and validating functional assays that will be essential to understand the role of nuclear positioning in
muscle function.
[email protected]
Interleukin 7 (IL-7) is a cytokine produced in the bone marrow, thymus and other
organs, required for normal T-cell development. However, IL-7 and the α subunit
of its receptor (IL-7Rα) have been implicated in T-cell acute lymphoblastic leukemia (T-ALL) development. In more than 70% of T- ALL cases, IL-7 induces proliferation of leukemia blasts. Moreover, 10% of T- ALL patients display IL-7Rα gainof-function oncogenic mutations. These data suggest that the IL-7/IL-7Rα axis can
be explored for the development of targeted therapies against T-ALL. Fully human
monoclonal antibodies are particularly useful for pharmaceutical applications as
they display reduced immunogenicity in humans. Using phage display technology
we now describe the isolation and initial characterization of a fully human antibody against IL- 7R.
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The life cycle of mutant mRNAs
R. Vaz-Drago1, N. Custódio1, A. Jesus1, M. Carmo-Fonseca1
1
[email protected]
Protein coding RNAs are synthesized and processed in the nucleus and subsequently translated in the cytoplasm to give rise to functional proteins. The presence of DNA mutations can interfere with the gene expression pathway, triggering
mutant mRNAs to nuclear and/or cytoplasmic RNA quality control. RNA surveillance mechanisms play an important role modelling the clinical phenotype of
human genetic diseases, particularly in diseases caused by disruption of splicing
or introduction of a premature termination codon (PTC), which correspond to
approximately one third of all genetic diseases. For deep understanding of the
molecular basis of genetic diseases is essential to study in detail the life cycle of
mutant mRNAs.
In my PhD project I have been particularly interested in studying the impact of cotranscriptional quality control mechanisms in human disease.
We used patient-derived lymphoblastoid cell lines as disease models to address
how the biogenesis of mRNAs is affected by splice site or PTC mutations.
Additionally, we have engineered a HEK293 cell line that stably expresses a single
copy of the human β-globin gene carrying a PTC mutation and MS2-GFP binding
sites enabling the life cycle of a mutant RNA to be followed, from its synthesis to
its degradation in the cytoplasm.
Taking advantage of both biochemical approaches and life cell microscopy we
found that splice-site mutations can lead to retention of transcripts in the nucleus,
co-transcriptional or post-transcriptional RNA degradation and down-regulation
of transcription.
These results suggest that transcriptional-coupled RNA quality control systems
contribute to eliminate abnormally spliced transcripts produced in patients with
genetic diseases. We also show that life-cell microscopy is a unique methodology
that allows us to obtain a unified picture of RNA life cycle.
Oth
NeSSLaSP The Neuropsychiatric Symptoms and
Syndromes in Late-Stage Parkinson’s Disease Study
I.Chendo1, J. Ferreira2, V. Voon3
Department of Neurosciences and Mental Health, Hospital Santa Maria, Faculdade de Medicina,
Universidae de Lisboa;
2
Department of Neurosciences and Mental Health, Hospital Santa Maria; Clinical Pharmacology
Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa; Laboratory
of Clinical Pharmacology and Therapeutics, Faculdade de Medicina, Universidade de Lisboa;
3
Department of Psychiatry; Behavioural and Clinical Neuroscience Institute; University of Cambridge
1
[email protected]
Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder after Alzheimer’s disease. PD has a worldwide age-adjusted prevalence
of 1.8%. The mean age of onset is 65 years, with prevalence rising from 0.6% at age
65–69 years to 2.6–3.5% at age 85–89 years.
PD is traditionally defined as a progressive disorder characterized by the triad of
rigidity, bradykinesia and tremor due to the degeneration of the dopaminergic
nigrostriatal system.
It has become increasingly recognized that nonmotor symptoms (NMS) are a prominent feature of PD. They can precede the motor onset of PD, increase in frequency
and severity in later disease stages and include sleep disorders, autonomic dysfunction and gastrointestinal, sensory and neuropsychiatric symptoms (NPS).
NPS (depressed or elevated mood, apathy, anxiety, anhedonia, psychosis, cognitive
impairment and dementia, obsessional behaviour and addictions) are common and
can be part of the process of PD itself or result from the pathological changes of
the disease, emotional reactions to parkinsonism or treatment-related side-effects.
More than 60% of patients (pts) with PD report one or more NPS at some point,
and they often cause disability.
Only a few studies address the prevalence of clinical features, motor and non-motor
complications in pts with late stage PD (LSPD) Differential diagnosis and evaluation of NPS like apathy, depression and even psychosis is difficult in this population.
A characterization of NPS and syndromes (NPSS) in LSPD has not been performed
and would be of importance for the promotion of better quality of life (QoL) for pts.
Therefore, the aim of the NeSSLaSP study is to establish the prevalence and characterize the NPSS in a sample of pts with LSPD and to identify the best instruments
for that evaluation.
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The fibrinogen-erythrocyte interaction as prognosis
biomarker in heart failure patients
A.F. Guedes1, F.A. Carvalho1, I. Malho1, N. Lousada2, L. Sargento2, N.C. Santos1
Portugal; 2Heart Failure Unit, Cardiology Department, Hospital Pulido Valente, Centro Hospitalar
Lisboa Norte, Lisboa, Portugal
1
[email protected]
High fibrinogen levels are a relevant cardiovascular risk factor, but the biological
mechanisms associated with pathologic alterations are not totally clear.
15 Ischemic and 15 non-ischemic CHF patients, as well as 15 healthy donors were
enrolled in the study. Fibrinogen-erythrocyte interactions were evaluated, at the
single-molecule level, by atomic force microscopy (AFM)-based force spectroscopy. These measurements were performed in buffer, with the protein covalently
attached to the AFM tip, and the erythrocytes on a poly-L-lysine coated-glass slide.
Clinical outcome was assessed during a 12-months follow-up.
Force spectroscopy data showed that CHF patients presented stronger specific
fibrinogen-erythrocyte binding forces than the control group (p= 0.038), despite
a lower binding frequency (p=0.003). According to etiology, ischemic patients
had higher binding forces than donors (p=0.004) and lower binding frequency
(p= 0.002). Ischemic patients presented increased fibrinogen-erythrocyte binding
forces relative to non-ischemic (p=0.021). Non-ischemic patients also had a lower
binding frequency than donors (p= 0.040). Their cell stiffness is also altered. These
variations were only statistically significant for the median, in which we observed
an increased cell stiffness (or decreased elasticity) on both groups of CHF patients,
with the larger variation being observed for the non-ischemic patients (390 Pa
for control group vs. 743 Pa for non-ischemic vs. 568 Pa for ischemic patients).
Follow-up data demonstrated that patients presenting higher fibrinogen-erythrocyte binding forces at the beginning of the study had a higher probability of being
hospitalized due to cardiovascular complications on the subsequent year.
As fibrinogen-erythrocyte interactions, evaluated by AFM, are modified in CHF
patients and associated with short-term clinical outcome, here we demonstrate
the power of this nanotechnology-based evaluation as potential biomarker for cardiovascular risk and patients’ clinical prognosis evaluation.
3
A Hierarchical Model of Behaviour Therapy
A. Portêlo1, Y. Shiban2, T.V. Maia1
1
Portugal; 2Department of Psychology (Clinical Psychology and Psychotherapy), University of
Regensburg, Germany
[email protected]
Objective: This work aims at developing a computational model for describing
the effects of Exposure and Response Prevention (ExRP) on patients’ response,
assessed by a subjective report scale (SUDS). Method: Twenty-eight spider-phobic patients were randomly assigned to 2 ExRP groups, where the stimulus was
presented either in a single context (SCE) or in multiple contexts (MCE). None of
the patients were involved in other psycho- or pharmaco-therapies.
Six candidate functions were fitted, at within- and between-exposure levels, and
selected according to their approximate likelihood and mean squared error deviance. Afterwards, a nonlinear hierarchical model of within- and between-exposure
habituation was assembled. Selected features were habituation rate and response
recovery. The model’s structure was validated by conceptual correspondence
between the model and the real process. The model’s behaviour was validated
using simulations and predictions. Predictions were assessed by their mean absolute scaled errors (MASE). Results: Within-exposure habituation follows an exponential decay in both groups. Habituation rate follows a logarithmic decay in both
groups. Response recovery is dependent on the context of stimulus presentation,
as expected, following an exponential decay and a linear decay in the SCE and
MCE groups, respectively. Our model correctly describes response habituation
throughout ExRP and generates correct patterns of future behaviour, according to
the validity tests. Predictions lie within the average decrease in SUDS per measurement of a given subject, yielding MASE<1 for 5 and 7 (half) of the subjects in
the SCE and MCE groups, respectively. Conclusions: Our model, is able to accurately describe the typical response of a patient undergoing habituation within
and between ExRP sessions, due to its hierarchical nature. Further adequacy tests
should be performed at this stage, such as including less subjective response
measures, like physiological signals. Nevertheless, our model makes an essential
contribution for describing nonlinear therapeutic effects on patients’ response.
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Oth
Glial cell-derived neuroregulators control type 3
innate lymphoid cells and gut homeostasis
2
Plasmodium-mediated bacterial awakening
D. Mukherjee1, A. Chora1, R. Ramiro2, J. Batista2, I. Gordo2, M.M. Mota1
Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal;
de Gulbenkian de Ciência, Portugal
1
S. Ibiza1 *, B. García-Cassani1 *, H. Ribeiro1, T. Carvalho1, L. Almeida1, R. Marques2 †,
A.M. Misic3 ‡, C. Bartow-McKenney3, D.M. Larson4, W.J. Pavan4, G.Eberl2, E.A. Grice3,
H. Veiga-Fernandes1
These authors contributed equally to this work
Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal;
2
Microenvironment and Immunity Unit, Institut Pasteur, 25-Rue du Docteur Roux, 75724 Paris,
France; 3 Department of Dermatology, Perelman School of Medicine, University of Pennsylvania,
421 Curie Blvd, 1007 Biomedical Research Building, Philadelphia, PA 19104, US;
4
Genetic Disease Research Branch, National Human Genome Research Institute, National
Institutes of Health, Bethesda, MD 20892, US20;
†
INSERM, U1163, Laboratory of Intestinal Immunity, and Université Paris Descartes-Sorbonne
Paris Cité and Institut Imagine, Paris, France
‡
Center for Host-Microbial Interactions, Department of Pathobiology, School of Veterinary
Medicine, University of Pennsylvania, Philadelphia, PA 19104, US
2
Instituto
[email protected]
*
1
[email protected]
Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and
infection at mucosal barriers1. ILC3 development and function have been
considered to be programmed. Nevertheless, how ILC3 perceive, integrate and
respond to local environmental signals remains unclear. Here we show that ILC3
sense their environment and control gut defence as part of a novel glial-ILC3-5
epithelial cell unit orchestrated by neurotrophic factors. We found that enteric
ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led
to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity,dysbiosis
and increased susceptibility to bowel inflammation and infection. Neurotrophic
factors directly controlled innate Il22, downstream of p38 10 MAPK/ERK-AKT
cascade and STAT3 activation. Strikingly, enteric ILC3 were adjacent to neurotrophic factor expressing glial cells that exhibited stellate-shaped projections
into ILC3 aggregates. Glial cells sensed commensal-derived cues in a MYD88
dependent manner to control neurotrophic factor expression and innate IL-22.
Accordingly, glial-intrinsic Myd88 deletion led to impaired ILC3-15 derived IL-22 and
pronounced propensity to gut inflammation. Our work sheds light into a novel
multi-tissue gut defence unit, revealing that glial cells are central hubs of neuron
and innate immune regulation via neurotrophic factor signals.
In malaria endemic areas, Plasmodium, the causative agent of malaria, and bacterial
co-infections are highly concurrent, often with fatal outcomes. Recently, it has
been shown in patients from Sub Saharan Africa that Plasmodium infection predisposes to high bacteremia (Were et al., 2011). However, the causal link between
Plasmodium infection and increased bacteremia as well as the putative mechanisms involved are currently unknown.
We report here that infection of C57BL/6 mice with Plasmodium parasites leads to
an increase in the bacterial load in the liver.
Two hypotheses can be put forward to explain this observation.
One hypothesis is that Plasmodium infection might compromise the integrity of
the intestinal epithelial barrier, thus allowing bacterial translocation from the gut
into the liver. However, increase in the hepatic bacterial burden is not associated
with increased intestinal permeability or with histological alterations in the intestinal barrier. In parallel, there are no significant changes in the gut microbiome of
malaria infected mice 5 days post infection when compared to non-infected controls.
Alternatively, malarial infection might impair immunosurveillance mechanism(s)
responsible for controlling hepatic bacterial burden. In keeping with this notion,
previous reports have shown that an ongoing blood stage of Plasmodium infection causes a global immunosuppression against subsequent antigens (Lundie et
al.,2010). Moreover, there are evidences that CD4 expressing cells may facilitate
the early clearance of bacteria by regulating neutrophils function in sepsis models
(Martignoni et al.,2008).
Concurrent with this hypothesis, in hosts defective in the adaptive immune system
(RAG2ko), the increase in hepatic bacterial load upon Plasmodium infection was
no longer noticeable; an effect paralleled by antibody-mediated depletion of host
CD4+T or CD8+T cells. All these results suggest that Plasmodium blood stagemediated immunomodulation may result in aberrant expansion of bacteria in different organs during malaria infection.
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3
Chemical Probes to uncover novel glycosyltransferases in Trypanosoma brucei
Adenosine A2A receptor overexpression – synaptic
trigger for early aging?
J.Bevan1 2, E.C. Lourenço2, M.R. Ventura2, J.A. Rodrigues1
M. Temido-Ferreira1, D.G. Ferreira1 2, R. Gomes1, J.E. Coelho1, H. Marie3, P.A. Pousinha3,
T.F. Outeiro2, L. V. Lopes1
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa,
Portugal; 2 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de
Lisboa, 2780-157,Oeiras, Portugal.
1
[email protected]
Department of Neurodegeneration and Restorative Research, University Medizin Göttingen, Germany;
3
Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique
(CNRS), Université de Nice Sophia Antipolis, France
1
2
[email protected]
Trypanosoma brucei (T. brucei) is a protozoan parasite, carried by the tsetse fly,
which causes African sleeping sickness in humans. Three types of glycoconjugates
shown to be essential to T. brucei are: variant surface glycoproteins, glycosylphosphatidyl-inositol anchors and “giant” N-linked complex glycoproteins. Due to their
essential roles in the parasite they are promising targets for therapeutics.1
Trypanosoma brucei requires several UDP-Gal-dependent glycosyltransferases
(GTs) to synthesise its range of glycoproteins. Currently, only 14 of a minimum
of 38 distinct T. brucei GTs have been identified using gene homology.2 In this
project we aim to identify further GTs through the use of synthetic probes that
target T. brucei galactosyltransferases. Chemically synthesised substrate UDP-Gal
and analogues are immobilised on a resin and binding GTs will be identified using
Mass Spectrometry based Quantitative Proteomics.
Aging and Alzheimer’s disease (AD) are associated with cognitive impairments and
hippocampal structural and functional alterations. There is compelling evidence of
hippocampal adenosine A2A receptors (A2AR) upsurge associated to cognitive deficits. Recently, we demonstrated that blockade of A2AR in aging and AD experimental models prevents, or even reverts, hippocampus-related impairments (Batalha et
al, 2013, Mol. Psychiatry; Laurent et al, 2015, Mol. Psychiatry). Consequently, hippocampal A2AR function dysregulation may drive part of the detrimental processes
leading to aging and AD. However, the underlying mechanisms are still unknown.
We generated transgenic rats with a neuronal-specific overexpression of the human
A2AR [tg(CAMKII-hA2AR)]. We then performed whole-cell patch-clamp recordings
in CA1 hippocampal neurons from transgenic versus wildtype (WT) animals.
The synthesis of the first two probes (UDP-Gal 1 and UDP-4-deoxy-4-fluoro-Gal
2) from methyl D-galactopyranoside will be presented. The main challenges of
these syntheses are: 1) selective addition of the SMCC linker at the 6-OH position
for subsequent coupling to the resin; 2) the sensitivity of the phosphate to acidic
conditions 3) selective fluorination at C-4. The characterisation of the compounds
attached to the Tentagel resin was performed directly using HR-MAS NMR, avoiding the need for cleaving the products from the solid support.
A2AR blockade decreased the mean amplitude of the excitatory post-synaptic currents (EPSCs) in tg(CAMKII-hA2AR) animals, suggesting an excitatory tonic effect
of overexpressed A2AR on synaptic transmission (n=4-5;P<0.05). To evaluate presynaptic effects, we measured Paired Pulse Ratios (PPRs) in 50-200ms intervals.
The PPR magnitude was reduced in tg(CAMKII-hA2AR) rats (n=10;P<0.05), suggesting that A2AR overexpression enhances neurotransmitter release probability. A2AR
blockade completely rescued these effects. To assess post-synaptic alterations, we
quantified the AMPAR/NMDAR ratio, which was decreased in tg(CAMKII-hA2AR)
animals (n=19-21;P<0.05). Current-voltage (I-V) relationships in pharmacologically
isolated NMDAR or AMPAR EPSCs revealed that AMPAR activation was decreased
(n=7;P<0.05), while the I-V profile of NMDAR was increased (n=7;P<0.05).
References:
1. L. Izquierdo et al., Mol. Microbiol., 2009, 71, 478–491.
2. M. Damerow et al., J. Biol. Chem., 2014, 289, 9328–9339.
Altogether these data show that neuronal A2AR overexpression, per se, shares some
electrophysiological features observed in AD and aging models, suggesting that
A2AR overexpression might be one of the key events in the AD- and aging-associated
glutamatergic synaptic dysfunction.
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The role of EXP-2 during liver infection of
Plasmodium parasites
2
Biophysical evaluation of the mechanisms of
action of antimicrobial peptides
J. Mello-Vieira1, T.F de Koning-Ward2, V. Zuzarte-Luís1, M.M. Mota1
M.R. Felício1, O. Franco 2 3, N.C. Santos1, S. Gonçalves1
2
School of Medicine, Deakin University, Waurn Ponds, Victoria, Australia
Instituto de Microbiologia and Instituto de Medicina Molecular, Faculdade de Medicina,
Universidade de Lisboa, Lisboa, Portugal;
2
Centro de Proteómica e Análise Bioquímica, Universidade Católica Brasília, Brazil;
3
S-inova, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco,
Campo Grande, Brazil
1
[email protected]
Malaria is a tropical disease, caused by infection of the protozoan parasites of
the genus Plasmodium. Disease is provoked when parasites infect red blood cells
(RBCs), multiplying inside them, eventually rupturing and exiting the RBC, starting
the cycle anew. During this cycle, the parasite develops inside a parasitophorous
vacuole (PV) but manages to remodel the infected RBC, placing its enzymes in the
RBC’s cytosol and its transporters at the membrane of the infected cell.
The translocation of proteins from the parasite to the RBC relies on the Exported
Protein 2 (EXP-2), which creates a pore in the PV membrane, through which
Plasmodium proteins are exported. Interestingly, while protein translocation is limited to the blood stage (BS), EXP-2 expression occurs throughout Plasmodium spp.
life-cycle. Recently, EXP-2 was found in two states, during the BS: in combination
with the other translocation components or alone. These findings lead to the hypothesis that EXP-2 may have another function: serving as an opening through which
small molecules, nutrients or toxins, are exchanged between host and parasite.
The aim of this project is to study the function of EXP-2 during the liver stage (LS)
of infection. EXP-2 seems to be essential for the BS because it is refractory to
gene disruption. Consequently, EXP-2 knock-out parasites cannot be propagated
to the mosquito. However, our collaborators have generated a conditional knockout parasite, where the EXP-2 gene is knocked-out only during the development of
the parasite in the mosquito, just before progression to the LS.
Preliminary results show that parasites without EXP-2 do not progress normally
through the LS, never establishing a BS infection. We plan to infect mice, as well
as hepatoma cells to characterize the pre-erythrocytic development of these parasites. Our current hypothesis is that the parasite cannot uptake nutrients and
therefore fails to develop inside hepatocytes.
1
[email protected]
Increased resistance to conventional antibiotics has become a major problem
worldwide. Existing antibiotics are increasingly ineffective as a result of resistance, becoming imperative to find new antibacterial strategies. One of the most
promising possibilities is the use of antimicrobial peptides (AMPs), which play
an important role in the innate immune response of different organisms. AMPs
are small cationic amphipathic molecules. Their mechanism of action is not
fully understood, but their physicochemical properties are determinant, namely
their amphipathic conformation upon interaction with biomembranes and their
positive net charge, which allows them to interact preferentially with negatively
charged biomembranes, such as those of Gram-negative bacteria and cancer cells.
Until now, AMPs demonstrate a low propensity for drug resistance development.
Two different AMPs (Pa-MAP 1.5 and 1.9) were synthetically designed based on
an antifreezing peptide from the Antarctic fish Pleuronectes americanus. Both
peptides showed promising therapeutic results against bacteria and cancer cells.
Using biophysical techniques, namely fluorescence spectroscopy (with the probes
di-8-ANEPPS, Laurdan, DPH and TMA-DPH), dynamic light scattering, zetapotential, surface plasmon resonance and atomic force microscopy, we assessed
the different mechanisms of action of these peptides, using lipid vesicles that
mimic the target membranes, as well as Escherichia coli cells. Flow cytometry
assays were also important to gain insight on the properties of both AMPs at
the cellular level. With the different peptide-membrane properties studied here,
it was possible to show that, even in peptides that are so closely designed, small
changes can affect their activity toward the target cells, and therefore, their potential application.
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Effort in neuropsychiatric disorders
C. Farinha1, T.V. Maia1
1
2
The neuropeptide neuromedin U regulates group 2
innate lymphoid cells
[email protected]
Motivation and effort are crucial for adaptive behavior in everyday life. However,
despite their importance, the neural mechanisms that regulate them are still far
from well-understood.
Evidences suggest that there are two key neuromodulators involved in these processes: dopamine (DA) and serotonin (5-HT). The former has a wealthy amount of
studies, showing how the modulation of DA influences the vigor and willingness
to make effort towards a reward. The significance of the serotonin is less clear but
it has been featured as having an opposite role to DA. So, while DA may regulate
situations where subjects have to balance the costs and benefits of reward, 5-HT
might play a similar role in aversive processing and behavioral inhibition (avoiding
punishments).
Since attention-deficit/hyperactivity disorder (ADHD) and obsessive-compulsive
disorder (OCD) have clinical features that suggest anomalies on how effort is
processed, and are known for their deficits in the dopaminergic and serotonergic
circuits, we hope that by assessing the performance of patients diagnosed with
these disorders in a novel computational task (related to effort and to the costbenefit tradeoff), we can improve the current knowledge about effort in these two
disorders.
The work done so far is mostly related to the optimization of the task. Through an
iterative process with several phases of data collection – first in healthy adults, and
then in healthy children with different ages – key parameters like task’s duration,
difficulty or instructions were optimized for the statistical analysis. The results
from this pilot phase show that the task is robust enough for our purposes, allowing us to find variability even between heathy subjects.
The next phase will consist on collecting data from healthy control, ADHD and
OCD children, together with complementary neuropsychiatric and personality
questionnaires.
V. Cardoso1, J. Chesné1, D. Fonseca-Pereira1, S. Arroz-Madeira1, N.L. BarbosaMorais1, H. Veiga-Fernandes1
1
Universidade de Lisboa, Lisboa, Portugal
[email protected]
Group 2 innate lymphoid cells (ILC2) are main regulators of helminth infection
and allergic diseases at barrier surfaces, such as the lung and the intestine. ILC2
produce type 2 cytokines, notably IL-5, IL-13 and amphiregulin, which empower
ILC2 with myriads of biologic functions. Despite recent progress in the understanding on ILC2 development, how ILC2 perceive, integrate and respond to
micro-environmental cues still poorly understood.
Neuromedin U (NmU) is a neuropeptide expressed in the gastrointestinal tract
and central nervous system (CNS). NmU interacts with two G protein-coupled
receptors, neuromedin U receptor 1 (Nmur1) at peripheral tissues and neuromedin U receptor 2 (Nmur2) at CNS. Nmur1 has been previously detected in
immune cell subsets, but the putative function of NmU in the immune system
remains elusive.
Herein, we demonstrated that lung and small intestine ILC2 express the neuropeptide receptor Nmur1. We found that stimulation of purified ILC2 with mouse
recombinant NmU23 peptide resulted in a robust increased production of the
type 2 cytokines IL-13, IL-5 and GM-CSF at protein and mRNA levels. Using pharmacological inhibitors we further demonstrated that calcineurin and extracellularsignal-regulated kinase signaling pathways are required for NmU-induced production of innate type 2 cytokines. Altogether, these data demonstrate a novel NmU/
Nmur1 pathway that might significantly controls ILC2 in relevant physiological
conditions. Thus, increased knowledge on this novel ILC2 cell surface receptor
and its respective signaling pathway may pave the way for new therapeutic avenues in infectious and allergic diseases that are major public health concerns.
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Training in novelty exploration tasks Prevents cognitive decline in a rat model of Temporal Lobe Epilepsy
Â. Amaro-Leal1 2, I. Rocha2, D. Cunha-Reis1 2
1
Portugal; 2Cardiovascular Autonomic Function Lab, Institute of Physiology & CCUL, Faculdade de
Medicina, Universidade de Lisboa, Lisboa, Portugal
[email protected]
Temporal lobe epilepsy (TLE), characterized by recurrent seizures with origin in
the temporal lobe, is highly refractory and associated with severe cognitive impairment. Since we previously showed that novelty training improves cognitive performance, we now tested if a novelty training program based on novel spatial distribution of objects can prevent cognitive decline in an animal model of TLE.
Male Wistar rats exhibiting spontaneous recurrent seizures (SRSs) (Li2+pilocarpine) and Sham rats were either subjected to a training program or were
without any training (naïve, N group). SRSs and Sham novelty trained (NT) animals were exposed to three objects always presented in a different spatial configuration in a holeboard for two weeks. As control for novelty training, SRSs and
Sham animals were also trained by exposure to either a fixed object’s spatial configuration (FT), or to the holeboard without any object (HT). All animals (trained
and naïve; SRSs or Sham) were subjected to radial arm maze (RAM) test to evaluate hippocampal-dependent spatial learning.
Regarding the training program, all trained SRSs rats had lower spatial exploratory
activity (rearings) and exploration of objects (nose-pokes) but a longer traveled
distance when compared to Sham rats. NT SRSs and Sham rats showed a higher
exploration ability (nose-pokes) when compared to FT and HT animals.
All trained animal groups (SRSs and Sham) were more efficient (committed less
total failures) in finding the baited arms when compared to the respective N group.
NT SRSs were more efficient in finding the baited arms than FT and HT SRSs. No
differences were observed between different trained Sham groups.
Our data shows that a training routine based on novel spatial distribution of
objects can improve SRSs rat cognitive performance in spatial learning tasks suggesting that novelty exposure may be useful for cognitive recovery in TLE.
Behavioral flexibility in obsessivecompulsive disorder and under
serotonergic modulation:
a reinforcement learning approach
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V. Conceição1, F.H. Petzschner2, S.Raman2, D.M. Cole2, K.E. Stephan3, T.V. Maia4
1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa; ETH Zürich,
Switzerland; 2 Translational Neuromodeling Unit (TNU), Institute for Biomedical Engineering (IBE),
University of Zürich; ETH Zürich, Switzerland; 3 TNU, IBE, University of Zürich; ETH Zürich,
Switzerland; Wellcome Trust Centre for Neuroimaging, University College London, London, UK; Max
Planck Institute for Metabolism Research, 50931 Cologne, Germany; 4 Instituto de Medicina Molecular,
Faculdade de Medicina, Universidade de Lisboa; Department of Psychiatry, Columbia University
[email protected]
Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder with an estimated prevalence of 1 to 3%. Its onset is associated to genetic and environmental
factors, and patients suffering from this disorder are typically characterized by the
manifestation of repetitive thoughts and behaviors. However, the pathophysiology
of this disorder is still far from well-understood.
OCD patients are standardly treated with selective serotonin reuptake inhibitors
(SSRIs) and cognitive behavioral therapy, but almost half of the patients are (at least)
partially refractory to treatment.
One of the cognitive features which has been most reproducibly reported in patients
with OCD is subpar performance during reversal learning contingencies. Nonetheless,
this might be explained by deficits both in the serotonergic and dopaminergic systems,
and existent reversal learning tasks suffer from caveats which do not allow to disentangle the contributions from these neuromodulatory systems to behavioral performance.
Aiming at disentangling these contributions, we have developed a new cognitive
task: a Go/NoGo task during which 6 different stimuli are shown and multiple
hidden-states per stimulus exist. We have currently applied this task in a doubleblinded randomized study to 50 healthy adults, who did this task in two sessions:
on- and off-SSRI administration.
Individual labels concerning drug-status have not been revealed yet. Nonetheless,
preliminary data analysis suggests that our task-design was successful: Subjects’
accuracies were significantly above chance (p-value < 0.001), and accuracies were
better during hidden-state revisiting (p-value < 0.05), suggesting that simple stimulus-response learning does not appropriately explain subjects’ behavior and that
higher-order cognitive processes, which we hypothesize to be impaired in OCD, are
necessary to achieve optimal performance.
To study subjects’ behavior in a more mechanistic way, multiple reinforcement
learning models were already fitted to the data. Preliminary comparison between
these further supports the aforementioned finding (exceedance probabilities > 0.99
in favor of models implementing higher-order state-learning).
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Single N-terminal phosphorylation modulates
mutant huntingtin aggregation and toxicity
J. Branco-Santos1 3, G.M. Poças2, Y. Pires-Afonso2, F. Giorgini3, P.M. Domingos2,
F. Herrera1, T.F. Outeiro4 5
Laboratory of Cell Structure and Function, Instituto de Tecnologia Química e Biológica (ITQB-UNL),
Oeiras, Portugal;
2
Laboratory of Cell Signaling in Drosophila, Instituto de Tecnologia Química e Biológica (ITQB-UNL),
Oeiras, Portugal;
3
Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom;
4
Department of Neurodegeneration and Restorative Research, University Medical Center
Goettingen, Waldweg 33, 37073 Goettingen, Germany; 5 Max Plank Institute for Experimental
Medicine, 37075 Goettingen, Germany
1
[email protected]
Huntington’s disease (HD) is an incurable neurodegenerative disorder caused
by a polyglutamine (polyQ) expansion in the N-terminal region of the huntingtin protein (HTT). The first 17 amino acids of the protein (NT17), immediately
preceding the polyQ tract, are a critical functional domain for HTT function and
HD pathogenesis. Recent studies suggest that double NT17 phosphorylation at
serines 13 and 16 reduces mutant HTT aggregation and toxicity. However, double
phosphorylation events are less likely to occur than single phosphorylation events
and require overexpression of specific kinases. Here, we analyzed the effect of
single NT17 phosphorylation events (at Thr3, Ser13 or Ser16) in HTT aggregation
and toxicity in cell and Drosophila models of HD. The generation of large aggregates, but not oligomeric species, was completely abolished by single phosphomimic mutations in living cells, while phosphoresistant mutants did not produce
overt phenotypes. Thr3 phosphorylation resulted in a strikingly decrease of HTT
aggregation in both larva and adult flies, and Ser13 or Ser16 phosphorylation ameliorated climbing impairments. On the other hand, NT17 single dephosphorylation events potentiated age-dependent motor dysfunction and shortened lifespan
of HD flies. Furthermore, specific protein phosphatase inhibitors decreased HTT
aggregation and toxicity both in vitro and in vivo. Our findings suggest that phosphorylation of the HTT N-terminal region plays a critical role in HD pathogenesis,
and support the targeting of specific NT17 phosphorylatable sites and protein
phosphatases for HD therapy.
Key words: Huntington’s disease, huntingtin, phosphorylation, bimolecular
fluorescence complementation, Drosophila melanogaster, neurodegeneration
2
PHYLOViZ Online: web-based phylogenetic data
analysis and visualization for allelic profiles and
SNP data
B. Ribeiro-Gonçalves1, A. Francisco2 , C. Vaz2 3, M. Ramirez1, J.A. Carriço1
Universidade de Lisboa, Lisboa, Portugal; 2INESC-ID / Instituto Superior Técnico, Universidade de
Lisboa, Lisboa, Portugal; 3Instituto Politécnico de Lisboa, Lisboa, Portugal
1
[email protected]
Next Generation Sequencing methods created a paradigm shift in Microbial Typing
and Genomic Epidemiology fields. The ability to sequence hundreds of strains created the need to find effective ways to represent phylogenetic relationships between
isolates that are scalable and robust. Single Nucleotide Polymorphism (SNP) analysis
and core genome MLST approaches create profiles with up to thousands of loci.
Their analysis can be done using traditional phylogenetic algorithms or Minimum
Spanning Tree (MST) like approaches, with the latter being able to cope with the
increasing number of isolates that are used in each study. PHYLOViZ software allows
the use of epidemiological data to annotate the resulting tree, but lacks a simple way
to share results between users or provide public data access to a given dataset. To
overcome that limitation we have developed PHYLOViZ Online, a user-friendly web
application for any profile based data analysis, visualization and sharing, which also
offers a RESTful API for automatic data submission and dataset visualization.
PHYLOViZ Online is a Node.js application written in a modular perspective, separating visualization from the data processing. The goeBURST algorithm is used
to define the MST and a webGL rendering engine allows displaying thousands of
isolates on a tree simultaneously in a web browser. Other novel features are: (a) an
interactive matrix-based heatmap with distances between user-selected nodes that
can be ordered by isolate auxiliary data; (b) the N Locus Variant (NLV) graph analysis, which generates clusters by linking all profiles at a given distance; (c) dataset
storage and sharing.
The PHYLOViZ Online web-application provides an online resource for sequencebased typing analysis that is modular and expandable. Its simple graphical interface
allows non-expert users to navigate through the application, while the RESTful API
opens doors from possible integration with other resources. PHYLOViZ Online can
be freely accessed at https://online.phyloviz.net.
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Characterization of neuromodulatory dysfunction
in Amyotrophic Lateral Sclerosis
N. Rei1 2, S.H. Vaz 1 2, M. Colino-Oliveira1 2, C.A. Valente 1 2, J.A. Ribeiro1 2, A.M.
Sebastião1 2
Institute of Pharmacology and Neurosciences, Faculdade de Medicina;2 Instituto de Medicina
Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal
1
2
Sonographic evaluation of fetal growth at the last
weeks of the third trimester of low-risk pregnancy
C. Policiano1, A. Fonseca1, J. Barros1, D. Martins2, N. Clode1, L.M. Graça1
Departamento de Ginecologia e Obstetrícia, Hospital de Santa Maria, Centro Hospitalar Lisboa
Norte; 2Hospital Beatriz Ângelo
1
[email protected]
[email protected]
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized
by the progressive degeneration of motor neurons in corticospinal tract, leading
to muscle weakness, atrophy, paralysis and death. Previous research provided evidence that neuromodulation by adenosine, is disturbed in ALS. Some recent studies support that vascular endothelial growth factor (VEGF) protects motor neurons
from degeneration in ALS models.
We now evaluated wether there are any changes in the levels of the inhibitory adenosine receptor, the A1R, at the cerebral cortex and spinal cord of a mice model of ALS
[SOD1(G93A)] at two disease states and if there is any changes in the mRNA expression levels of VEGF receptors (VEGF-R1 and VEGF-R2) at pre-symptomatic phase.
Ligand binding assays were performed to evaluate the expression of A1R in cortex and spinal cord. To evaluate the mRNA levels of VEGF receptors in same areas,
through disease progression, we performed a quantitative Real-time PCR.
There was a decrease of the maximum number of specific A1R binding sites (Bmax)
in the cortex of ALS mice in both phases, when compared with age-matched wild
type (WT) mice (n=5-7; p<0.05). No statistically significant differences were found
while using membranes isolated from the spinal cord. These findings suggest that
an early dysfunction at adenosine signaling that occurs even before disease onset is
accompanied by a decrease in the expression of the inhibitory A1R.
There is a significant decrease of VEGF-R2 mRNA levels in cortex of pre-symptomatic mice but no statistically significant differences were found for the VEGF-R1
mRNA expression (n=5; p<0.05). On the other hand, in spinal cord there is a significant increase of both VEGF receptors mRNA levels in pre-symptomatic phase (n=5;
p<0.05), suggesting that there is a variation in the expression of VEGF receptors
mRNA and that variation differs between cortex and spinal cord of ALS mice model.
Fetal growth restriction (FGR) without prenatal diagnosis is associated with a higher
risk of adverse neonatal outcomes (Apgar score at 5 minutes <4, neonatal seizures, acidosis and neonatal death) compared with fetuses whose diagnosis of
FGR was made antepartum. Moreover, a significant proportion of term stillbirths
correspond to undiagnosed late FGR. Sonographic fetal weight estimation at the
last weeks of third trimester in low-risk pregnancies is an effective method for
diagnosis of FGR permitting a close surveillance and timely delivery. The need
for a systematic ultrasound evaluation at the last weeks of a low-risk pregnancy
and the best time to perform it remains controversial. The most commonly used
clinical screening tool in this population is the serial measurement of symphysisfundus distance, which is a method of a variable and low sensitivity. The aim of
this study is to evaluate the importance of an ultrasound assessment performed
in the last weeks of a low-risk pregnancy as a screening method for late FGR.
The first task is to evaluate the incidence, in late (35-366 / 7 days weeks) low-risk
pregnancies, of fetuses below the 3rd percentile, 5th percentile and 10th percentile, with and without abnormal umbilical and middle cerebral arteries Doppler
indices. At the same time, we intend to compare the sensitivity of the assessment
of uterine symphysis-fundus distance with sonographic fetal weight estimation in
predicting infants of low birth weight. At this stage, it will be also evaluated intraand interobserver variability in the sonographic evaluation.
The second task is the prospective randomized study comparing the perinatal outcomes between a study group (with an ultrasound scan at 35-366 / 7 days weeks
for fetal weight estimation and Doppler flowmetry) with a control group with FGR
screening by serial measurements of symphysis-fundus distance.
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Double retrograde anatomical tracing unravels
an ego- to allocentric functional gradient in the
midline cortex
2
Revealing the uis4 protein interacting network and
its role during the malaria liver stage infection
V. M’Bana1, K. Slavic1, M-M. Mota1
1
E. Ferreira-Fernandes1, M. Remondes1
1
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal
Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal
[email protected]
[email protected]
The midline cortex (cingulate and retrosplenial divisions) is a central hub for the
integration of egocentric, allocentric and memory-related information, in the service of choice behaviors. The structural and functional details of the intrinsic and
extrinsic projections of the midline cortex are, however, poorly characterized, hindering our ability to causally test their relevance in freely behaving rats.
To establish the boundaries of cingulate and retrosplenial divisions, based on their
structural connectivity, and clarify whether there exist local microcircuit specificities, as well as differential distribution of bottom-up projections along its anteriordorsal extent, we performed double retrograde tracing experiments at all levels
of this cortical structure. Our results show abundant intrinsic projections connecting the anterior cingulate and retrosplenial divisions, as well as differential
targeting of such divisions by extrinsic projections. The anterior cingulate division
is enriched in somatosensory inputs and is selectively targeted by dorsal lateral
entorhinal cortex, dorsal intermediate entorhinal cortex, and thalamic nuclei mediodorsal, ventral anterior and ventrolateral. The retrosplenial division is enriched
in visual inputs and is selectively targeted by caudomedial entorhinal cortex, subiculum, and thalamic nuclei anterodorsal and laterodorsal. We also found a novel
population of neurons with collateralized projections in the midline cortex, thalamus and ventral claustrum, simultaneously targeting cingulate and retrosplenial
divisions, and a novel population of putative long-range inhibitory neurons in the
hippocampus, directly targeting the midline cortex. Together, our results suggest
the division of the midline cortex in two connectivity modules. The cingulate division is in strategic position to act as an integrative hub for somatosensory information in self-referenced perception, whereas the retrosplenial division may act
as an integrative hub for visuospatial information in allocentric-referenced perception. The populations of neurons with collateralized projections and long-range
inhibitory neurons are suggested as candidates to control dynamic synchronization, required for the flexible communication among these brain regions.
Malaria is a vector born-disease caused by Plasmodium parasites that are transmitted to vertebrate host through the bites of infected Anopheles mosquitoes.
It is one of the major global health threats. Despite the great effort to eliminate
malaria, it remains, one of the most prevalent infectious diseases worldwide,
with an annual death toll close to one million. The infection is initiated when the
sporozoites (the mammalian infectious Plasmodium stage) are injected into the
skin of the host during the mosquito blood meal. Hepatocyte infection is the first
natural step to the establishment of a malaria infection. However, relatively little is known about the molecular interactions established between the parasite
and the host during the liver stage of infection. A subset of proteins encoded
by the “Upregulated in Infective Sporozoites” (UIS) genes are exported to the
Parasitophorous Vacuole Membrane (PVM) and seem to be essential for the parasite survival, namely UIS4. The biological function of UIS4 remains to be discovered. Our hypothesis is that UIS4 protect the parasite by inhibiting the accidification. We also hypothesize that this protein does not works alone, but in a complex
network interaction. In this project I propose to study the biologic role of UIS4 by
revealing its interaction network.
To accomplish this task, I’m using the gene knock out and phenotype rescue
experiment. The identification of the putative that are interacting with UIS4 is
being done by proximity-dependent biotin labeling approach, named BioID.
BioID is an approach for identification of physiologically relevant protein-protein
candidate interactions that occur in the living cells. The mechanism is based on
the use of the biotin protein ligase (BirA), which is fused to a protein of interest
and when expressed in cells, upon addition of the biotin, leads to the biotinylation
of the neighbor proteins.
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Anticoagulation in patients with cirrhosis and
portal vein thrombosis is associated with increased
portal vein recanalization and better prognosis
C.N. Ferreira1, T. Rodrigues2, A.J. Pedro3, P. Ferreira1, M.S. Dias1, A. Gonçalves4,
L.X. Brito1, F. Serejo1, R.T. Marinho1, C.B. Costa1, N. Fatela1, H. Cortez-Pinto1,
F. Ramalho1, P. Alexandrino1, J.F. Velosa1
Serviço de Gastrenterologia e Hepatologia, Hospital de Santa Maria, Centro Hospitalar Lisboa
Norte, Lisboa, Portugal;
2 Laboratório de Biomatemática, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
3 Serviço de Medicina II, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, Lisboa, Portugal;
4 Serviço de Imagiologia, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, Lisboa, Portugal
1
21(0-376) months. At the end of follow-up, 29(42%) patients died, with no deaths
due to anticoagulation related bleeding.
By Cox regression analysis, factors associated with mortality at the end of followup were: Age (HR 1.040, 95% C.I. 1.002–1.078, p=0.037), CP score (HR 1.35, 95%
C.I. 1.18–1.55, p<0.001),MELD score (HR 1.14, 95% C.I. 1.08–1.21, p<0.001), creatinine (HR1.52, 95% C.I. 1.06–2.16, p=0.021).
Anticoagulation significantly decreased mortality at the end of follow-up even after
adjusting for VB at diagnosis of PVT (HR 0.30 95% C.I.0.11–0.82, p=0.019). KM
survival analysis confirmed that patients with cirrhosis and PVT given anticoagulation had better outcome compared to those not given anticoagulation(p=0.025).
Portal vein recanalization was more frequent in patients on anticoagulation than
no anticoagulation (61% vs 22%) (p=0.005).
[email protected]
Introduction: Cirrhosis is recognized as a prothrombotic state. Aims: To analyze
the effect of anticoagulation on recanalization of non-tumoral PVT in patients with
cirrhosis and its effect on prognosis.
Conclusions: Anticoagulation in patients with cirrhosis and PVT seems to be safe
and associated with higher portal vein recanalization rates and significantly lower
mortality.
Methods: 69 consecutive patients with cirrhosis diagnosed with non-tumoral PVT
were studied. Decision to start anticoagulation was taken at the discretion of the
clinician managing the patient. The effect of anticoagulation on PVT recanalization and mortality was analyzed.
Results: The average age was 58.6±11.8 years and 44(64%) were males. Severity
of cirrhosis: Median(Range) Child–Pugh(CP) score: 8(5-15), MELD score:13(6-35).
CP class: A-15(22%), B-32(46%), C-22(32%). At diagnosis of PVT, 55(80%) were
symptomatic. Variceal bleeding(VB) in 30(46%) and abdominal pain in 19(29%)
were the main clinical presentations.
Anticoagulation (LMWH–9, warfarin–16) was administered in 25(36%) patients
one of whom with cavernoma. Patients with VB were less likely to be given anticoagulation (p=0.037).
Recanalization of PVT was assessed by at least one imaging study in 60 patients
and recanalization was documented in 22(37%) patients. Median follow–up was
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Transcutaneous Spinal Direct Current Stimulation
on the Cervical Spine Region
S.R. Fernandes1 2, R. Salvador1, C. Wenger1, M. A. de Carvalho2, P.C. Miranda1
Instituto de Biofísica e Engenharia Biomédica (IBEB), Faculdade de Ciências da Universidade de
Lisboa (FCUL), Lisboa, Portugal;
2
1
2
West-Nile virus capsid protein interacts with host
intracellular lipid droplets and VLDL
A.S. Martins1, F.A. Carvalho1, A.F. Faustino1, I.C. Martins1, N.C. Santos1
1
Portugal;
[email protected]
[email protected]
Transcutaneous spinal direct current stimulation (tsDCS) is a recent technique
with promising neuromodulatory effects on spinal neuronal circuitry related with
motor and sensory functions. Electrode configuration, geometry and stimulation parameters can influence the electric field distribution and, consequently,
result on different modulation effects on the transmembrane neuron potential.
Computational studies on tsDCS using realistic human models may be useful to
establish optimal stimulation settings for specific therapeutic purposes.
The main objective of the present study was to perform a finite element analysis
of the electric field distribution in tsDCS in the cervical spine region, with varying
electrode configurations and geometry.
The electric field distribution for cervical tsDCS predicted higher magnitude values when considering the target and return electrodes placed over the C3 and T3
spinous processes, respectively, reaching a maximum of 0.71 V/m in the spinal
white matter and 0.43 V/m in the spinal grey matter, whereas the values are >
0.15 V/m in the region of the spinal circuits related with upper limb innervation.
Considering the configuration applied in most exploratory studies on cervical
tsDCS (target electrode over the C7 spinous process and return electrode over the
right deltoid muscle), the values were found to be < 0.15 V/m through the entire
spinal cord.
West-Nile and dengue viruses are closely related flaviviruses, originating mosquito-borne viral infections for which there are no effective and specific treatments. Their capsid proteins sequence and structure are particularly similar,
forming superimposable α-helical homodimers. The measurement of proteinligand interactions at the single-molecule through nanobiophysics techniques
yields detailed information of use to nanomedicine and nanotechnology. In this
works such an approach enabled measuring at the nanoscale the West-Nile capsid protein biologically relevant interactions with host lipid structures. Since we
already reported that dengue virus capsid protein binds intracellular lipid droplets (an essential step of viral replication) and blood plasma very-low density lipoproteins (which may prompt the formation of highly infectious lipoviroparticles),
the related West-Nile virus capsid protein could be expected to also interact with
these host lipid systems. Here, we directly tested West-Nile virus capsid protein
interaction with these lipid systems. Zeta potential studies, dynamic light scattering measurements and single-molecule atomic force microscopy-based force
spectroscopy show that the interactions with lipid droplets and with lipoproteins
are strong, specific and require K+ ions as well as surface proteins of these host
lipid systems. West-Nile virus capsid protein binds very-low density lipoproteins
but not low-density lipoproteins. The previously proposed model of dengue virus
capsid protein interaction with host lipid systems seems thus to be identical in
West-Nile virus and, possibly, in other closely related flaviviruses.
Computational studies using realistic models may be a powerful tool to predict
physical effects of tsDCS on the cervical spinal cord and to optimize electrode
placement focused on specific neurologic patient needs related with upper limb
function.
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The influence of vitamin D on the BMD of
hyperthyroid patients with and without silent
vertebral fractures
A.P. Barbosa1 4, M.R. Mascarenhas1 4, J. Janeiro5, I. Távora5, N. Duarte4, M. Bicho2,
F. Sampaio4, J. Monteiro4
University Clinic of Endocrinology
2
Environmental Health Institut-Faculdade de Medicina de Lisboa;
3
CEDML – Clinic of Endocrinology, Diabetes and Metabolism of Lisbon Lda.;
4
Fracturary Osteoporosis Outpatient Clinic- Endocrinology, Diabetes and Metabolism Department;
5
Imagiology Department, Hospital Santa Maria, CHLN-EPE, Lisboa, Portugal.
No patient was previously treated for hyperthyroidism nor osteoporosis/low bone
mass.
According the US Endocrine Society of Medicine, the 25(OH)vitamin D blood
levels (ng/mL) were divided in deficiency (<20), insufficiency (21-29) and normal
(>30) groups.
Adequate statistical tests were used, with P <0.05 significant.
1
[email protected]
In hyperthyroidism, changes in vitamin D metabolism can be associated to nutrients malabsorption and to an increase urinary calcium loss and may contribute to
the increase risk of falls. Moreover, the increase in bone resorption originates a
marked BMD reduction, osteoporosis and fragility fractures.
Objectives
To evaluate the relations between vitamin D, thyroid hormones and BMD in
patients with hyperthyroidism, with and without silent vertebral fractures.
Materials and Methods
We evaluated 44 patients with hyperthyroidism, 21 men and 23 premenopausal
women.
Results
The mean 25(OH)vitamin D levels were similar between the groups of patients with
and without fractures, in both sexes. 25(OH)vitamin D in men: deficiency-43%,
insufficiency-33%, normal-24% and in women: deficiency-35%, insufficiency-39%
and normal-26%.
Significant correlations in men: 25(OH)vitamin D vs. BMD at L1-L4 (CC= 0.690;
p=0.0022), femoral neck (CC=0.503; p=0.0398) and total femur (CC=0.554;
p=0.0210) and in women: 25(OH)vitamin D vs. BMD at distal radius (CC=0.04927;
p=0.0378).
Conclusions
This study suggests that vitamin D blood levels are inadequate in about 75% of
the hyperthyroid patients.
Moreover, vitamin D blood levels seem to influence the cortical bone in both
sexes and the trabecular bone just in men, as a result of an increase bone remodeling and subsequent bone mass loss, which can conjugate for the occurrence of
asymptomatic vertebral fractures.
Fast blood collection was performed to measure fT3, fT4, TSH, iPTH, calcium,
phosphorus and 25(OH)vitamin D.
BMD (g/cm2) at L1-L4, proximal femur, distal radius and whole body, lean and fat
body masses (Kg) were evaluated by DXA.
Vertebral fractures were evaluated by “vertebral fracture assessment” (VFA).
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RNA modifications and antigenic variation in
Trypanosoma brucei
I. Viegas1, J. Rodrigues 1, J. Nina1, F. Aresta-Branco1, L. Figueiredo1
1
[email protected]
Trypanosoma brucei is a unicellular eukaryote parasite that causes sleeping sickness in humans. In this parasite, transcription is mainly constitutive and regulation of gene expression is essentially post-transcriptional. In other eukaryotes,
N6-methyladenosine (m6A) is an RNA modification involved in post-transcriptional gene regulation. We have discovered that this RNA modification is present
in Trypanosoma brucei RNA, representing around 0,06% of total adenosines
(similar to other eukaryotes) . Using immunoblotting and RNA digestion mapping
analysis, we found that m6A is enriched in the polyA tail of the variable surface
glycoprotein (VSG) transcript. This transcript is extremely important for parasite
infection in the mammalian host. It is an essential component of antigenic variation, a mechanism that allows the parasite to escape from the immune system.
Antigenic variation depends in the monoallelic expression of one VSG gene and
the ability to switch the VSG expressed. It has been known for many years that
VSG transcript is very stable with a half-life of 4.5hr, while the median half-life of
T. brucei transcripts is 12 min. The enrichment of m6A in the poly (A) tail of VSG
transcript opens the possibility that this methylation has a role in antigenic variation by stabilizing the transcript or facilitating its translation.
2
Experimental Autoimmune Encephalomyelitis
induces de novo generation of IL-17-producing
γδ T cells
P. Papotto1, N. Gonçalves-Sousa1, J. Ribot1, N. Schmolka1, S. Mensurado1,
B. Silva-Santos1
1
[email protected]
More than 30 years after their discovery, γδ T cells are still a very puzzling population of lymphocytes. Part of the unconventional biology of γδ T cell resides in their
thymic developmental program, which differs from their γδ counterparts. Whereas
the latter need to go through peripheral processes of activation and differentiation, the former exit the thymus with fully mature effector programs. γδ17 cells,
particularly, seem to be strictly dependent on thymic preprogramming, and their
generation is thought to be constrained to a tight window during embryonic development. These innate-like features contradict the adaptive characteristics recently
attributed to peripheral γδ17 cells. Therefore, in the present study we hypothesized that environmental triggers are able to peripherally promote de novo generation of γδ17 cells. In order to test this hypothesis, bone marrow chimeras (BMC;
which lack γδ17 cells) were challenged with pro-type-17 stimuli and peripheral
generation of γδ17 cells was assessed. Both intraperitoneal Lipopolysaccharide
(LPS) injection and Listeria monocytogenes infection were not able to induce γδ17
cell responses. Strikingly, using the experimental autoimmune encephalomyelitis
(EAE) model, γδ17 cells were found mainly in the brains and spleens on days 14
and 18 post-immunization – during the peak of central nervous system (CNS)
inflammation. However, on day 7 post-immunization gd17 cells were only found in
the draining lymph nodes (dLN), indicating that their generation does not occur in
the target tissue. Moreover, these cells were found to be Vγ4+ and their generation
is coupled with proliferation. Altogether, our findings show that Vγ4+ γδ17 cells
are generated in the early stages of EAE in the dLN and migrate to the inflamed
tissue thereafter.
89
P42
3
Low doses of ionizing radiation
induce angiogenesis: therapeutic
implications
F.G. Marques1, C. Cardina1, J. Ferreira1, T. Freitas1, J. Malaquias3,
J.C. Mendes de Almeida2 3, M. Jorge4, F. Pina4, E. Poli 4, M. Mareel5, S.C.R. Santos1 2
Angiogenesis Unit, Centro Cardiovascular da Universidade de Lisboa, Lisboa, Portugal;
Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal,
3
Departamento de Cirurgia, Hospital de Santa Maria, CHLN, Lisboa, Portugal;
4
Serviço de Radioterapia, Hospital de Santa Maria, CHLN, Lisboa, Portugal;
5
Department of Radiotherapy, Ghent University Hospital, Ghent, Belgium
P43
2
The Economic Impact of Early Retirement Caused
by Rheumatic Diseases
1
2
[email protected]
During radiotherapy, peri-tumoral tissues are exposed to sub-therapeutic doses of
ionizing radiation (IR). The molecular and biological effects of such radiation remain
unexplored. Using animal models, we demonstrated that low doses of IR (LDIR)
promote tumour growth and metastasis by enhancing angiogenesis1. Therefore, we
are interested in validating these findings in humans.
A collaboration between bench researchers, physicists and clinicians has been
crucial to carry out this work. Two distinct peritoneal biopsies exposed or not to
LDIR from patients with rectal cancer that received neoadjuvant radiotherapy were
collected and immunostained for CD31 followed by endothelial cell (EC) isolation
by laser capture microscopy. The expression of several pro-angiogenic targets was
assessed by qRT-PCR. Our results suggest that their expression is upregulated by
LDIR, 8 weeks after the end of the radiotherapy treatment.
Furthermore, other cell types in the microenvironment may also be modulated by
LDIR. Knowing that adipocytes produce multiple factors that stimulate angiogenesis,
we are also interested in investigating if LDIR regulate the adipocyte differentiation
and/or the angiogenic switch by modulating the expression of angiogenic factors in
adipocytes. Pre-adipocytes were exposed or not to LDIR at different time points of
the differentiation process. The expression of several angiogenic factors was measured by ELISA and Zymography. The conditioned medium from adipocytes differentiated from irradiated pre-adipocytes was assessed in the chick embryo chorioallantoic membrane (CAM) assay. Our results show that LDIR significantly increase the
secretion of Vegf by mature adipocytes when pre-adipocytes were exposed to LDIR,
leading to a pro-angiogenic response in vivo.
The results herein will have the potential to reveal new mechanisms and understand
the pro-metastatic effect of IR, leading to significant breakthroughs and advancing
the state of the art in the field. Our findings will provide a new rationale basis to the
improvement of current radiation oncology protocols.
P. Laires1, H. Canhão1, M. Gouveia2
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,Lisboa,
Portugal;2 Católica Lisbon School of Business
1
[email protected]
Objectives: To measure early retirement caused by self-reported rheumatic and
musculoskeletal diseases (RMD) and to estimate the respective indirect costs and
years of working life lost (YWLL).
Methods: We used individual level data from the national, cross-sectional, population-based EpiReumaPt study (Sep2011-Dec2013) where 10,661 inhabitants
were randomly surveyed in order to capture and characterize all cases of RMD
within a representative sample of the Portuguese population. In this analysis we
used all participants aged between 50 and 64, near the official retirement age. A
national database was used to calculate productivity values by gender, age and
region, using the human capital approach. YWLL were estimated as the difference between each participant’s current age and the respective retirement age,
while the potential years of working life lost (PYWLL) were given by the difference
between official and actual retirement ages.
Results: 29.9% of the Portuguese population with ages between 50 and 64 were
retired with 13.1% self-reporting retirement due to RMD. The estimated annual indirect cost following premature retirement caused by RMD was €910 million (€555 per
capita; €1,625 per self-reported RMD patient and €13,592 per early retiree due to
RMD). Females contributed with 84% for these costs (€766 million; €882 per capita
versus €187 from males). We observed a total number of 389,939 accumulated YWLL
(228 per 1000 inhabitants) and 684,960 PYWLL (401 per 1000 inhabitants). RMD
patients with higher values of disability have the highest risk of early retirement.
Conclusions: Early retirement caused by self-reported RMD amounts to approximately 0.5% of the national GDP in 2013, due to large YWLL. Both the public health
concern and the economic impact highlight the need to prioritize investments in
health and social protection policies targeting patients with rheumatic conditions.
References: 1 Sofia Vala I, Martins LR, Imaizumi N, Nunes RJ, Rino J, et al. (2010) Low Doses
of Ionizing Radiation Promote Tumor Growth and Metastasis by Enhancing Angiogenesis.
PLoS ONE 5(6): e11222.
91
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P45
2
Dullard and Eps8L2 as modulators of nuclear
movement
3
CellectAb - Intracellular Targeted Evolution for
Discovery of Therapeutic Antibodies
C. Janota1, D. Osório2, Y. Jiao2, J. Diaz2, E. Gomes1 2
J. Ministro1 2, S. Corte-Real2, F.A. da Silva3, S. Oliveira2, J. Gonçalves1
Portugal; 2 Université Pierre et Marie Curie, UPMC, Paris
1
1
[email protected]
Nuclear positioning in the rear of the cell is important for cell migration in multiple
contexts and we identified Dullard as an activator of that movement. Interestingly,
it was first described to be expressed in neural tissues in Xenopus, where its
depletion caused neural tube defects (Satow et al. 2006). We performed a Yeast2-Hybrid screen and found a new binding partner for dullard, Eps8L2, an actin
regulator. We hypothesized that perinuclear regulation of Eps8L2 by dullard could
be involved in nuclear movement and centrosome orientation.
To test our hypothesis, we first validate the screen by knocking-down dullard
using iRNA and performed a wound assay. We found it dullard to be required for
nuclear movement, but not for centrosome position. Furthermore, we observed
that knock-down of Eps8L2 per se can block nuclear movement. Then we aimed
to understand if these proteins actin organization. We observed that depletion of
dullard or Eps8L2 did not interfere with actin organization however, Eps8L2, but
not dullard, siRNA disrupts dorsal actin cables. Then, we asked if dullard-induced
nuclear movement was dependent of dullard phosphatase activity. We knockeddown dullard and then performed rescue experiments with full length dullard and
dullard lacking phosphatase activity. We concluded that dullard-induced nuclear
movement is dependent of its phosphatase activity. Then, we focused on the biochemical characterization and identified two different Eps8L2 regions that bind to
dullard.
In summary, our work shows for the first time the involvement of dullard and
Eps8L2 in nuclear movement. Our data suggest that in the absence of dullard,
other proteins might dephosphorylate Eps8L2 and create actin dorsal cables, however it does not lead to nuclear movement. According to this, now we intend to
study the role of both proteins on the actin dynamics at the perinuclear regions
and its implications for nuclear movement.
iMed – Research Institute of Medicines, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal;
TechnoPhage;
3
Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal
2
[email protected]
Hybridoma technology and phage display have revolutionized the field of antibody
discovery. However, with the increasing numbers of antibodies reaching the market there is a need to develop new technologies that stem from the limitations of
current discovery platforms. No current technologies integrate all the discovery
steps in a single cell platform to optimize, reduce cost and increase productivity
of new biotherapeutics.
CellectAb answers these needs with a technology that mimics B-cell development
of antibodies and uses engineered human T-cells to perform all steps of therapeutic protein discovery and development. This platform presents several advantages
over other discovery methods: a) intracellular generation of diversity in potentially
any antibody format, mimicking B-cell diversity mechanisms and expanding the
paratope repertoire of synthetic libraries; b) antibodies are expressed in native
conditions which also provides a new method for discovery of stable antibodies; c) it
is designed to select binders or neutralizing antibodies against native membrane
proteins (e.g. difficult targets such as GPCR or ion channels) therefore capable of
functional activity selection; d) increase speed of discovery and development; and
e) increase odds of favorable manufacturability.
Here we present the initial outcomes of the proof-of-concept for the CellectAb
platform, how we generate variability in antibodies using our knowledge on DNA
specific recognition sites, how we can select binders or neutralizing antibodies
against a membrane protein, and how we can bring all these assumptions in one
single-cell platform.
This novel technology of antibody discovery can drastically improve the biophysical
and functional properties of new immunotherapies increasing effectiveness and
reducing the time to enter in clinical trials, and consequently the time-to-market.
93
P46
P47
Oth
Exercise extreme beneficial in obesity?
I. Alonso1, A. Matos1, C. Afonso1, A.Gil1, A.P. da Silva1, C. Cardoso2, M.Bicho1 3
Laboratory of Genetics, Faculdade de Medicina, Universidade de Lisboa;
Clinical Analysis Laboratory, Group . Dr. Joaquim Chaves, Lisboa;
3
Institute for Scientific Research Bento da Rocha Cabral, Lisboa
2
Going Deep in the Brain with alternative Splicing
in Parkinson’s disease
1
2
M.Bordone1, N.L. Barbosa- Morais1
1
[email protected]
[email protected]
Physical exercise is associated with physical, psychological and social benefits,
relating positively with obesity. However, the extreme exercise may not be so beneficial due to exposure to high oxidizing factors responsible for highly inflammatory processes that are limiting the physical condition. Were evaluated 55 athletes
in a mountain bike race, which included 20 nationalities, of both sexes in most
males aged from 25-56 years. Anthropometric variables, haematological and liver
function were evaluated by the conventional methods were measured several
inflammatory parameters such as haptoglobin by nephelometry, CRP (C-reactive
protein) and Gammaglutamil transferase.
Regarding the start of the race it can be seen that some individuals have had an
inflammatory condition. After the test there was an increase both in the hematological parameters of hepatic function in the general population of the cyclists.
Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal
Alternative splicing (AS) is a process whereby different types of RNA are produced
from a single gene through the usage of different splice sites in the RNA. The
majority of the human genes undergo AS and brain is known to be the organ
where AS is more frequent and evolutionarily conserved. Therefore it is not surprising that abnormal AS has been reported in many human diseases, namely
neurodegenerative ones.
Our work is focused on Parkinson’s disease (PD), the second most common
human neurodegenerative disorder. Little is known about the mechanisms that
underlie this disorder, being the majority of PD cases idiopathic. PD-specific AS in
brain areas has been previously detected for genes already described as playing a
role in the PD, namely SNCA, LRRK2, PARK2, VPS35, and SRRM2.
We therefore hypothesize that deregulation of AS plays a key role in the molecular aetiology of PD and that there is a genome-wide AS signature for PD in brain
tissues. More specifically, we hypothesize that PD-specific changes in the expression of trans-acting AS regulators and/or in the sequence of splicing cis-acting
elements alter the splicing regulation of genes with key functions in the brain
and that alteration is involved in PD pathogenesis. This hypothesis is based on
our analysis of microarray data showing a significant downregulation of neuronalspecific splicing factors such as CELF4 in the substantia nigra of PD patients when
compared to controls. We expect our project to bring new insights on PD’s aetiology
and identify novel molecular candidates for therapeutic targeting.
95
P48
P49
3
Force transmission and endothelial cell
rearrangements during vascular remodelling
J. Carvalho1, C. Fonseca1, A. Filomena1, N. Santos1, C.A. Franco1
1
[email protected]
Formation of new blood vessels through sprouting angiogenesis involves a series
of tightly coordinated processes that allow them to invade avascular tissues
and expand the pre-existing network. Yet, to become a functional hierarchically
branched network, blood vessels undergo extensive vascular remodeling. We
previously showed that non-canonical Wnt signaling prevents premature vessel
regression, an essential step in vascular remodeling. Strikingly, this pathway regulates endothelial cell polarity against the blood flow direction in vivo, controlling
the threshold of sensitivity to flow-induced shear stress. However, the mechanism
downstream non-canonical Wnt signaling is not understood.
We now discovered that endothelial cells use a specific non-canonical Wnt ligand
(Wnt5a) to control collective cell polarity, through activation of the ROR2 receptor.
We discovered that Wnt5a-depleted endothelial cells tend to form quiescent lowforce reticular junctions, rather than discontinuous junctions associated to active
junctional remodeling. This suggests that force transmission, through adherens
junctions, seems to be compromised in the absence of Wnt5a. In accordance,
using atomic force microscopy, we found that the strength of cell-cell interactions
in Wnt5a-depleted cells is significantly reduced, demonstrating that Wnt5a controls force transmission at adherens junctions. Moreover, our preliminary observations suggest that rather than affecting the levels of junctional-associated proteins, Wnt5a could control the robustness of the association of cadherin to actin
filaments.
Our study shows for the first time that adherens junction-based force transmission can be a direct regulator of endothelial cell response to blood flow, which in
turn could control how these cells behave collectively. Our results have a direct
impact in our understanding of the way that blood vessels establish a hierarchical
and functional blood vessel network, which could have important translational
applications in cancer and ischemic disorders.
2
Molecular mechanisms regulating endothelial
cell axial polarity during vascular morphogenesis
P. Barbacena1, C.A. Franco1
1
[email protected]
The establishment of a functional patterned vascular network is crucial for development, tissue growth and physiology. The mis-patterning or dysfunction of this
network is associated to stroke, cancer, and arteriovenous malformations. In contrast to sprouting angiogenesis, the molecular mechanisms regulating vascular
patterning are poorly understood. While both blood flow and VEGF are recognised as regulators of vascular patterning, very little is known about the cellular
and molecular mechanisms that regulate endothelial cell behaviour in response
to both these cues.
In order to characterise the dynamic behaviour and polarity patterns of endothelial cells in response to blood flow during vascular patterning, we generated a
construct enabling dual expression of mCherry in the Golgi complex and eGFP in
the nucleus. We are now using this reporter to produce both an inducible and a
constitutive endothelial-specific reporter mouse lines.
In parallel, preliminary data shows that the Par complex – a main regulator of
cell polarity – is essential for both VEGF and flow-dependent polarisation, thus
Par3 could constitute a central signalling hub for vascular patterning. We are,
currently, using Par3 and PrKCi iEC-KO mouse models to study the importance
of flow-dependent axial polarity and motility in determining stability of vessels
and overall patterning of the vascular network. Next, we will study in detail the
dynamic activation and molecular composition of the Par complex upon flow and
VEGF stimulation using microfluidic devices, to unravel how these two opposing
polarity cues regulate polarisation and, therefore, vascular patterning.
Given the physiological relevance of vascular patterning in health and disease,
our approach will give insights in defining the cellular and molecular principles
involved in vascular patterning.
97
P50
P51
3
Induction And Maintenance Of Atrial Fibrillation
In A Sympathoexcitatory Background As Metabolic
Syndrome
3
Epigenetic drivers of genomic instability and
intratumoral heterogeneity
M.R. Matos1, I. Posa1, A.R. Grosso1, S.F. de Almeida1
1
N. Rosa1 2
Instituto de Fisiologia, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
1
2
[email protected]
Objectives: Evaluate the effects of acute vagal stimulation on atrial conduction, atria
and pulmonary veins (PV) refractoriness and AFib inducibility in a MetS rabbit model.
Methods: MetS was induced in male NZW rabbits, 8 weeks, by a high sucrose diet
given for 6 months, after which, under anaesthesia, a thoracotomy was performed
to expose the heart. An array of 5 microelectrodes was placed in PV vicinity and in
the atrial epicardium to record cardiac electrograms. The right vagus nerve was prepared for electrical stimulation (1ms, 50Hz, ~100µA). ECG electrodes were placed in
3 of the 4 limbs. The epicardial recordings were made in sinus rhythm. Stimulation
bursts (10s, 50Hz) were used, alone or combined with vagal stimulation, in the right
atrial appendage, left atrial appendage and PV to evaluate AFib inducibility. The
effective refractory periods (ERP) and conduction times from the high-lateral right
atrium to the high-lateral left atrium and PV were quantified before and after vagal
stimulation. Heart rate variability using Fast Fourier Transform (FFT) was applied
on autonomic evaluation. A control group matching age and sex was used.
Results: AFib inducibility was greater in MetS-rabbits with a 50Hz pacing (38±7% vs
21±7%) and after vagal stimulation (53±6% vs 33±4%). The evoked AFib duration
was longer in MetS rabbits than in controls and increased significantly after vagal
stimulation. ERPs were lower in MetS rabbits and decreased at all evaluated sites during vagal stimulation. MetS-rabbits had an higher interatrial conduction time than
controls (22±1 vs 11±1ms, p <0.05). FFT analysis confirmed a sympathoexcitatory
condition in MetS comparing to controls (0.40±0.09 vs 0.11±0.06mmHg2, p<0.05).
[email protected]
Genetic alterations and disruption of epigenetic regulatory mechanisms are hallmarks of cancer. Nevertheless, while the genetic contribution for cancer is easily illustrated by mutations in tumor suppressors or oncogenes, the epigenetic
involvement is far more complex and has only recently become a focus of cancer
research. In this study we aimed at investigating the impact of epigenetic deregulation on two important tumour features - genomic instability (GI) and intratumor heterogeneity (ITH) - in a panel of 16 distinct carcinomas represented in The
Cancer Genome Atlas. GI, defined as the number of point mutations and INDELs,
was measured in 2802 tumour/normal paired samples. The ITH, defined as the
genetic heterogeneity of the primary tumour sample, was retrieved from the same
set of samples. Our analysis revealed that mutations in epigenetic modifiers associate significantly with increased GI and ITH in most cancers. A detailed inspection of clear cell renal cell carcinoma (ccRCC), the cancer that presents the highest frequency of mutations in epigenetic modifiers, revealed that the DNA and
histone methyltransferase genes DNMT1, DNMT3A and SETD2 are the stronger
determinants of GI and high ITH. Our study discloses a novel link between epigenetic deregulation, GI and ITH that is prevalent in several cancers. Our on-going
in vivo studies will clarify the molecular details of the involvement of the identified
candidate genes (DNMT1, DNMT3A and SETD2) in these tumour features. Our
findings have the potential to define new molecular targets for therapeutic interventions and to shape existing chemotherapeutic schemes used to treat cancer.
Conclusions: Despite MetS-rabbits have an increased basal sympathetic activity
which favoured AFib induction, a simultaneous increased vagal tonus seems to
be important not only for the inducibility but also for the maintenance of AFib in
this animal model.
99
NOTES
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1649-028 Lisboa
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985 100
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Students Meeting - Faculdade de Medicina da Universidade de Lisboa

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