Abstract book

Transcription

Abstract book

BioMediTech
Research Day 2015
Abstract book
Contents
2-3 Table of contents
28 Matti Annala
4-5 Programme
29 Kirsi J. Granberg
6-7 Exhibition
30 Kirsi J. Granberg
8 Keynote Speakers
31 Tommi Rantapero
- Arto Urtti, U. of Helsinki
32 Kirsi M. Kaukoniemi
- Jukka Westermark, U. of Turku
33 Susanna Teppo
34 Ebrahim Afyounian
Abstracts
35 Suvi Luoto
1 Maria Teresa Calejo
36 Edouard Mobarak
2 Elina Talvitie
37 Hanna Juhola
3 Jennika Karvinen
38 Fabio Lolicato
4 Jenny Parraga
39 Jake Lin
5 Jette-Britt Naams
40 Sampo Kukkurainen
6 Janne Koivisto
41 Marja-Leena Linne
7 Äyush Mishra
42 Magdaléna von Essen
8 Aleksi Hänninen
43 Suresh Palanivel
9 Markus Karjalainen
44 Antti-Juhana Mäki
10 Kati Juuti-Uusitalo
45 Eeva Laurila
11 Juha Määttä
46 Kimmo Kartasalo
12 Anzhelika Karjalainen
47 Mikko Peltokangas
13 Sanna Auer
48 Kalle Lehto
14 Javier Gracia
49 Mira Valkonen
15 Jenni Leppiniemi
50 Dhanesh Rajan
16 Niila Saarinen
51 Zuzet Martinez Cordova
17 Soili Lehtonen
52 Sanna-Kaisa Harjula
18 Joonas Tuominen
53 Mirva Järvelä-Stölting
19 Heini Kallio
54 Leena-Maija Vanha-aho
20 Minna Ampuja
55 Melina Ahonen
21 Elisa Vuorinen
56 Markus Ojanen
22 Defne Us
57 Saara Aittomäki
23 Liisa Sjöblom
58 Mirja Niskanen and Henna Myllymäki
24 Sini Eerola
59 Meri Uusi-Mäkelä
25 Annika Kohvakka
60 Risto-Pekka Pölönen
26 Mauro Scaravilli
61 Antti Ahola
27 Hanna Karvonen
62 Marlitt Viehrig
63 Hannu Välimäki
64 Joose Kreutzer
65 Ville Rantanen
66 Virpi Alarautalahti
67 Anne Skogberg
68 Antti Vehkaoja
69 Cagri Yalgin
70 Sina Saari
71 Katharina Bremer
72 Priit Joers
73 Suvi Vartiainen
74 Jack George
75 Marten Szibor
76 Ana Andjelkovic
77 Tanja Ilmarinen
78 Iina Vainio
79 Paavilainen Tanja
80 Sanni Virjula
81 Miina Ojansivu
82 Heidi Hongisto
83 Tiina Joki
84 Laura Hyväri
85 Musammir Khan
86 Mitra Akbari
87 Mäkinen Meeri
88 Muhammad Waqas Ahmad Khan
89 Teemu Ihalainen
90 Rolle Rahikainen
Programme
Numbers in front of the speakers indicate their abstract numbers found in the abstract book. The speakers do not have a poster at the event.
8.30–8.45
Opening words
8.45–10.00
Scientific Session 1
Chair: Saara Aittomäki, (LS A1)
1. Maria Teresa Calejo: Porous films for the treatment of retinal disease
21. Elisa Vuorinen: Karyopherin alpha 7 (KPNA7) mediated nuclear transport in cancer
37. Hanna Juhola: The Role of Synaptic Cell Membrane in Neurotransmitter Entry into its Receptor
54. Leena-Maija Vanha-aho: Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps
in Drosophila melanogaster
86. Mitra Akbari: Characterization of Flexible Graphene Oxide Film Reduced by Pulsed Xenon Flash
10.00–10.30
Coffee Break
10.30–11.00
Chair: Dr Waldemar Kulig, (LS A1)
26. Mauro Scaravilli: A comprehensive repertoire of tRNA-derived fragments in prostate cancer
67. Anne Skogberg: Nanocellulose surfaces as cell culture substrates
11.00–11.45
Keynote 1: Arto Urtti, University of Helsinki
11.45–13.00
Lunch (at one’s own cost)
13.00–13.45
Keynote 2: Jukka Westermark, University of Turku: “Discovery of CIP2A, the universal human oncoprotein”
13.45–14.15
Chair: Susanna Valanne, (LS A1)
90. Rolle Rahikainen: Destabilized talin-1 mutants for studying cellular mechanosensing
61. Antti Ahola: Analysis tool for human induced pluripotent stem cell derived cardiomyocyte beating dynamics
using video imaging
14.15–15.00
Coffee Break
15.00–16.15
Chair: Soile Nymark, (LS A1)
30. Kirsi J. Granberg: Strong FGFR3 staining is a surrogate marker for FGFR3 fusions and poor prognosis in diffuse
gliomas
3. Jennika Karvinen: Hydrazone crosslinked hyaluronan based hydrogels for neural application
60. Risto-Pekka Pölönen: Orientation analysis of hiPSC derived cardiomyocytes exposed to uniaxial mechanical
stretching using CytoSpectre software
65. Ville Rantanen: Device for Studying Reanimation of Unilateral Facial Paralysis
79. Tanja Paavilainen: Role of astrocytes in formation of functional neuronal networks in vitro
16.15–16.30
Closing words
16.30–18.00
Poster session, researchers available at their posters (“portrait hall”)
BioMediTech Research Day 2015 is organized on Friday, 4th of December at University of Tampere, 08:20–16:50, main building
(LS A1).
During the lunch and coffee breaks there is a possibility to check the corporate and poster exhibition (9:30-15:00).
Registration is needed for the seminar: https://elomake3.uta.fi/lomakkeet/15665/lomake.html by latest 26th November.
Note: Wireless Internet connection available during the event!
BioMediTech Research Day 2014
Networking Session
BioMediTech organizes a parallel session for BMT researchers and group leaders during Friday 4th December between 11:45
-13:00 in UTA main building seminar room A3 with lunch serving. The purpose of the lunch seminar is to inspire, stimulate and
encourage attendees to think out of the box and generate new ideas not forgetting the networking opportunity offered.
Programme
11.45
Opening words,
BioMediTech Institute, Program Director Juho Väisänen D.Sc.
11.50
Revenio Research Oy, R&D Manager Ville-Pekka Seppä D.Sc.
”Turning technology to innovation – Story of Tide Medical”
12.05
Modulight Oy ,CEO Petteri Uusimaa D.Sc.
”Lasers in immunotherapy”
12.20
Protorhino Oy, CTO Pasi Karppinen MSc
“Potentials of Speckle-imaging technology”
12.35
Biomedicum Functional Genomics Unit (FuGU), University of Helsinki, Martyn James PhD
“Genome profiling services at the Biomedicum Functional Genomics Unit, Univ.of Helsinki”.
12.50
Genome Biology Unit (GBU), University of Helsinki, Kaisa Laajanen MSc
“Research services by Genome Biology Unit”
During the lunch and coffee breaks there is a
possibility to check the corporate and poster
exhibition (9:30-15:00).
K
S
e
t
o
n
y
e
s
r
e
peak
11.00–11.45
Arto Urtti
University of Helsinki
13.00–13.45
Jukka Westermark
University of Turku
“Discovery of CIP2A, the
universal
human oncoprotein”
S
B
A
S
T
C
A
R
T
Biomaterials
Author: Maria Teresa Calejo ([email protected])
Research group leader: Minna Kellomäki
Co-author(s): Tanja Ilmarinen, Hatai Jongprasitkul, Heli Skottman, Minna Kellomäki
Affiliation(s): BioMediTech, Tampere University of Technology; University of Tampere
Porous films for the treatment of retinal disease
Keyword(s): Retinal pigment epithelium; honeycomb films; permeability; porosity; protein adsorption; tissue
engineering; pluripotent stem cells
Abstract:
Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised
by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts
with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to
treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold
materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the
conditions during film preparation, it was possible to produce films with homogeneous pore distribution and
adequate pore size (~3-5 µm), i.e. large enough to ensure high permeability but small enough to enable cell
adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous
adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface.
hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in
pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers.
These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue
engineering, importantly enabling the free flow of ions and molecules across the material.
Biomaterials
Author: Elina Talvitie ([email protected])
Co-author(s): Inari Lyyra (1), Darren Whitaker (2), Konrad Mulrennan (2),
Marion McAfee (2) and Minna Kellomäki (1)
Affiliation(s): (1) Department of Electronics and Communications Engineering, Tampere University of Technology,
Tampere, Finland (2) Centre for Precision Engineering, Materials & Manufacturing, Institute of Technology Sligo, Sligo,
Ireland
Real-time monitoring of polymer degradation during extrusion
Keyword(s): degradation, extrusion, real-time monitoring
Abstract:
Bioresorbable polymers are processed into various shapes (e.g. rod, sheet, fibre) using melt processing.
The melt processing of bioresorbable polymers is challenging and there is a risk of polymer degradation
during processing. Conventionally, the quality and properties of the processed products are evaluated in
laboratory after processing. In addition, variations in raw material batches or processing conditions might
result in the inconsistent product properties and hence require additional processing runs. These abovementioned factors will increase the development and production costs of bioresorbable polymer products.
Real-time monitoring of the degradation during the melt processing will help in controlling the production
and thus saves time and decreases the expenses. PLDLA 96/4 multifilament fibres have been produced by
melt spinning and the real-time monitoring of the degradation during fibre production has been studied.
The processing trials have been carried out to induce degradation by increasing the extrusion temperatures
and residence time. The extruder has been equipped with two miniature pressure transducers with
thermocouples, two miniature thermocouples and two fibre optic sensors connected to a near infrared (NIR)
measurement system. The collected sensor data (pressure, temperature, NIR spectra) has been correlated to
the molecular weight, viscosity, and monomer content of the produced fibres which have been characterised
after processing. According to the performed studies, the models built using the processing data can be
utilised in the qualitative detection of the degradation during the extrusion of PLDLA 96/4.
Biomaterials
Author: Jennika Karvinen ([email protected])
Co-author(s): Janne Koivisto, Rami Pääkkönen, Tiina Joki, Teemu Ihalainen,
Teresa Calejo, Susanna Narkilahti, Minna Kellomäki
Affiliation(s): Tampere University of Technology, University of Tampere, BioMediTech
Hydrazone crosslinked hyaluronan based hydrogels for neural application
Keyword(s): Hyaluronan, hydrazone, injectable, hydrogel, neural
Abstract:
Central nervous system deficits resulting from neuroregenerative disease or traumatic injury cause severe
dysfunctions to patients. Transplantation of cells incorporated into supportive biomaterials, comparing
to medical strategies used, offer a better way to cure these problems. Hydrogels are considered the most
relevant biomaterial type for neural application due to their ECM mimicking nature. Also, injectability
enables non-invasiveness. Hyaluronan (HA) is a major glucosaminoglucan component in brain ECM and has
similar properties to brain tissue. In this study, injectable, quickly gelated, transparent hydrazone crosslinked
hyaluronan based hydrogels with variety of properties were produced. Polymers were modified with
complementary reactive aldehyde and hydrazide groups and three types of hydrogels (HA-HA, HA-Gellan
gum and HA-Poly(vinyl alcohol)) were formed by simply mixing the components together. The properties of
hydrogels were changed by altering the degree of substitution and molecular weight of polymers, the polymer
concentration of hydrogel and ratio of hydrogel components. Hydrogels swelled double to hundreds times
the original weight in water and PBS. The mesh size approximation based on the rheological and diffusion
studies showed that specific sized components can be blocked inside the gel. The degradation studies with
hyaluronidase enzyme showed that the gels could be degraded in vivo. Also, brain mimicking mechanical
properties were achieved. The aim of the study was to find a suitable starting material for neural application,
thus the biocompatibility of the hydrogels using neural cells was confirmed. The chemical structure of these
hydrogels enables further functionalization with proteins, peptides etc. to enhance the cell adhesion ability.
Biomaterials
Author: Jenny Parraga (a,b) ([email protected])
Research group leader: Minna Kellomaki
Co-author(s): Janne Koivisto (a,b,c), Jennika Karvinen (a,b), Minna Kellomäki (a,b)
Affiliation(s): a. Biomaterials & Tissue Engineering group, Department of Electronics and Communications Engineering,
Tampere University of Technology b. BioMediTech, Tampere, Finland c. Heart group, BioMediTech, University of
Tampere
Interpenetrating network hydrogels based on gelatin and gellan gum for tissue engineering
Keyword(s): gellan gum, gelatin, interpenetrating polymer network, hydrogel
Abstract:
Hydrogels produced by semi- or interpenetrating polymer networks (IPN) technology are cross-linked
polymeric networks with a hydrophilic polymer chain or network in their structure. The formation of IPN
can improve the biocompatibility, biodegradability and stability of the materials ensuring the mechanical
strength. In this work, we are preparing and characterizing IPN hydrogels based on gellan gum and gelatin
using the bioamine spermidine as a cross-linker. Gelatin has gained much attention for its biodegradability,
excellent functional and filmogenic properties. However, gelatin is weak in mechanical strength which limits
its applications. On the other hand, gellan gum is a biopolymer with excellent mechanical properties recently
used for several applications in the tissue engineering field. Some modifications were performed in these
biomolecules in order to increase the interaction of gelatin molecule into the network during the hydrogel
formation. Thus, vicinal diols in the gellan gum molecule where oxidized and gelatin was modified by amidation
allowing the generation of Schiff bases. In order to establish the biological impact of the increase of positive
charge in the gelatin, we evaluated the cytocompatibility in WI38 human fibroblasts. After demonstrating the
cytocompatibility of the aminated gelatin as a coating, IPN hydrogels made of ionically crosslinked network
are produced to investigate parameters that control its mechanical properties. The resulting IPN hydrogel
exhibited appropriated mechanical properties and support the attachment of WI38 fibroblast in vitro. The
preliminary results indicate that this hydrogel has promising potential and it is expected to function as cell
support in the field of tissue engineering.
Biomaterials
Author: Jette-Britt Naams (1,2) ([email protected])
Co-author(s): Janne Koivisto (1,2,3), Minna Kellomäki (1,2)
Affiliation(s): 1. Biomaterials & Tissue Engineering group, Department of Electronics and Communications Engineering,
Tampere University of Technology 2. BioMediTech, Tampere, Finland 3. Heart group, BioMediTech, University of
Tampere
A rapid cell culture protocol for screening material cytocompatibility
Keyword(s): hydrogel, 3D cell culture, cytocompatibility, BioStation CT
Abstract:
Hydrogels differ from many other cell culture substrates in that they provide a three-dimensional, soft
environment to cells, opposed to a two-dimensional surface on which cells are traditionally cultured. This
geometry better represents the in vivo environment of cells. In addition, hydrogels are easily functionalized
for enhanced cell response. Therefore, new hydrogels are continuously developed for cell culture and tissue
engineering purposes. Applicability of a hydrogel as cell culture substrate depends on several chemical and
physical attributes. Together they affect cytocompatibility of the hydrogel, i.e. cell attachment, survival
and cell specific response. The objective of this thesis was to create a protocol for first-step, short-time cell
culture screening of new hydrogels. Compared to 2D culturing systems, cytocompatibility testing methods
need to be adjusted for macroscopic 3D hydrogels. Another challenge in this screening is adequate stability
and representativeness of cells. An interview was conducted with Regenerative Medicine professionals in
BioMediTech working with cardiomyocytes; bone and cartilage cells; corneal and retinal epithelial cells;
and nerve cells. Knowledge was gathered on the types and properties of representable cell models of each
cell type and on tissue-specific aspects of analyzing. A commercial human embryonic fibroblast cell line
WI-38 was chosen and acquired for the initial cytotoxicity testing. WI-38 cells were cultured on top and
encapsulated in hydrogels. The suitability of Nikon BioStation CT for the screening was evaluated. Although
this image based analysis method was not as high-throughtput as desired, it was possible to estimate cell
attachment on hydrogels and cell survival in hydrogels.
Biomaterials
Author: Janne Koivisto ([email protected])
Co-author(s): Janne Koivisto(1,2), Nick Walters(3), Jenny Parraga(1), Minna Kellomäki(1)
Affiliation(s): 1 Biomaterials & Tissue Engineering Group, Electronics and Communications Engineering Dept.,
BioMediTech,Tampere University of Technology; 2 Heart Group, BioMediTech, University of Tampere; 3 Adult Stem
Cell Group, BioMediTech, University of Tampere
Mechanical Characterisation of Hydrogels in Macro- and Micro Scale
Keyword(s): Hydrogel, Compression testing, Atomic force microscopy, 3D Cell Culture
Abstract:
In addition to soluble cues, such as growth factors, stem cell differentiation is influenced by mechanical
interactions with the extracellular matrix via integrin-mediated mechanotransduction. Traditionally, cell
culture is performed on very stiff, two-dimensional substrates which induce a very spread morphology,
non-representative of most cell types in their native tissue. This artificial environment can alter numerous
cellular behaviours, including adhesion, proliferation, migration, gene expression, electrophysiology and
differentiation. Three-dimensional culture platforms which more closely mimic the niche of specific cell
types would provide cells with a more suitable microenvironment and be used for disease modelling,
developmental biology and tissue engineering applications. Hydrogels with precisely defined, biomimicking
mechanical properties and which enable encapsulation of cells in 3D are therefore under development.
Various techniques are employed to determine mechanical properties of hydrogels. Compression testing
determines the elastic modulus under static or dynamic conditions and measures fracture strength and
deformation at break. This technique, however, requires that the specimen is freestanding and has a
distinct macroscopic shape, making it unsuitable for weak gels or costly nanogels. By contrast, rheological
analysis determines the shear properties under dynamic conditions. These bulk measurements, however,
are unable to determine localised variations, such as gradients in strength and porosity. Atomic force
microscopy can therefore be employed to measure the localised elastic modulus of hydrogels on a micron
scale. This should give a more representative estimation of how the cells actually perceive the mechanics of
their microenvironment. Here we review these complementary hydrogel characterisation techniques and
discuss their advantages and disadvantages.
Biomaterials
Author: Äyush Mishra ([email protected])
Co-author(s): 1) Jean Rocherullé, 2) Jonathan Massera
Affiliation(s): 1)Equipe Verres et Céramiques, UMR-CNRS 6226, Sciences Chimiques de Rennes, Université de Rennes
I, F-35042 Rennes Cedex, France 2)Electronics and Communications Engineering, Tampere University of Technology,
Korkeakoulunkatu 10, FI-33720 Tampere, Fi
Characterization of Ag, Cu and Fe doped phosphate bioactive glasses
Keyword(s): glass, phosphate, bioactive, immersion, structural and thermal properties
Abstract:
Phosphate based glasses within the composition 50P2O5-20CaO-20SrO-10Na2O have been found to react in
biological media and form a dicalcium phosphate di-hydrate (DCPD) layer at their surface upon immersion.
However, the high phosphate content is responsible for the fast initial dissolution rate. As a consequence,
human gingival fibroblasts cultured at their surface were found to have difficulties in attaching and
proliferating during the first 3 days of culture. For longer immersion time the cells were found to proliferate
at a rate similar to the typical silicate bioactive glass S53P4. In order to reduce the dissolution rate and
confer additional properties to the glass, the base glass was doped with Ag, Cu or Fe. The newly obtained
glasses were characterized in light of their thermal, structural and dissolution properties. DTA was used to
measure the glass transition, onset of crystallization and crystallization peak temperatures, in order to assess
if these glasses have potential for hot forming (scaffold sintering, fiber drawing). The in vitro bioactivity
was studied by immersing the glass powders (particle size 125 - 200 mm) in TRIS buffer solution for various
immersion time. Changes in glass structure, liquid chemical composition and the formation of a Ca-P layer
were investigated using FTIR, ion chromatography and SEM-EDX, respectively.
Biomaterials
Author: Aleksi Hänninen ([email protected])
Co-author(s): Minna Kellomäki, Susanna Miettinen, Kaarlo Paakinaho
Affiliation(s):
Seeding porous polymer-based scaffolds with human adipose-derived stem cells
Keyword(s): Cell seeding, adipose-derived stem cells, biomaterials, tissue engineering
Abstract:
One common strategy in tissue engineering is to seed porous biodegradable scaffolds with cells and culture
the constructs in vitro, after which they are implanted into the defect site. In order to enable homogenous
tissue formation inside the scaffold, cell distribution should be uniform throughout the scaffold. Along with cell
distribution, the seeding results should be reviewed in terms of efficiency and cell viability. Other variables to
consider include the amount of time consumed and equipment needed. In this study, 6 different cell seeding
methods were compared using human adipose-derived stem cells (hASC) and 4 different types of scaffolds.
In addition, a cell seeding model based on iron-labeled microspheres detected from inside the scaffold by
micro-computed tomography was proposed. The microsphere model proved to provide useful information
on seedability of supercritical carbon dioxide (scCO2) processed scaffolds made from either poly(L-lactideco-µ-caprolactone) 70/30 (PLCL) or a composite of PLCL and beta-tricalcium phosphate ceramic. In the actual
hASC seeding experiments also knitted and non-woven scaffolds, both made from poly-L/D-lactide 96L/4D
were studied. Due to their high porosity, both of them were easily seeded using a basic static seeding method,
where the cells are pipetted on top of the scaffold. In the case of scCO2 processed scaffolds, the uniformity of
the cell distribution was enhanced by forcing the cell suspension into the scaffold with a syringe.
Biomaterials
Author: Markus Karjalainen ([email protected])
Research group leader: Professor Jukka Lekkala
Co-author(s): Osmo Anttalainen, Joonas Melin, Niku Oksala, Jukka Lekkala
Affiliation(s): 1 Department of Automation Science & Engineering, Tampere University of Technology, Tampere, Finland,
[email protected], [email protected], [email protected] 2 Environics Oy, [email protected]
3 Department of Surgery, Medi
PRINTABLE THERMOPLASTIC COMPONENTS COMPATIBILITY WITH DIFFERENTIAL MOBILITY
SPECTROMETER
Keyword(s): plastic DMS instrumentation permeation adsorption desorption
Abstract:
Ion mobility spectrometry (IMS) is a method used to measure and detect volatile chemical compounds.
Compared with mass spectrometry (MS), it is simpler and robust. It is also possible to use the method in
ambient air. The method is mainly developed for detecting warfare agents, explosives and drugs. Later
more developed method has been introduced a Differential Mobility Spectrometry (DMS). For additional
measurement dimension, it simulates the temperature with a strong radiofrequency field. In recent years, the
IMS and DMS technology has spread the medical field for disease diagnostics. Diseases such as clostridium
difficile [1], prostate cancer [2] and lung cancer [3] are detected with good results. Ion mobility is affected
on factors such as humidity and temperature. When method is used on new applications fast prototyping
helps resolving hidden challenges. We tested the compatibility of some common 3D-printed thermoplastics
with DMS. [1] A. Roine, E. Veskimäe, A. Tuokko, P. Kumpulainen, J. Koskimäki, et al. (2014) Detection of
prostate cancer by an electronic nose: a proof-of-principle study. J Urol. doi: 10.1016/j.juro.2014.01.1 [2]
Rapid, Accurate, and Onsite Detection of C. difficile in Stool Samples, M. Bomers, F. Menke, R. Savage, C.
VandenbrouckeGrauls, M. van Agtmael, J.Covington, Y. Smulders, Am J Gastroenterol. 2015~110(4):588594
[3] H. Handa, A. Usuba, S. Maddula, J.I. Baumbach, M. Mineshita, T. Miyazawa, Exhaled Breath Analysis for
Lung Cancer Detection Using Ion Mobility Spectrometry , PLoS One. 2014; 9(12): e114555. Published online
2014 Dec 9
Biotechnology
Author: Kati Juuti-Uusitalo ([email protected])
Research group leader: Skottman
Co-author(s): Muranen J, Lähdekorpi H, Pajula E, Uusitalo H, Kaarniranta K, Skottman H
Affiliation(s): BioMediTech, University of Tampere; Department of Ophthalmology, University of Tampere; TAYS Eye
Center, Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Department of
Ophth, Kuopio University Hospital
An in vitro model of outer blood-retinal barrier based on human embryonic stem cell
derived retinal pigment epithelial cells and human retinal microvascular endothelial cells
Keyword(s): retinal pigment epithelial cell, hESC-RPE, oBRB, in vitro model,
Abstract:
The objective was to create an in vitro outer blood-retinal barrier (oBRB) cell co-culture model using
highly pigmented and mature human embryonic stem cell (hESC) -RPEs and human retinal microvascular
endothelial cells (hREC). The long-term co-culture effects on the barrier properties, morphology and function
of hESC-RPE cells was further assessed. The hESC- RPE cells (Regea08/017, Regea08/023 or Regea11/013)
were seeded on other side of perforated polyester filter inserts than primary human retinal microvascular
endothelial cells (ACBRI181), and thereafter co-cultured together up to 6 weeks. The barrier function was
examined by measuring the trans-epithelial resistance (TER) using a voltohmmeter and the permeability
of a 4 kDa fluorescein using the Ussing chamber system. The expression and the localisation of RPE and
endothelial marker proteins were analysed with the confocal microscopy, and the fine structure with
transmission electron microscopy (TEM). The effects of the co-culture on growth factor secretion of the
vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were analysed
with ELISA. The TER assessment revealed that co-culture increased the barrier function in hESC-RPE cells.
The co-culture had only a modest effect on the cumulative permeability of FD4 in hESC-RPE. In confocal
microscopy we saw that RPE specific CRALBP and endothelial cell specific vWF proteins had cell type specific
localisation. TEM revealed a cell line specific effect on the extracellular matrix deposition. The effect of coculturing on VEGF and PEDF secretion was also cell line specific. VEGF secretion in RPE cells differentiated
from Regea08/023 cell line but increased in Regea08/017 and Regea11/013 cell lines. The secretion of PEDF
was increased after co-culture in RPE cells differentiated from Regea 08/017 and Regea 08/023 but not in
Regea 11/013 cell line. The long-term co-culture of hESC-RPE cells with hRECs has effects on the matrix
deposition, barrier function and the functionality of hESC-RPE cells. Furthermore the data suggests - hESCRPE line specific differences in growth factor secretion.
Biotechnology
Author: Juha Määttä ([email protected])
Research group leader: Vesa Hytönen
Co-author(s): Niklas Kähkönen
Affiliation(s): BioMediTech, University of Tampere, Biokatu 6, 33520, Tampere, Finland
Protein Technologies
Keyword(s): Protein expression, protein purification, protein analysis
Abstract:
Protein Technologies core offers recombinant protein expression, protein purification and biophysical
characterization for life science research. The proteins may be used in structural biology, protein-ligand
and protein-protein interaction studies, and in development of diagnostics. In vitro, bacterial and insect
cell expression systems are situated in BioMediTech (BMT, Tampere) and the mammalian expression system
is operated by Haartman Institute (Helsinki). The facility offers hands-on counseling concerning protein
expression methods and expression vectors, but customer is typically responsible for the preparation of the
DNA plasmid. Typical workflow includes pilot scale protein production and purification, scale-up to liter scale,
protein isolation and purification and finally, protein characterization by various biochemical and biophysical
methods including interaction assays.
Biotechnology
Author: Anzhelika Karjalainen ([email protected])
Research group leader: Meenakshisundaram Kandhavelu
Co-author(s): Phuong DOAN, Ossi SANDBERG, Yli-Harja O, Nuno Rafael CANDEIAS and
Kandhavelu Meenakshisundaram
Affiliation(s): Molecular Signaling Lab, Computational Systems Biology Research Group, Department of Signal Processing,
Tampere University of Technology. Institute for Systems Biology, WA. BioMediTech, Biokatu 10 33520 Tampere
NOVEL SMALL MOLECULES ARE FOUND TO EXHIBIT CYTOTOXIC EFFECT ON OSTEOSARCOMA
CELLS
Keyword(s): Osteosarcoma, cancer, apoptosis, phenols, indolines, morpholines,
Abstract:
Cancer is a devastating disease that causes millions of deaths every year. The diversity of cancer incidences
and the rapid adaptation of tumour cells to provided treatment compels the field of drug development to
continuously seek for novel anticancer agents. In this work we have investigated the conjectural cytotoxic
effect of newly synthesized derivatives of phenols, indolines and morpholines to osteosarcoma cells. By
performing cytotoxicity and migration assays, we found that two compounds efficiently induced cell death
in a dose- and time-dependent manner. Based on the obtained IC50 values we further evaluated the cell
death mechanism triggered by the two small molecules. Having analysed the DNA content morphology and
the expression of specific apoptotic marker protein we concluded that both compounds induce apoptosis.
Furthermore, from the measurement of mitochondrial calcium concentration changes at the single cell level,
we hypothesised that mitochondrial calcium independent apoptotic pathway was triggered by the studied
molecules. Our study has proved that two novel small molecules have a potential to be developed into new
chemotherapeutical agents.
Biotechnology
Author: Sanna Auer ([email protected])
Co-author(s): Vesa Hytönen
Affiliation(s): BioMediTech, University of Tampere
Protein immobilisation for improved biofunctionality
Keyword(s): Biotin, avidin, protein immobilisation, Histidine-tags, protein engineering
Abstract:
Proteins need to be immobilized in oriented manner for example in diagnostic and biosensing applications.
Proteins can be immobilized via their amino-groups, but this leads to random orientation and might result
in loss of biological acivity or other functionality. Controlled and oriented immobilisation can be achieved
by embedding genetically engineered immobilisation sites, such as histidine-tags or biotinylation sites, into
the protein structure. Biotinylated proteins can then be further bound via avidin- proteins and respectively
His-tagged proteins can be attached via chelated metal ions (Ni2+ or Co2+). The third option for site-specific
protein immobilization are surface cysteines. Free thiol-groups can directly react with metal surfaces, such
as gold or silver, or they can be used for covalent attachment. Examples of all these immobilization types are
presented related to our efforts to immobilize proteins on biosensors, nanoparticles and carbon nanotubes.
Biotechnology
Author: Javier Gracia ([email protected])
Research group leader: Jari Viik
Co-author(s): Ville-Pekkä Seppä
Affiliation(s): Tampere University of Technology
Multilead impedance pneumography and forced oscillation technique for assessing lung
tissue mechanical properties
Keyword(s): Transfer impedance, lung mechanics, impedance pneumography, multilead impedance pneumography,
forced oscillation technique.
Abstract:
Forced oscillation technique measuring transfer impedance (Ztr) is a traditional method to assess mechanical
properties of the respiratory system. Lung tissue and airways mechanical properties are of clinical relevance, but
difcult to distinguish from the Ztr measurand. We propose a novel method that replaces the plestymographyc
chamber in Ztr by a multilead impedance pneumography (MLIP) system. We hypothesise that mechanical
properties of the lung tissue should be more evident in the new method that in Ztr. We applied pressure
oscillations at the mouth from 2 to 27 Hz in one sitting healthy subject holding breath at residual volume.
Thoracic electrical resistivity changes where simultaneously recorded at three different heights of the thorax
by a MLIP system. Lung volume changes were extract from the MLIP signals by subtracting the cardiac
component calculated by means of ensemble averaging. Consequently, mechanical impedances at the three
thorax levels were calculated as the complex ratio of mouth pressure (Pao) and the time integral of the lung
volume MLIP signals (QIP ): ZIP = Pao/QIP. We found a decrease in the real part of ZIP from the upper to the
lower areas of the chest. This is coherence with the known pleural pressure gradient distribution. Moreover,
we observed ZIP to share similarities with Ztr at low frequencies, (i.e. same resonance frequency). However,
ZIP for the upper and lower regions of the thorax differed greatly with Ztr for frequencies over 10 Hz. Results
need deeper investigation and larger clinical study samples. Nevertheless, this preliminary study shows the
potential of ZIP for assessing lung tissue mechanical properties.
Biotechnology
Author: Jenni Leppiniemi ([email protected])
Co-author(s): Riitta Mahlberg2, Panu Lahtinen2, Vesa P. Hytönen1 and Inger Vikholm-Lundin1
Affiliation(s): 1. BioMediTech, University of Tampere, Biokatu 6, Tampere, Finland; 2. VTT Technical Research Centre of
Finland, VTT P.O. Box 1000, FI-02044 Espoo, Finland
IMMOBILIZATION OF BIOMOLECULES ONTO NANOCELLULOSE
Keyword(s): biofunctionalization, biomolecule, avidin-biotin technology, nanocellulose, surface plasmon resonance
Abstract:
We studied the biofunctionalization methods of native nanocellulose and TEMPO-oxidized nanocellulose
with biomolecules such as avidin, which allows further functionalization with biotinylated-molecules. Thin
layers of native nanocellulose were spin coated on gold-coated glass slides. The layers were activated with
a bifunctional crosslinker molecule and chimeric avidin with surface-exposed cysteine groups (ChiAvd-Cys)
was covalently coupled to the activated nanocellulose layer. The formed avidin layer was used to capture
biotinylated antibody Fab -fragments towards C-reactive protein (CRP) and the binding was determined with
surface plasmon resonance (SPR). We demonstrated that nanocellulose functionalized with avidin and a
biotinylated-anti-CRP antibody could be used to detect CRP. We also used dot blot analysis on filter paper to
study the interaction of chimeric avidin with TEMPO-oxidized nanocellulose. The functionalized nanocellulose
may find use in the development of printable immunostrips on paper or as scaffold material for cell culturing
or wound dressings.
Biotechnology
Author: Niila Saarinen ([email protected])
Co-author(s): Olli Laitinen, Heikki Hyöty, Vesa Hytönen
Affiliation(s): BioMediTech and Medical School, University of Tampere
Development of diagnostics tools for enteroviruses Immunology
Keyword(s): Enterovirus, Diagnostics, Diabetes, Recombinant protein
Abstract:
Enteroviruses cause number of life-threatening diseases such as dilated cardiomyopathy. In addition, certain
enterovirus species, namely Coxsackievirus group B (CBV) viruses, are common in children developing
diabetes and they have been shown to infect pancreatic islet beta-cells in vitro and in vivo. Therefore it
would be important to recognize disease-associated enteroviruses and to study the molecular mechanisms
of their pathogenesis. However, there are only a limited amount of reagents available for the detection of
enteroviruses and especially the reagents capable of differentiating between different enterovirus serotypes
are missing. Thus we aim to develop improved antibody based tools for the specific detection of enteroviruses
from clinical samples. In order to develop sensitive and specific antibodies it is important to characterize
enteroviral antigens. The screening will be done using recombinant viral coat proteins as antigens and patient
sera will be screened for antibodies against them. So far we have produced four recombinant CBV3 VP1
capsid protein forms to test, which part of the VP1 are recognized by the CBV infected patient sera. It was
shown that a known serotype cross-reactive epitope in the N-terminus of the VP1 is the immunodominant
part of the protein, but antibodies recognizing other parts of the protein are also present in tested patient
sera. The other VP1 regions could be more species and serotype specific allowing more accurate and specific
diagnosis. In order to test this hypothesis we will produce recombinant VP1-proteins for several different
enteroviruses and produce monoclonal antibodies against them.
Biotechnology
Author: Soili Lehtonen (1) ([email protected])
Research group leader: Markku Kulomaa
Co-author(s): Tiina A. Riihimäki (1,2), Kristen Kurtzeborn (1,3), Jenni Leppiniemi (1), Magdalena von Essen (1), Markku
S. Kulomaa (1) and Vesa P. Hytönen (1)
Affiliation(s): 1) BioMediTech, University of Tampere, 2) VTT Expert Services Ltd., 3) Technical University of Tampere
Artificial multispecific protein enables spacial orientation and signal amplification
Keyword(s): Single chain avidin, multi-specificity, small molecule recognition, alternative scaffold
Abstract:
Avidin-biotin technology has been utilized for decades for detection, labeling, and drug delivery purposes. In
recent years, the technology has spanned other areas of the life sciences including cell biology, proteomics,
and catalysis. Multispecific proteins in nature often target receptors on the same cell surface, but they can
also be engineered and used to bring two different molecules (cells, enzymes, therapeutic moieties) into
close proximity. Bispecific proteins have been engineered recently to bring gene- or drug-carrying viruses
or nanoparticles into contact with diseased cells. Modifications to (strept)avidin are limited because they
are both homotetrameric proteins and are coded by single genes. Any modifications to the genes therefore
results in the modification of all four subunits of the mutant protein. To overcome this, different topological
modifications of avidin have been engineered, including single and dual chain avidins, which enable the
creation of avidins with one to four different active binding sites per tetramer. We have now generated new,
avidin structure-based protein tools with multiple specificities in a single protein molecule. These protein
tools enable spacial orientation and signal amplification to be used in diagnostic and biomedical applications.
Cancer
Author: Joonas Tuominen ([email protected])
Research group leader: Matti Nykter
Co-author(s): Ebrahim Afyounian, Sergei Häyrynen, Kristiina Nordfors,
Joonas Haapasalo, Hannu Haapasalo, Kirsi Granberg, Matti Nykter
Affiliation(s): BioMediTech, Tampere University Hospital, Tampere University of Technology, University of Tampere
Exome sequencing of choroid plexus papillomas reveals various copy number alterations
Keyword(s): brain cancer, next-generation sequencing, copy number alterations
Abstract:
Choroid plexus tumors are rare neoplasms which effect both adults and children. Choroid plexus tumors
are usually divided to papillomas, atypical papillomas, and carcinomas. The detailed information about
molecular evolution in choroid plexus tumors is still limited. There are some common gains in choroid plexus
tumors, including chromosome 14q21-q22 (harboring OTX2), chromosome 7q22 (LAMB1), and chromosome
9q21.12. In addition, choroid plexus papillomas harvest many known copy number alterations such as +5q,
+6q, +7q, +9q, +15q, +18q, and 21q. We exome sequenced five choroid plexus papillomas three of which
had also blood samples available, and identified nonsynonymous mutations and copy number alterations
from the resulting data. Previous reports have not identified many recurrent mutations, besides TP53
mutations, in the choroid plexus tumors and overall mutations rate has been low. Our exome sequencing
showed similar results. We had 2 cases carrying TP53 mutations in our cohort and, after applying strict
exclusion criteria, we had altogether 8 genes mutated within samples with blood control. Out of these 8
genes SLC12A5 is known to harbor mutations in colon cancer. We also found typical copy number gains
such as +5q, +7q, +9q, 15q and +18q. We also detected novel gains of chromosomes 12 and 17. Our results
suggest that choroid plexus papillomas are driven rather by copy number alterations than by mutations in
specific genes. This is case at least in the choroid plexus papillomas. More research is needed to find the
driver copy number alterations among the broken landscape of chromosomes.
Cancer
Author: Heini Kallio (1) ([email protected])
Research group leader: Tapio Visakorpi
Co-author(s): Matti Annala(1), Anniina Brofeldt(1), Reija Hieta(1), Kati Kivinummi(1), T
euvo Tammela(2), Matti Nykter(1), Hans G. Lilja(1), G. Steven Bova(1), and Tapio Visakorpi(1)
Affiliation(s): (1)Prostate Cancer Research Center, BioMediTech, University of Tampere and Fimlab Laboratories,
Tampere University Hospital, Tampere (2) Prostate Cancer Research Center, Department of Urology, Tampere University
Hospital and School of Medicine
Next generation sequencing panels to predict response to hormonal therapy in prostate
cancer
Keyword(s): Prostate cancer, androgen, biomarker, NGS, personalized treatments
Abstract:
Prostate cancer (PC) is the most common malignancy and third most common cause of cancer-related death
among men in Europe. Although most PCs grow slowly, 2025% of the patients believed to have organ-confined
disease will experience biochemical recurrence already during 5-years of follow-up. The standard treatment
against advanced PC is androgen deprivation. Unfortunately, androgen deprivation treatment eventually fails
leading to the emergence of castration resistant PC (CRPC) that is lethal. However, also CRPCs are dependent
on androgens. Owing to this understanding, several drugs have recently emerged for the treatment of CRPC
including enzalutamide and abiraterone, but approximately 2040% of patients have no response to these
agents. One explanation to this could be the expression of constitutively active androgen receptor splice
variants (AR-Vs). The aim of our project is to improve diagnostics of prostate cancer by developing NGS-based
biomarker panels and thereby help to tailor personalized treatments for the needs of each patient. The first
two panels have been set up using Agilent s SureSelect Target Enrichment system and they allow detection of
all AR-Vs as well as AR mutations, copy number variations and rearrangements. In addition, other interesting
genes that have shown significance in PC are included in the assays. In the next phase the most convenient
and accurate method for obtaining liquid biopsies from PC patients will be developed. The final aim is to
utilize liquid biopsies as material for NGS-based biomarker panels. In future, these panels could be used to
monitor patient s response to treatment in real-time.
Cancer
Author: Minna Ampuja ([email protected])
Research group leader: Anne Kallioniemi
Co-author(s): Alarmo EL, Owens P, Havunen R, Gorska AE, Moses HL, Kallioniemi A
Affiliation(s): University of Tampere, BioMediTech, Tampere, Finland; Department of Cancer Biology, Vanderbilt-Ingram
Cancer Center, Vanderbilt University, Nashville, TN, United States of America.
The effect of BMP4 on breast cancer metastasis in a mouse model
Keyword(s): Breast cancer, BMP4, in vivo, metastasis
Abstract:
Breast cancer is the most common cancer in women worldwide. Bone morphogenetic protein 4 (BMP4) is
a developmental regulator of cell proliferation, differentiation and migration. In breast cancer cells, BMP4
has been shown to reduce proliferation both in vitro and in vivo. Interestingly, in vitro certain cell types also
respond to BMP4 with induced migration and invasion. Thus we set out to investigate whether BMP4 has
an effect on metastasis using a xenograft mouse model. Luciferase-expressing MDA-MB-231 breast cancer
cells were injected to mouse heart and BMP4 or control was administered through tail vein three times
a week. Metastasis formation was followed with bioluminescence imaging. After seven weeks, the mice
were sacrificed and metastases collected. Mice that were treated with BMP4 developed metastases slightly
earlier, but the difference was not statistically significant. The number of overall metastases was similar (13
in BMP4 group vs. 12 in control), most occurring in bone and adrenal glands. In BMP4-treated mice bone
metastases were more common (10 vs. 7), but adrenal gland metastases less frequent (1 vs. 5) as compared
to control group. Immunostaining showed that there was no difference in signaling activation (pSMAD1/5/9),
proliferation (Ki67), blood vessels (MECA32), EMT (vimentin) or cancer-associated fibroblasts (±-SMA),
between the groups. Interestingly osteoclast marker (Tartrate-resistant acid phosphatase, TRAP) staining
was found in the cancer cells in both groups. In conclusion, there may be a small difference in metastasis
formation due to BMP4 but more work is needed to uncover the extent of the effect.
Cancer
Author: Elisa Vuorinen ([email protected])
Research group leader: Anne Kallioniemi
Co-author(s): Nina Rajala, Hanna Rauhala, Anne Kallioniemi
Affiliation(s): BioMediTech, University of Tampere, Finland
Karyopherin alpha 7 (KPNA7) mediated nuclear transport in cancer
Keyword(s): KPNA7, cancer, nuclear import, protein pull-down, nuclear morphology
Abstract:
Eukaryotic cells are divided into two compartments by the nuclear envelope, thus requiring a specific
machinery for the bidirectional transport of macromolecules. Its malfunction results in incorrect localization
of proteins, ultimately leading to variety of diseases including cancer. Karyopherin alpha 7 (KPNA7) is a
member of the karyopherin alpha family of nuclear importers. We previously showed that KPNA7-silencing
in pancreatic cancer cell lines with high endogenous expression dramatically reduced cell proliferation,
through a G1 cell cycle arrest and p21 induction. Here, we further explored the functional importance of
KPNA7 in a large panel of pancreatic and breast cancer cell lines and identified its cargo proteins that may
mediate the observed phenotype. KPNA7 was silenced in six pancreatic and breast cancer cell lines with low
to medium expression levels using siRNAs. Cell numbers were determined 72 to 96h after transfection. To
isolate KPNA7 cargos, stable KPNA7-overexpressing cell lines were established. An affinity-based protein pulldown was performed and KPNA7-interacting partners identified with mass spectrometry. KPNA7-silencing
led to decreased proliferation in all cell lines studied, implicating a role not restricted to pancreatic cancer.
In some cell lines, a distinct change from round to lobular nuclear morphology was observed, suggesting a
role for KPNA7 in the maintenance of nuclear architecture. The protein pull-down yielded multiple proteins
co-purifying with KPNA7. Many of these were involved in the regulation of cell cycle and the p21 pathway,
consistent with the phenotype observed in the functional studies. These results emphasize the importance
of KPNA7 in cancer pathogenesis.
Cancer
Author: Defne Us ([email protected])
Research group leader: Ulla Ruotsalainen
Co-author(s): Salla Ylipää,Ulla Ruotsalainen
Affiliation(s): Department of Signal Processing, Tampere University of Technology
Optimization of an axial PEM design
Keyword(s): axial PET, Monte Carlo simulations, breast cancer, PET reconstruction, positron emission mammography
Abstract:
As the breast cancer cases are increasing all over the world, positron emission mammography (PEM) offers
a high sensitivity imaging alternative for small and early stage lesions due to its proximity to the breast.
Although many PEM scanners are under ongoing research in universities, none of them except for one is
currently available on the market. This indicates the need for optimization of PEM structures. In this study,
we aimed to achieve a new design for an axial PEM scanner using Monte Carlo simulations. AvanTomography,
developed initially in CERN, is an alternative PEM scanner design that uses an axial geometry instead of radial
configuration used by conventional radial PET geometry. Axial PET configuration makes it possible to calculate
the 3 dimensional depth-of-interaction accurately, free of parallax error. The focus was put on optimizing the
parameters that determine the scanner geometry such as crystal and plastic scintillator lengths and number of
crystal layers. According to the results of the simulations, a 20x20 cm2 was found to be the optimal PEM plate
size. This geometry was then used in phantom simulations in order to evaluate the scanner characteristics
such as spatial resolution and sensitivity. Image reconstruction was carried out with iterative reconstruction
methods using the data collected from the simulations. The initial phantom simulation results showed that
axial PET geometry can achieve a 2.1 mm and a 4% sensitivity at the center of the field-of-view.
Cancer
Author: Liisa Sjöblom ([email protected])
Co-author(s): Outi Saramäki (1,2), Matti Annala (1), Katri Leinonen (1,2),
Janika Nättinen (1), Teemu Tolonen (3), Tiina Wahlfors (1), Matti Nykter (1),
G. Steven Bova (1), Johanna Schleutker (1), Teuvo LJ Tammela (4), Hans Lilja (1,5,6,7)
Affiliation(s): 1Prostate Cancer Research Center, Institute of Biosciences and Medical Technology (BioMediTech),
University of Tampere, 2Fimlab Laboratories, Tampere University Hospital, 3Department of Pathology, Fimlab
Laboratories, Tampere University Hospital, and 4Prostate Cancer Research Center and Department of Urology,
University of Tampere and Tampere University Hospital, Tampere, Finland, 5Departments of Laboratory Medicine,
Surgery, and Medicine, Memorial Sloan Kettering Cancer Center, New York, USA, 6Nuffield Department of Surgical
Sciences, University of Oxford, Oxford, UK, and 7Department of Translational Medicine, Lund University, Malmö,
Sweden.
Microseminoprotein-beta expression in different stages of prostate cancer
Keyword(s): MSMB, MSP, prostate cancer, biomarker
Abstract:
Microseminoprotein-beta (MSP, MSMB) is an abundant secretory protein contributed by the prostate, and
is implicated as a prostate cancer (PC) biomarker based on observations of its lower expression in cancer
compared with benign prostate epithelium. However, as the current literature on MSP is inconsistent,
we assessed the expression of MSP at the protein and mRNA levels in a comprehensive set of different
clinical stages of PC. Immunohistochemistry was used to study MSP protein expression in tissue specimens
representing prostatectomies (n=261) and in diagnostic needle biopsies from patients treated with androgen
deprivation therapy (ADT) (n=100), and in locally recurrent castration-resistant PC (CRPC) (n=105) and CRPC
metastases (n=113). The transcript levels of MSMB, nuclear receptor co-activator 4 (NCOA4) and read-through
MSMB-NCOA4 fusion were examined by qRT-PCR in prostatectomy samples and by RNA-sequencing in benign
prostatic hyperplasia, PC, and CRPC samples. We also measured serum MSP levels and genotyped the single
nucleotide polymorphism rs10993994 using DNA from the blood of 369 PC patients and 903 controls. MSP
expression in PC was more frequent than in CRPC (p<0.0001). The MSMB-NCOA4 transcript was detected at
low levels in PC. Adjusted MSP levels in serum were significantly associated with PC risk (p<0.001). Serum
levels of MSP in both PC patients and controls were significantly associated with the rs10993994 genotype
(p<0.0001). Decreased expression of MSP parallels the clinical progression of PC and adjusted serum MSP
levels are associated with PC risk.
Cancer
Author: Sini Eerola1 ([email protected])
Co-author(s): Santio Niina2, Tuomela Johanna3, Rainio Eeva-Marja4,
Moreau Pascale5, Anizon Fabrice5, Corthals Garry6, Härkönen Pirkko6 &
Koskinen Päivi2
Affiliation(s): 1Institute of Biosciences and Medical Technology, University of Tampere, Tampere, Finland; 2Department
of Biology, University of Turku, Turku, Finland; 3Pharmatest Services Ltd, Turku, Finland; 4Turku BioImaging, Turku,
Finland; 5Université Blaise Pascal, Clermont-Ferrand, France; 6Turku Centre for Biotechnology, University of Turku and
Åbo Akademi, Turku, Finland; 7Institute of Biomedicine and Anatomy, University of Turku, Turku, Finland
Pim kinases and Pim inhibitors in the regulation of prostate cancer cell migration and
invasion
Keyword(s): PIM kinases, prostate cancer, migration, invasion, phosphorylation
Abstract:
PIM kinases (Proviral Integration Site for Moloney Murine Leukemia Virus) are well known oncoproteins which
have been shown to regulate cell survival, proliferation and motility. In addition, their expression is often upregulated in human hematopoietic malignancies and solid tumors. Since PIM kinases promote cancerous
behavior of cells, they have become attractive targets for drug development. We have previously identified
a novel PIM-selective kinase inhibitor 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9). Here
we show the potential of DHPCC-9 to efficiently prevent PIM-dependent functions both in cell-based assays
and in mouse models. To elucidate signaling mechanisms via which PIM kinases regulate cell motility, we
studied the involvement of the previously known PIM substrate NFATC1 (Nuclear Factor of Activated T Cells).
Here we show that NFATC1 may play a role as a downstream mediator of PIM1 signaling in promoting cancer
cell migration. Prostate cancer cell migration and invasion were efficiently decreased by either PIM inhibition
by DHPCC-9 or silencing of both PIM1 and PIM2 by RNA interference. Stable PIM1 or PIM3 overexpression
promoted prostate tumor growth, while PIM inhibitor DHPCC-9 treatment decreased tumor size. Stable PIM
overexpression induced PC-3 cells to metastasize into the prostate- draining lymph nodes and lungs, and
treatment with PIM inhibitor DHPCC-9 decreased the amount of both types of metastases. Mutagenesis
of all PIM1 phosphorylation target sites results in decreased NFATC1 phosphorylation and transcriptional
activity, along with a reduced potential of NFATC1 to promote prostate cancer cell migration.
Cancer
Author: Annika Kohvakka ([email protected])
Co-author(s): Kati Kivinummi, Antti Ylipää, Ville Kytölä, Matti Annala,
Matti Nykter, Tapio Visakorpi
Affiliation(s): Prostate Cancer Research Cancer, BioMediTech, University of Tampere, Tampere, Finland
Expression of novel prostate cancer associated long noncoding RNAs correlates with
progression-free survival in prostate cancer patients
Keyword(s): Prostate cancer, long noncoding RNAs
Abstract:
Prostate cancer (PC) is the second most frequently diagnosed cancer in men worldwide. 10-20 % of the PC
patients develop castration-resistant prostate cancer (CRPC) that has no curative therapies. There are also no
effective prognostic markers to predict emergence of CRPCs. Long noncoding RNAs (lncRNAs) are a recently
found group of RNAs that are not translated into proteins. Many of them are found to be differentially expressed
in cancer, and shown to have a regulative role in tumorigenesis and tumor development. In addition, some
lncRNAs have been associated with cancer progression and/or survival, making them potentially interesting
as prognostic markers. Previously, we performed RNA sequencing of 28 hormonally untreated PC, 13 CRPC
and 12 non-cancerous, benign prostatic hyperplasia tissue samples, out of which 145 novel PC-associated
lncRNAs (PCATs) were discovered. To validate this, expression of 39 PCATs were analyzed in 87 hormonally
untreated PC samples by qRT-PCR on Fluidigm Biomark HD, and the results were associated with clinical data.
Some of the PCATs had a significant correlation with progression-free survival in prostate cancer patients.
One of these PCATs, PCAT-2-180961, was also found to have a significant effect on viability of PC cells in vitro
after siRNA knockdown. In near future, expression of PCATs will be studied in hormonally untreated PC as
well as in CRPC tissues by RNA in situ hybridization and in blood samples of both hormone-naïve PC and CRPC
patients with qRT-PCR.
Cancer
Author: Mauro Scaravilli ([email protected])
Research group leader: Prof. Tapio Visakorpi
Co-author(s): Michael Olvedy *1, Mauro Scaravilli *2,3, Youri Hoogstrate 1,
Tapio Visakorpi 2,3, Guido W. Jenster 1 and Elena S. Martens-Uzunova 1
Affiliation(s): 1Department of Urology, Erasmus MC, Rotterdam, The Netherlands, 2 Institute of Biosciences and
Medical Technology-BioMediTech, University of Tampere, Tampere, Finland, 3 Fimlab Laboratories, Tampere University
Hospital, Tampere, Finland
A comprehensive repertoire of tRNA-derived fragments in prostate cancer
Keyword(s): tRNA-derived fragments (tRFs), prostate cancer (PCa), RNA-sequencing, non-coding RNA, biomarker
Abstract:
Prostate cancer (PCa) is the most common cancer among men in developed countries. Although its genetic
background is thoroughly investigated, rather little is known about the role of small non-coding RNAs (sncRNA)
in this disease. tRNA-derived fragments (tRFs) represent a new class of sncRNAs, which are present in a
broad range of species and have been reported to play a role in several cellular processes. Here, we analyzed
the expression of tRFs in fresh frozen patient samples derived from normal adjacent prostate and different
stages of PCa by RNA-sequencing. We identified 598 unique tRFs, many of which are deregulated in cancer
samples when compared to normal adjacent tissue. Most of the identified tRFs are derived from the 5 and
3 end of mature cytosolic tRNAs, but we also found tRFs produced from other parts of tRNAs, including pretRNA trailers and leaders, as well as tRFs from mitochondrial tRNAs. The 5 -derived tRFs comprise the most
abundant class of tRFs in general and represent the major class among upregulated tRFs. 3 -derived tRFs
types are dominant among downregulated tRFs in PCa. We validated the expression of three tRFs using qPCR.
The ratio of tRFs derived from tRNA LysCTT and tRNA PheGAA emerged as a good indicator of progressionfree survival and a candidate prognostic marker. This study provides a systematic catalogue of tRFs and their
dysregulation in PCa and can serve as the basis for further research on the biomarker potential and functional
role of tRFs in this disease.
Cancer
Author: Hanna Karvonen ([email protected])
Research group leader: Daniela Ungureanu
Co-author(s):
Affiliation(s): BMT/Cancer Signaling
ROR1 mechanism is hematological cancers
Keyword(s): ROR1, leukemia, lymphoma, drug-resistance
Abstract:
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen. Because of its expression
on the cell surface of leukemia (CLL) and lymphoma (MCL, MM) cells, but not on normal B-cells or other
postpartum tissues, ROR1 is an attractive candidate for targeted therapies. RORs belong to the family of
receptor tyrosine kinase proteins as well as pseudokinases. They are evolutionary conserved proteins among
different species and are primarily expressed during embryogenesis where they are required for skeletal,
respiratory and cardiac development. While the ligand for ROR2 has been identified as Wnt5a, ROR1 remains
yet an orphan receptor. In human adult tissue ROR1 expression is linked to malignant transformation,
especially in the hematological system. Humanized antibody Cirmtuzumab-Vedotin developed by UCSD
researchers and now in Phase I clinical trials is a potential treatment for ROR1-positive leukemia and solid
tumors, and these monoclonal antibodies were not only efficacious against specific cancer types but also
played a role in the epithelial-mesenchymal transition (EMT), which is considered important in tumor
progression. We will make use of ROR1 exclusive expression on the surface of leukemia cells and fully explore
its therapeutic potential. Using parental cell lines and drug-resistant cells we are investigating a) what are the
survival pathways engaged by ROR1, b) if ROR1 expression is linked to developing drug resistance in leukemia
and c) if blocking ROR1 can improve or complement targeted drug therapy. This will help us to define to what
degree targeting ROR1 will improve the current treatments in hematological cancers.
Cancer
Author: Matti Annala (1) ([email protected])
Co-author(s): Arun Azad (2), Stanislav Volik (3), Kevin Beja (3), Brian McConeghy (3),
Anne Haegert (3), Evan W Warner (3), Fan Mo (3), Sonal Brahmbhatt (3),
Robert Shukin (3), Stephane Le Bihan (3), Martin E Gleave (3), Matti Nykter (1),
Kim Chi (3), Alexander W Wyatt (3)
Affiliation(s): (1) BioMediTech, University of Tampere, Finland; (2) Department of Medical Oncology, British Columbia
Cancer Agency, Canada; (3) Vancouver Prostate Centre, University of British Columbia, Canada
Genomic alterations in circulating tumor DNA reveal mechanisms of enzalutamide resistance
in castration resistant prostate cancer
Keyword(s): prostate cancer, castration resistance, enzalutamide, ctdna
Abstract:
The molecular drivers of resistance to the AR antagonist enzalutamide in metastatic castration resistant
prostate cancer (mCRPC) are currently unclear. Since mCRPC tissue biopsies are often impractical in the clinic,
we collected circulating cell-free DNA (cfDNA) from 65 mCRPC patients before, during and after enzalutamide
treatment and performed integrated genomic profiling. AR mutations and/or copy number alterations
were detected in 46% of pre-treatment and 66% of post-treatment samples. AR amplification associated
with primary enzalutamide resistance, as did heavily mutated AR (2+ mutations). AR mutations exhibited
clonal selection during enzalutamide treatment, with loss of bicalutamide-sensitive W742 mutants and an
increase in glucocorticoid-sensitive L702H and enzalutamide-sensitive F877L mutants. L702H mutants only
arose in patients with prior abiraterone treatment, implicating continued glucocorticoid administration in
enzalutamide resistance. cfDNA sequencing also revealed alterations in TP53, MYC, PTEN, PIK3CA, FOXA1
and CTNNB1 in multiple patients. Actionable DNA repair alterations in BRCA2 and PALB2 were identified in 3
patients. These data demonstrate that clinically informative genomic profiling of cfDNA is feasible in nearly
all mCRPC patients and provides important insights into treatment resistance.
Cancer
Author: Kirsi J. Granberg*1,2 ([email protected])
Co-author(s): Matti Annala*2, Joonas Haapasalo3,4, O. Yli-Harja2, Hannu Haapasalo,
Wei Zhang6, Matti Nykter1
Affiliation(s): 1. Institute of Biosciences and Medical Technology University of Tampere, Tampere, Finland; 2. Department
of signal processing, Tamperre University of Technology, Tampere, Finland; 3. Unit of Neurosurgery, Tampere University
Hospital, Tampere, Finlan
Gatekeeper inactivation drives glioma progression into secondary glioblastoma
Keyword(s): deep sequencing, cancer progression,
Abstract:
Glioblastoma (GBM) is the most lethal form of brain tumors called gliomas. Median survival is 15 months
with best available treatment. Most GBMs arise de novo (primary GBM), but 510% progress from lower grade
gliomas (secondary GBM). As progression of low grade glioma into secondary GBM significantly impacts
prognosis, a better understanding of this process is paramount for treatment and monitoring of affected
patients. In this study, we applied whole-genome and transcriptome sequencing to low-grade glioma and
relapsed secondary GBM tissue from seven patients with progression. All primary gliomas carried IDH1
mutation, which was inherited by the secondary GBM. ATRX alterations in all five astrocytomas and TERT
promoter mutations in both 1p19q-codeleted oligoastrocytomas were also inherited in progressed tumors.
In five patients, progression was associated with increased genomic instability, whereas mutation load was
significantly increased in two other patients. One of them exhibited a hypermutation signature caused by a
mutation in the proofreading domain of DNA polymerase epsilon, while the second had lost both copies of
the DNA mismatch repair protein MSH2. In addition, both oligoastrocytomas had acquired focal inactivating
deletions of protein tyrosine phosphatase PTPRD at progression, suggesting a novel driver mechanism
for GBM progression. Most common progression-related genomic alterations were TP53 mutations and
deletions in CDKN2A, RB1, PTEN, and genes crucial to double strand break repair pathway. Taken together,
progression into secondary GBM was significantly related to tumor suppressor gene deletions and TP53
mutations. Disruption of these gatekeepers appears to be a significant mechanism for glioma progression.
Cancer
Author: Kirsi J. Granberg1,2,3 ([email protected])
Co-author(s): Matti Annala1,2, Birgitta Lehtinen1,2, Juha Kesseli1, Joonas Haapasalo4,5,
Olli Yli-Harja2, Tapio Visakorpi1,4, Hannu Haapasalo4,6, Matti Nykter1*, Wei Zhang3*
Affiliation(s): 1) Institute of Biosciences and Medical Technology (BioMediTech), University of Tampere, Tampere,
Finland, 2) Department of Signal Processing, Tampere University of Technology, Tampere, Finland,3) Department of
Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA, 4) Fimlab Laboratories ltd., Tampere
University Hospital, Tampere, Finland, 5) Unit of Neurosurgery, Tampere University Hospital, Tampere, Finland, 6)
Department of Pathology, University of Tampere, Tampere, Finland, *) Shared last authorship
Strong FGFR3 staining is a surrogate marker for FGFR3 fusions and poor prognosis in diffuse
gliomas
Keyword(s): Diagnostics, prognostic factor, FGFR3-TACC3, FGFR3 gene fusion
Abstract:
Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option
for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled coil 3 (TACC3)
have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded
well to FGFR inhibitors. As high FGFR3 expression has been detected in fusion positive tumors, we sought
to determine the clinical significance of FGFR3 protein expression level and its potential to indicate FGFR3
fusions. We thus performed FGFR3 IHC on tissue microarrays containing 676 grade II-IV astrocytoma and
116 grade II-III oligodendroglial tumor specimens. Selected cases were further analyzed using targeted
sequencing. Moderate-to-strong FGFR3 staining was detected in all tumor grades, was more common in
females, and associated with poor survival on both univariate and multivariate analyses. Targeted sequencing
identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 12 strongly stained cases (staining
specificity 86%), whereas no fusions were found in 12 negatively-to-moderately stained cases (staining
sensitivity 100%). Fusion-positive cases were predominantly female and negative for IDH, TP53, and EGFR/
PDGFRA/MET alterations. Importantly, FGFR3 staining revealed intratumoral heterogeneity, with subclonal
negative staining in a subpopulation of fusion-positive cases. Taken together, strong FGFR3 protein expression
is indicative of FGFR3 fusions and may serve as a cost-effective predictive marker for FGFR-inhibitor-based
treatment regimens. FGFR3 fusion positive cases are typically female and void of alterations in other growth
factor receptors, IDH genes and TP53.
Cancer
Author: Tommi Rantapero ([email protected])
Co-author(s): Minna Ampuja, Alejandra Rodriguez-Martinez, Emma-Leena Alarmo,
Maaria Palmroth, Matti Nykter, Anne Kallioniemi
Affiliation(s): University of Tampere, BioMediTech, Tampere, Finland
Uncovering gene regulatory basis of differential BMP4 response in breast cancer cell lines
Keyword(s): Breast cancer, BMP4, RNA-seq, DNaseq
Abstract:
Bone morphogenetic proteins (BMPs) are a group of growth factors that have been shown to have a role in
breast cancer progression. It has been shown that BMP4 reduces proliferation in multiple breast cancer cell
lines in vitro, while simultaneously inducing migration in a subset of the cell lines. Our study aims to uncover
the early BMP4 regulatory target genes and characterize the chromatin landscape in order to gain insight
into the underlying basis for the different BMP4 response in breast cancer cell lines. In this study, response
to BMP4 stimulation in two breast cancer cell lines MDA-MB-231 (responds to BMP4 by increased migration)
and T-47D (responds by decreased proliferation) were studied. RNA-seq and DNase-seq were conducted for
both cell lines after 3 h stimulation with BMP4 and untreated control. DNase I hypersensitive sites (DHS,
which correspond to regulatory sites within the genome) and differential DHS sites were detected from the
DNase-seq data. Furthermore, digital footprinting and transcription binding site prediction were conducted
for all DHS-sites. RNA-seq data revealed altogether 92 differentially expressed genes in MDA-MB-231 and
204 differentially expressed genes in T-47D. A subset of differentially expressed genes were selected and
validated with qPCR. In addition, a detailed inspection of the open chromatin sites in the promotor regions
of upregulated genes in MDA-MB-231 revealed enrichment of several transcription factor binding sites,
including SMAD4 which is a known mediator of BMP4 signaling. Further analysis and experiments will reveal
a more detailed view of the transcriptional regulation
Cancer
Author: Kirsi M. Kaukoniemi ([email protected])
Co-author(s): Hanna E. Rauhala, Matti Annala, Antti Ylipää, Kati Kivinummi,
Matti Nykter, and Tapio Visakorpi
Affiliation(s): Institute of Biosciences and Medical Technology - BioMediTech, University of Tampere, Tampere, Finland.
HOXC4-6 genes in the development of prostate cancer
Keyword(s): Prostate Cancer, HOXC4-6 genes
Abstract:
Conserved HOX genes have a major role during vertebrate embryogenesis. In addition, they regulate various
processes in cells, including apoptosis, receptor signaling and angiogenesis, which suggest that they might be
important also in the carcinogenesis. HOX genes encode for transcription factors, which bind to DNA using
DNA-binding domain called homeodomain. Humans have 39 HOX genes organized into four clusters A-D.
HOXC cluster includes nine genes, HOXC4-6 and HOXC8-13. Previously, it has been suggested that HOXC4-8
genes may have a role in the development of prostate cancer. By using RNA-seq, we demonstrated here that
the expression of HOXC4-6 genes is increased in prostate cancer. Using MeDIP-seq we found that the HOXC46 locus is also hypermethylated in prostate cancer. The sequencing findings were validated with qRT-PCR and
MethylMiner-qPCR. In addition, we have shown that the growth of LNCaP cells is reduced when transiently
transfected with siRNAs targeting HOXC4 and HOXC6. Next, we will interrogate the regulation of HOXC4-6
expression by methylation in more details.
Cancer
Author: Susanna Teppo ([email protected])
Research group leader: Olli Lohi
Co-author(s): Toni Grönroos, Thomas Liuksiala, Juha Mehtonen, Matti Nykter,
Merja Heinäniemi, Olli Lohi
Affiliation(s): Tampere Center for Child Health Research, University of Tampere and Tampere University Hospital;
BioMediTech, University of Tampere; Institute of Biomedicine, University of Eastern Finland
Hunting for sources and targets of leukemia progression
Keyword(s): pediatric leukemia, functional genomics
Abstract:
Acute lymphoblastic leukemia (ALL) impacts thousands of children s lives worldwide each year and despite
the relatively good overall prognosis of the rapidly progressing cancer, a part of patients still relapse. Long
cytotoxic treatment may also leave adverse life-long health effects. Pediatric ALL incidents generally involve
specific recurrent genetic rearrangements of which many affect important genes encoding for hematopoietic
transcription factors (agents in blood cell differentiation process). We focus on functional genomics and
molecular biology behind leukemia initiation and progression by combining the forces of cell biology and
bioinformatics. We attempt to clarify the consequences of the non-random alterations in genome: How do
they influence global gene transcription, regulatory region activity, or signaling networks? What kinds of
environmental factors may trigger disease progression? What are the driving downstream targets in each
subtype, how do they function in the cells, and could these processes be targeted by therapy? In testing the
role of genetic changes in leukemia, we have performed overexpression, silencing, or inhibition of specific
genetic elements or transcripts in cells, and measured changes in cell phenotype and molecule levels. We have
performed next generation sequencing experiments and utilized data in repositories to decipher functional
genomic processes following the alterations in patients and in disease models. In determining physiological
triggers in leukemia initiation, we have used a zebrafish model featuring the most common genetic alteration
suggested to serve as first-hit in leukemia progression. The presentation introduces examples of our work.
Computational biophysics and systems biology
Author: Ebrahim Afyounian ([email protected])
Co-author(s): Kirsi Granberg (1,2), Mari Saarinen (1), Matti Annala (1), Niina Paunu (3),
Hannu Haapasalo (4,5), Matti Nykter (1)
Affiliation(s): (1) BioMediTech, UTA (2) Tampere University of Technology, Department of signal processing (3)
Department of oncology, Tampere University Hospital (4) Fimlab Laboratories ltd., Tampere University Hospital (5)
Department of Pathology, UTA
Identification of Predisposing Germline Variants in Familial Glioma
Keyword(s): Familial glioma, Germline variant analysis, Whole-exome sequencing
Abstract:
Gliomas are generally sporadic but occasionally several family members are affected by these tumor types.
Except for Li-Fraumeni syndrome, little is known about predisposing factors for familial forms of the disease.
In this study, we aimed at identifying glioma susceptibility variants. We thus performed exome sequencing of
14 persons within four families using blood-derived DNA. Eight of the samples were from the affected persons
and the remaining samples were from the other family members. An in-house tool was used to call germline
variants. Variants common to the Finnish population of the 1000 genomes and the sequencing initiative
Suomi (SISu) projects were discarded. Identified variants were further filtered based on their annotation, a
bimodality test, and pathogenicity analysis. Moreover, gene expression of normal brain, glioblastoma, and
low grade glioma were extracted for genes harboring the variants. Results were then manually inspected
and filtered by removing sequencing artefacts, genes not expressed in brain or glioma as well as variants not
shared by the affected family members. Resulting 98 germline variants were validated by Sanger sequencing.
In addition, copy number analysis was performed. After manual filtering based on the presence of copy
number variation in affected family members, four regions were selected for further validation. We detected
no TP53 or CHEK2 variants typical of Li-Fraumeni syndrome. Previously reported POT1 variants were not
observed either, suggesting that novel factors are increasing the glioma susceptibility in Finnish families.
Next, we will study the detected variants in larger familial glioma cohort using targeted sequencing.
Author: Suvi Luoto ([email protected])
Co-author(s): J. Kesseli1, M. Nykter*1, K. J. Granberg*1,2
Affiliation(s): 1)Institute of Biosciences and Medical Technology, University of Tampere, Tampere, 2)Department of
Signal Processing, Tampere University of Technology, *) Shared last autorship
Different immune cell responses are associated with glioblastoma subclassification and
typical genetic alterations
Keyword(s): glioblastoma, tumor microenvironment, immune response, tumor-inflitrating lymphocytes, regression
analysis, non-negative least squares, Markov cluster algorithm
Abstract:
Interactions between components in tumor microenvironment and dysregulated immune responses play
important roles in cancer development. To better understand the role of immune cells in tumor pathogenesis
and destruction, we have computationally analysed the microenvironment of an aggressive brain tumor
glioblastoma (GBM). We downloaded GBM patient RNA-seq data from the Cancer Genome Atlas (TCGA).
Using cluster analysis, we identified 16 clusters, containing 10-933 genes that show a statistical enrichment
of immune response related gene ontology terms. Utilizing a panel of RNA-seq data from normal cell types,
we constructed regression models to characterize the expression profiles of GBM samples in the clusters of
interest as linear combinations of normal cell and reference GBM expression profiles. This was done using
non-negative linear regression that results in a sparse solution for the model coefficients. Simulated data
was used to validate that the regression model coefficients accurately reflect the contributions of normal cell
types to the expression profiles of tumor samples. We were able to uncover high variability in the composition
of microenvironment across the TCGA cohort, suggesting diverse immune responses in tumors. Association
analysis revealed higher proportion of estimated macrophage and/or CD8+ T lymphocyte proportions in
mesenchymal and neural subtypes as well as in tumors with EGFR and NF1 alterations. Taken together,
our analysis provided a characterization of the immunomicroenvironment in GBM and linked immune cell
responses to typical GBM alterations. More detailed characterization of diverse immune responses will
facilitate patient stratification and might provide tools for personalized immunotherapy in the future.
Author: Edouard Mobarak ([email protected])
Research group leader: Prof. Ilpo Vattulainen
Co-author(s): Dr. Moutusi Manna, Dr. Tomasz Ròg, Prof. Ilpo Vattulainen
Affiliation(s): Tampere University of Technology, Department of Physics, Biological Physics Group
Studying the Regulation and Behaviour of Proteins Involved in the Immune System and
how Lipids Affect Them
Keyword(s): Innate Immune System, Toll Like Receptor 4, Lipopolysaccharide, Glycolypids
Abstract:
The innate immune system is the first line of defense against foreign agents. In mammals, Toll-Like Receptors
(TLRs) are responsible for the activation of the innate immune system, and they are expressed on the surface
of sentinel and dendritic cells. TLR4 is activated by the presence of Lipopolysaccharides (LPS) found on
the outer membrane of Gram-negative bacteria. After activation, signaling cascades are engaged, leading
to downstream release of inflammatory modulators. Our experimental collaborators have uncovered that
glycolipids play a role on TLR4 activation, and this project aims to gain knowledge on the effects glycolipids
have on the behaviour and conformation of TLR4. The TLR4 model was inserted into an asymmetric lipid
bilayer. Two variants were used for the extracellular leaflet: either with or without glycolipids, and two variants
of the TLR4 complex were built: either with or without LPS. Thus, 4 different systems were constructed
and simulated for 2 microseconds. When interacting with LPS, the extracellular part of TLR4 periodically
bends towards the membrane. Without LPS making contact with TLR4, this phenomenon is not observed.
When glycolipids are present in the membrane, the phenomenon is more intense and more frequent.
We hypothesized that when TLR4 makes contact with LPS, it starts to probe the membrane looking for an
interaction site, and that this process might be part of the TLR4 activation. The presence of glycolipids in the
membrane intensifies this phenomenon, which is consistent with the experimental findings that glycolipids
play a role in TLR4 activation.
Author: Hanna Juhola ([email protected])
Research group leader: Ilpo Vattulainen
Co-author(s): Sami Rissanen, Pekka A. Postila, Tomasz Róg
Affiliation(s): Department of Physics, Tampere University of Technology
The Role of Synaptic Cell Membrane in Neurotransmitter Entry into its Receptor
Keyword(s): Molecular Dynamic Simulation, Neurotransmitter, Synaptic Cell Membrane
Abstract:
While there is growing awareness that membrane phospholipid composition could play a role in several
neuropathologies, the interactions between neurotransmitters and synaptic cell membranes are not fully
known. However, it has been suggested that lipid membranes could compete with receptors embedded
in membranes for ligand binding, highlighting the potential role of lipids in the ligand binding process. In
practice, the given neurotransmitter could enter the receptor’s binding site either directly from bulk water or
through binding to the membrane surface from which it would further migrate to the binding site via lateral
diffusion in the membrane plane. The aim of the work was to determine whether the synaptic membrane
affects the aden-osine entry process with the adenosine 2A receptor. In order to gain insight into the role
of the synaptic membrane, computational methods were used. The studied system was composed of a
lipid bilayer mimicking the outer leaflet of the postsynaptic cell membrane. A2A receptor was inserted into
the membrane with adenosine molecule in the binding pocket located in the central cleft of the receptor s
transmembrane domain. The system contained also water molecules. The entry pathways of adenosine were
probed with atomistic molecular dynamic (MD) simulations and steered MD simulations. The free energy for
the binding of neurotransmitter entry was determined through umbrella sampling simulations.
Author: Fabio Lolicato ([email protected])
Research group leader: Ilpo Vattulainen
Co-author(s): Tomasz Rog
Affiliation(s): Tampere University of Technology, Department of Physics, Biological Physics Group
Non-vesicular Trafficking of Sphingolipids: A Study on the Mechanism of Intracellular
Transfer of Ceramide-1-Phosphate
Keyword(s): Non-vesicular Trafficking, Sphingolipids transfer proteins , Molecular dynamics,
Abstract:
Sphingolipids are highly bioactive compounds that play crucial roles in the regulation of cell growth. They
have also been shown to act as signaling molecules for different cellular processes and in the regulation
of patho-biological mechanisms such as cancer, cardiovascular and neurodegenerative disorders, as well
as inflammatory or infectious diseases. In particular, phosphorylated sphingolipid ceramide-1-phosphate
(C1P) seems to act as a key regulator of cell growth, survival and migration, and it contributes to disease
pathogenesis such as asthma, atherosclerosis and thrombosis. C1P is a negatively charged sphingolipid
containing a phosphomonoester headgroup that in mammalian cells is synthesized from ceramide by means
of the ceramide enzyme kinase. It has recently been discovered the protein that carries this important
sphingolipid inside human cells, namely, the Ceramide-1-Posphate transfer protein (C1TP). The present study
examines the Ceramide-1-Posphate intracellular transfer mechanism through a computational approach.
In order to investigate the source of the selectivity of this phenomenon and how the binding specificity
takes place, the interaction between two different membrane compositions have been studied to mimic the
Protein-Membrane interactions with the outer leaflet of Golgi and the inner layer of Plasma membranes. The
aim is to investigate how C1P is transported inside the cells. Furthermore, since the lipid composition of a
given endomembrane may be controlled by the transport of individual molecular lipid species, understanding
how the specific protein-sphingolipid interactions take place, acquires a considerable importance.
Author: Jake Lin ([email protected])
Co-author(s): Sergei Häyrynen, Thomas Liuksiala, Petri Pölönen, Kirsi Granberg,
Olli Lohi, Merja Heinäniemi, Matti Nykter
Affiliation(s): BioMediTech, University of Tampere; Virtanen Institute for Molecular Sciences, University of Eastern
Finland
Computational characterisation and visualisation of blood cancer cell lineages
Keyword(s): Gene expression normalising, Leukemia, AML, mutations, visualisation
Abstract:
Methods and tools for categorizing and visualizing biological data are in demand as the adoption and
decreased cost of high throughout technologies have resulted in a big data reality. There is great incentive
within systems medicine in applying novel machine learning methods to existing public data with discovery
of potential new genetic and lineage reasoning insights and drug markers. Approximately 10,000 blood
cancer samples with gene expression profiles with curated annotations are assembled and 2 dimensional
maps generated with dimension reduction using t-SNE. The maps, including the TCGA acute myeloid study,
and their annotations can be explored on the web along with advanced search functions and box plotting
of custom sample normalised expression profiles for selected genes. Virtual classification, introduced here
as eStain of the samples are enabled using gene expression profiles, pathways and drug pathway signatures
using gene set variation analysis. Pairwise associations between clinical and genetic features are computed
and accessible, including eStain of clinical categories and annotation groups. These visualisations allowed
for cluster identifications and map to annotation and statistical summaries are integrated. Other advanced
features currently being worked on are functional mutation analysis and remapping from user custom
uploaded sample sets. The custom remapping is imagined to be web based and a novel computational tool
for interactive visual data mining and characterising in the context of published blood cancer and healthy
donor cell lineages and expressions. The HTML5 application is available at: http://compbio.uta.fi/hemap
Please contact authors for access.
Author: Sampo Kukkurainen 1 ([email protected])
Co-author(s): Adam OrBowski 2, Latifeh Azizi 1, Bernhard Wehrle-Haller 3,
Tomasz Róg 2, Ilpo Vattulainen 2, Vesa Hytönen 1
Affiliation(s):
Molecular dynamics of the talin head domain
Keyword(s):
Abstract:
The integrin-talin interaction mediates signals both outside-in and inside-out of the cell. The head domain
of talin binds the ²-integrin cytoplasmic tail and participates in integrin activation. Acidic phospholipids are
important for talin-mediated integrin activation, but the exact activation mechanism is not known. The talin
head has multiple sites that are thought to bind acidic phospholipids. We therefore studied the interactions
of the talin head domain with integrin and lipids in long, atomistic molecular dynamics simulations. We are
currently performing experiments to evaluate the model.
Author: Marja-Leena Linne ([email protected])
Research group leader: Marja-Leena Linne (Computational neuroscience)
Co-author(s): Tiina Manninen, Riikka Havela, Heidi Teppola,
Mikko Lehtimäki, Ausra Saudargien
Affiliation(s): Computational Neuroscience Group, Dept. of Signal Processing, Tampere Univ. of Technology; Dept. of
Informatics, Vytautas Magnus University, Lithuania; European Institute of Theoretical Neuroscience, Paris, France;
BioMediTech, Univ. of Tampere
MODELING CORTICAL NEUROPLASTICITY AND SYNAPTIC FUNCTIONS: IMPLICATIONS FOR
FUTURE NEUROSCIENCE, MEDICINE AND COMPUTING
Keyword(s): Neuroplasticity, neural network, synapse, computational modeling, the Human Brain Project
Abstract:
A subfield of Neuroinformatics, Computational Neuroscience, uses mathematical modeling, analysis and
computer simulation to study the structure and function of the nervous system. Our research group uses
knowledge and data from intracellular signaling pathways, particularly those underlying plasticity, biochemical
and bioelectrical properties of neurons and glial cells, as well as information on connectivity of cells, to assess
how neural networks process information, learn, store and recall information. Our work includes studies in
both healthy conditions and in different disease states. Our research belongs to The Human Brain Project
(HBP) which is a European Commission Future and Emerging Technologies (FET) Flagship, aiming to achieve
a multi-level, integrated understanding of brain structure and function with the help of novel information
and communication technology. The new technology is expected to enable large-scale collaboration and data
sharing, reconstruction of the brain at different biological scales, federated analysis of clinical data to map
diseases of the brain, and the development of brain-inspired computing systems. The results are expected
to accelerate progress towards effective diagnosis and treatment of brain diseases and the development
of novel robotics and information technology that mimics the human brain. In this presentation, we will
describe the recent research of the Computational Neuroscience Research Group at TUT, specifically the
work towards understanding cortical neuroplasticity and synaptic functions. Furthermore, we present the
goals of HBP and discuss the potential impact of the HBP on the neuroscience and society in general.
Health informatics
Author: Magdaléna von Essen ([email protected])
Co-author(s): Leo-Pekka Lyytikäinen, Terho Lehtimäki
Affiliation(s): Protein Dynamics, BioMediTech, Dep. of Clinical Chemistry, Fimlab Laboratories, Pirkanmaa Hospital
Atherosclerosis and Single Mutation in Mechanosensitive Proteins
Keyword(s): Atherosclerosis, mechanosensitive proteins, mechanostability
Abstract:
Introduction. Atherosclerosis is a disease with complex ethiology developing over great periods of time.
Despite intensive research, the causatives of early onset and progress of the disease or the development of
unstable plagues remain unclear. Our previous studies have shown that the expression of several proteins
involved in the cellular mechanostability is affected in later stages of atherosclerotic plague formation. Here
we focused on the single functional mutations in mechanosensitive proteins as a single disease causative.
Methods. We have analyzed SNP data of cardiovascular patients of the Pirkanmaa region (TVS, FINCAVAS
and ANGES collections). Core Exome and Illumina Chips were used for the SNP analysis. The data was
consequently imputed to GWAS 2000 with impute2 software. We have scanned the genotype for over
300 genes coding for mechanosensitive proteins filtering functional mutations. Patient genotypes with
atheroslerotic phenotype were compared to Finnish individuals from the same region. Statistical analysis
and correlation was performed with R ver.3.0.2. Results. The analysis of the atherosclerotic genotype for
functional mutations in mechanosensitive proteins did not uncover any mutation significantly correlating
with atherosclerosis development (p=0.05, FDR correction).
Health informatics
Author: Suresh Palanivel ([email protected])
Research group leader: Olli Yli-Harja
Co-author(s):
Affiliation(s): Suresh Palanivel, Olli Yli Harja, and Meenakshisundaram Kandahvelu*
Investigation of small molecule effects in human embryonic kidney cell lines
Keyword(s):
Abstract:
Cancer is one of the major burdens in the world, where the mortality is raising continuously and predicted
to be doubled in year 2030. Even though, extensive range of treatments is available, the survival is yet
questionable. Many novel drugs are being synthesized for the cancer treatment. Every drug has specific mode
of action for specific cancer. In our study, newly synthesized drug azo-derivatives of ²-diketones were screened
for its cytotoxic property. We studied the effect of azo-derivatives of ²-diketones to human embryonic kidney
cell line (HEK293T) and analysed its apoptosis and necrotic induction. The level of cytotoxicity increases over
the dose and time. Using live and dead cell assay, DNA content morphology analysis and apoptosis protein
expression analysis we identified that novel azo derivatives can efficiently inhibit the growth of HEK293T
cells. Moreover, from the single-cell microscopy analysis, we studied the cell death transition mechanism at
single cell level. In the present report, we will discuss the results of novel compounds cytotoxicity effect and
its mode of regulation at the single cell level.
Images, signals and models
Author: Antti-Juhana Mäki ([email protected])
Research group leader: Pasi Kallio
Co-author(s): Tomi Ryynänen, Jarmo Verho, Jukka Lekkala, Pasi Kallio
Indirect temperature measurement in microfluidic cell culture devices using identification
techniques
Keyword(s): cell culture, microfluidics, modeling, system identification, thermal analysis
Abstract:
Correct temperature level is considered to be vital for a successful long-term cell culturing. Because of
possible temperature gradients in the culture system, it is important to measure temperature as near as
possible from the cell area. However, implementing a sensor close to cells can be challenging because of
several practical reasons, for example limited space or microscopic inspection requirements. Furthermore,
the sensor located near cells can interfere with them and negatively affect e.g. cell growth. To overcome this
problem, we propose here a method to measure temperature indirectly using identification method. Briefly,
the idea is to measure temperature from a suitable location (e.g. outside the cell culture device), and then
use the developed mathematical model to estimate temperature in the desired location. For identificationbased model development and validation, a large set of experimental data using a commercial temperature
heater and a house-made sensor plate were performed. Obtained mean error between measured and
approximated temperature was always below 0.1C in every measurement. Furthermore, a maximum shortterm error of between measured and modeled temperature was monitored to be always less than 0.6C, thus
demonstrating the usefulness of the model also during larger temperature changes. It can be conclude that
the results from the model are consistent with the experimental values in a reasonable accuracy.
Author: Eeva Laurila ([email protected])
Research group leader: Katriina Aalto-Setälä
Co-author(s): Antti Ahola, Jari Hyttinen, Katriina Aalto-Setälä
Affiliation(s): University of Tampere, BioMediTech and School of Medicine, Tampere, Finland; Tampere University
of Technology, Department of Electronics and Communications Engineering, BioMediTech, Tampere, Finland; Heart
Hospital, Tampere University Hospital, Tampe
Novel methods for the functional analysis of stem cell derived cardiomyocytes
Keyword(s): disease modeling; stem cell derived cardiomyocytes; video microscopy
Abstract:
Since discovery of induced pluripotent stem cells, the number of iPSC based disease models for genetic cardiac
disorders has been rapidly increasing. Functional in vitro analysis of cardiomyocytes is essential for both
understanding the pathogenic mechanisms of cardiac disorders and performing preclinical safety studies for
novel drugs. The traditionally and commonly used electrophysiological analysis methods have been recently
accompanied by several novel video microscopy based methods for analyzing the mechanical beating behavior
of the cardiomyocytes. We have developed a semi-automated method for video based analysis of stem cell
derived cardiomyocytes. It is based on a high quality video recording of a beating cardiomyocyte, which is
quantified to a numerical signal, representing the mechanical activity of the cell. High resolution and frame
rate allow us to obtain detailed data on contraction-relaxation dynamics. The method has been successfully
used for the detection and analysis of the beating abnormalities of cardiomyocytes derived from patients
with genetic cardiac disorders, such as long QT syndrome. Although video microscopy based methods do not
allow analysis of the cell electrophysiology, they are non-invasive and non-terminal for the cells, as well as
affordable and have potential for high throughput. Taken together, these methods provide a promise for a
new faster yet reliable method for cardiomyocyte functional analysis. Development of the methodology not
only serves the basic research on the disease models, but could also provide the much needed efficient first
phase screening method for cardiac safety toxicology.
Author: Kimmo Kartasalo ([email protected])
Co-author(s): Pekka Ruusuvuori, Leena Latonen, Matti Nykter
Affiliation(s): University of Tampere, BioMediTech, Tampere, Finland
Benchmarking of image registration algorithms for 3D tissue reconstruction
Keyword(s): 3D reconstruction, image registration, histopathology
Abstract:
Current histological evaluations of tumors are subjective and laborious in nature, allowing only a small part of
each specimen to be analyzed. The result is highly dependent on the pathologist, complicating the unification
of diagnostic criteria across different hospitals or biobanks. Moreover, the 3D structure of the tissue is mostly
disregarded. Digital pathology can circumvent some of these issues by adopting computational approaches.
To address these issues and to better understand the heterogeneity and 3D organization of prostate cancer,
we aim to develop a three-dimensional computational model of the cancer. Digitized tissue slices will be
used to reconstruct image volumes of cancerous prostates, allowing the study of morphological features in
a 3D setting. A crucial step of the model-building process is forming an accurate 3D reconstruction of the
tissue based on the 2D tissue slice images. This involves a series of 2D to 2D image registration operations to
transform all of the tissue slices into a common coordinate system. Due to anatomical and technical variations
between adjacent slices, obtaining an accurate registration is highly challenging. Evaluating the accuracy of
the result is perhaps even more challenging due to the lack of a reliable ground truth. Using a combination
of manually annotated correspondences and indirect measures of reconstruction accuracy, we performed a
comprehensive benchmarking of several image registration algorithms for this task. The results will not only
support our aim of modeling prostate cancer in 3D but they will also be useful for other algorithm developers
in the future.
Author: Mikko Peltokangas ([email protected])
Research group leader: Jukka Lekkala
Co-author(s): Antti Vehkaoja, Jukka Lekkala
Affiliation(s): Department of Automation Science and Engineering, Tampere University of Technology.
Non-invasive methods for the detection of the markers of the vascular health
Keyword(s): Atherosclerosis, PPG, EMFi, classification, pulse wave analysis
Abstract:
Valuable information on the vascular health and the state of the local circulation is obtained by analyzing
non-invasively recorded arterial pulse wave (PW) signals. An individual PW is a superposition of the heartbeat induced percussion wave and its reflections. The propagation velocity of these waves depends on the
elasticity of the arterial wall. The arteries tend to degenerate due to ageing but also due to unhealthy lifestyle.
These differences are seen in the parameters determined from the PW curves. The study aims in developing
methods for analyzing the multi-channel PW signals recorded with the sensors made of Electromechanical
Film (EMFi) and optical photoplethysmographic (PPG) transducers. The development of the analysis methods
includes clinical investigations rst with atherosclerotic patients.The analysis of PW signals measured at
multiple locations provides methods that help in the diagnosing of the vascular diseases. For example, a
simple linear discriminant classifier discriminates healthy control subjects from atherosclerotic patients with
a specificity of 86.9 and a sensitivity of 77.4. The applications of the PW measurements are not only limited
to the vascular screening: currently there is not an established method for monitoring the circulation of
the patients that have undergone free ap surgery for various reasons. If the vascular failure occurs after
operation, the free ap can be salvaged if the complication is observed early enough. Optical PPG sensors are
a potential option for the microvascular monitoring of free aps. The nal objective of the study is to develop a
user-friendly system for automated vascular screening and monitoring.
Author: Kalle Lehto (3) ([email protected])
Research group leader: Jari Hyttinen
Co-author(s): Jing Zou (1,2), Markus Hannula (3), Hao Feng (1), Aula A.S (3,4),
Hyttinen J. (3), Pyykkö Ilmari (1)
Affiliation(s): 1. Hearing and Balance Unit, Field of Oto-laryngology, School of Medicine, University of Tampere, Tampere,
Finland; 2. Department of Otolaryngology-Head and Neck Surgery, Center for Otolaryngology-Head & Neck Surgery of
Chinese PLA, Changhai Hospital, Second Military Medical University, Shanghai, China; 3. Department of Electronics and
Communications Engineering, Tampere University of Technology, BioMediTech, Tampere, Finland; 4. Department of
Medical Physics, Imaging Centre, Tampere University Hospital, Tampere, Finland
Contrast Enhanced X-ray Microtomography in Cochlear Imaging
Keyword(s): X-ray microtomography, cochlear implantation, contrast enhancement
Abstract:
Cochlear implantation (CI) is an effective state-of-the-art treatment for profound hearing loss, but there are
no means to investigate the position of the cochlear electrodes in high resolution in vivo. In this report, nondestructive contrast enhanced X-ray microtomography (¼CT) was applied ex-vivo for studying the electrode
positioning after CI. Nine human temporal bones were studied by inserting a CI electrode using the round
window approach. The temporal bones were imaged using ¼CT (Zeiss Xradia microXCT-400) with and without
10 mg/ml iodine dissolved in pure ethanol as contrast agent. All of the specimens were imaged using a
0.4x and 4x objectives resulting in a 22.6 ¼m, 13.4 ¼m and 3.6 ¼m isotropic voxel sizes. Subsequently, the
image stacks were reconstructed, visualized and analyzed. Results indicate that the cochlear fine structures,
including e.g. Reissner’s membrane, stria vascularis, spiral ligament, basilar membrane, spiral limbus, osseous
spiral lamina, and Rosenthal’s canal that hosts the spiral ganglion cells, were clearly delineated; the electrode
array avoided the lateral wall of the scala tympani in the hook region and then ran along the lateral wall of
the scala tympani without any exception. In conclusion, the current ex-vivo ¼CT imaging system produced
high-quality images that could demonstrate the fine cochlear structures faithfully and verify the implantation
efficacy of the electrode array. The acquired image sets can be used for 3D histology without physically
sectioning the sample.
Author: Mira Valkonen ([email protected])
Co-author(s): Matti Nykter (1), Leena Latonen (1), Pekka Ruusuvuori (1,2)
Affiliation(s): (1) Institute of Biomedical Technology, BioMediTech, University of Tampere, Tampere, Finland, (2)
Tampere University of Technology, Pori, Finland
Learning based detection of early neoplastic changes in histological images
Keyword(s):
Abstract:
Digital pathology combined with image processing and machine learning allows quantitative analysis of tumor
morphology and histopathologic classification. Compared to visual inspection of histological images, automated image
analysis in digital pathology can utilize significantly more data and detect smaller molecular alterations connected
to early neoplastic changes. Our objective was to implement fully automated image analysis process for extracting
multidimensional feature data to describe a section of tissue and utilize these features to detect early neoplastic
changes. We used hematoxylin-eosin stained whole slide images of mouse prostatic intraepithelial neoplasia (mPIN)
confined in prostate. Set of these neoplastic lesions of abnormal epithelial cell growth and glands of normal tissue
were segmented by an expert and used in this study. The implemented image analysis pipeline consists of four main
steps: pre-process, segmentation, feature extraction and classification. First, equalization of colors was performed
through the image stack of regions of interest to get comparable feature data. After this, unwanted regions and cell
nuclei were segmented. Multidimensional feature data was extracted to describe qualities such as shape, texture and
spatial arrangement of nucleus or lesion. Logistic regression classifier was used to detect early neoplastic changes and
spatial locations of lesions utilizing the feature data. The classifier was trained with the set of expert-defined lesions
representing mild changes versus advanced changes. The implemented model estimates the probability to belong
to either of these groups. Learning-based method was able to capture lesions with visible neoplastic changes and
indicate representative features that could potentially be linked with neoplastic changes. Finding the relevant features
considering prostate cancer is useful for future studies. Additionally, detection of early lesions is important both in
studying events leading to cancerous changes in tissue and to develop digital pathology tools.
Author: Dhanesh Rajan ([email protected])
Co-author(s): Heimo Ihalainen, Jukka Lekkala
Affiliation(s): Department of Automation Science & Engineering, TUT
Concept of simultaneous cell imaging and image based optical chemical measurements Cell
Imaging, Image based sensing
Keyword(s): Digital microscopy, Image based pH sensing
Abstract:
Human induced pluripotent stem (iPS) cell-derived cells eg. cardiomyocytes are frequently cultured on Multielectrode arrays (MEA) for different applications including characterization of electrophysiological effects of
drug molecules. In many cases, besides the conventional studies of physiology, pathology and cell circuitconnectivity on MEA, a routine microscopy is essential. Additionally, video microscopy and image analysis are
used for functional characterization of cells. MEA s, for all these, usually need to be moved to a conventional
microscope, causing intervention of the cell s natural growth environment unless special arrangements are
provided. This situation can be avoided with an all-in-one setup where a MEA module, Imaging module, Sensor
module etc. are integrated for eg. as a modular cell culture environment. The imaging module captures HD
images and videos. With some effort, pH, pO2, can also be measured optically by numerical analysis of images
of dissolved/ immobilized chemical calorimetric indicators. We put forward the possibility of simultaneous
cell imaging and image based chemical measurement, eg. pH as a starting point. In cell culture, pH is a critical
control parameter for maintaining cell viability and tissue functions. Monitoring pH during cell culture and
differentiation provides, valuable information on metabolic processes and the overall environment. Though
electrochemical pH sensors are fast and easy to use, they require sample physical contact and a relatively
large sample volume. Optically, pH can be computed from images of phenol red culture medium which can
facilitate a fully non-contact pH monitoring suitable for a long term continuous operation.
Immunology
Author: Zuzet Martinez Cordova ([email protected])
Research group leader: Marko Pesu
Co-author(s): Anna Oksanen1, Ville Kytölä2, Valentina Taverniti3, Sanna Hämäläinen1, Ilkka Junttila4, 5, Antti Ylipää2,
Matti Nykter2, Marko Pesu 1,5
Affiliation(s): 1 Immunoregulation, Institute of Biosciences and Medical Technology (BioMediTech), University of
Tampere, FI-33014 Tampere, Finland
Myeloid Cell Expressed Proprotein Convertase FURIN Attenuates Inflammation
Keyword(s): cytokine, LysM, M1-type, macrophage, TGF-²1
Abstract:
A proprotein convertase enzyme FURIN converts immature proproteins into functional products. FURIN is
upregulated in activated T lymphocytes and monocytes and regulates peripheral tolerance along with Th1/
Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and processing
viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional
deletion of FURIN in myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of
neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1² and reduced numbers
of splenocytes. LPS injection resulted in an accelerated mortality, elevated serum pro-inflammatory cytokines
and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis
revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages.
Moreover, FURIN inhibited iNOS and promoted Arg1 expression, which implies the regulation of the M1/M2type macrophage balance. FURIN was required for the normal production of bioactive TGF-²1 cytokine, but it
inhibited the maturation of inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has
an anti-inflammatory function in LysM+ myeloid cells in vivo.
Immunology
Author: Sanna-Kaisa Harjula ([email protected])
Research group leader: Mika Rämet
Co-author(s): Anni Saralahti (1), Markus Ojanen (1), Jenna Ilomäki (1),
Heather Mathie (1), Nicholas Halfpenny (1), Tommi Rantapero (1),
Kirsi Granberg (1), Matti Nykter (1), Olli Lohi (2), Mataleena Parikka (1),
Mika Rämet (1,3,4,5)
Affiliation(s): (1) UTA, BioMediTech (2) TACC, UTA Medical School and TAUH (3) Department of Children and Adolescents,
OUH (4) PEDEGO Research Unit, Medical Research Center Oulu, UO (5) Department of Pediatrics, TAUH
Genes affecting mycobacterial infection in zebrafish
Keyword(s): zebrafish, Mycobacterium marinum, tuberculosis
Abstract:
In 2013, one and a half million people died of tuberculosis and nine million developed the disease.
Mycobacterium marinum, a natural zebrafish pathogen, causes a systemic infection in zebrafish. This infection
is partly similar to tuberculosis. Thus M. marinum infection in zebrafish is a potentially good model for
tuberculosis. The aim of this study is to identify genes underlying defence mechanisms against M. marinum
infection in zebrafish and to study their role during this infection. To achieve our aim we carry out a genebreaking transposon (GBT)-based forward genetic screen. In addition, we study genes selected based on
the literature. For the genetic screen, we have injected GBT RP2 construct (a generous gift from Professor
Stephen C. Ekker s laboratory (Mayo Clinic, Rochester, USA)) with synthetic mRNA of Tol2 transposase into
fertilized wild type zebrafish eggs to induce random mutations. Successfully injected embryos have been
raised and crossed further. In F3 generation heterozygous and homozygous zebrafish for the mutation and
wild type zebrafish are screened for resistance against M. marinum. For the screen, we have infected 111
out of 130 mutant zebrafish lines. Four of these lines have repeatedly showed altered resistance against M.
marinum infection compared to the wild type. Currently, we have identified some of the mutated genes in
those four lines with targeted sequencing and inverse-PCR. We have also infected zebrafish with a mutation
in genes selected based on the literature. We expect this study to provide novel information about the host
mechanisms involved in resistance against mycobacterial infection.
Immunology
Author: Mirva Järvelä-Stölting ([email protected])
Co-author(s): Susanna Valanne1, Henna Myllymäki1, Sanna-Kaisa Harjula1 & Mika Rämet1,2
Affiliation(s): 1Laboratory of Experimental Immunology, BioMediTech, University of Tampere, Finland 2 PEDEGO
Research Center and Medical Research Center Oulu, University of Oulu and Department of Children and Adolescents,
Oulu University Hospital, Oulu, Finland
Brahma complex modulates immune signalling in Drosophila melanogaster
Keyword(s): Drosophila melanogaster, innate immunity, NFkappaB, Toll, negative regulation
Abstract:
Drosophila is a widely used model animal in immunology research. Drosophila defence against yeast and
bacteria is mainly mediated by two evolutionarily conserved NF-kappaB signalling pathways, the Toll and
the Imd pathway. Although the core Toll signalling is well-known, the negative regulation of Toll signalling is
poorly understood. In a luciferase reporter-based genome-wide RNA interference (RNAi) in vitro screen we
identified 14 genes negatively regulating the Toll pathway activity. We investigated the in vivo significance
of these 14 genes using the UAS-GAL4 method to silence genes in specific tissues in Drosophila. The UASRNAi flies were crossed to the C564-GAL4 driver, to silence the expression of these genes mainly in the
fat body. The progeny flies were infected first with gram positive bacteria, Micrococcus luteus, to activate
the Toll pathway. 24h post infection, flies were further infected with more virulent gram positive bacteria,
Enterococcus faecalis. In our assays, silencing the expression of at least two members of the Brahma complex
(brahma and osa) creates a similar phenotype both in vitro and in vivo. Survival of brahma and osa RNAi
flies from gram positive bacterial infection was enhanced compared to controls. Drosomycin expression was
elevated in M. luteus infected osa RNAi flies compared to controls. These results suggest that brm and osa
have a function in negative regulation of the Toll pathway indicating that DNA modification may play a role in
Drosophila Toll signalling. Further studies will elucidate the more specific function of the Brahma complex in
Drosophila immune response.
Immunology
Author: Leena-Maija Vanha-aho(1) ([email protected])
Co-author(s): Ines Anderl (2,3), Laura Vesala(2), Dan Hultmark(2,3),
Susanna Valanne(1), Mika Rämet(1,4)
Affiliation(s): (1)Laboratory of Experimental Immunology, BioMediTech, University of Tampere, Finland (2)Laboratory
of Genetic Immunology, BioMediTech, University of Tampere, Finland (3)3 Department of Molecular Biology, Umeå
University, Umeå, Sweden (4)PEDEGO Rese
Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in
Drosophila melanogaster
Keyword(s): Drosophila, immunity, parasitism, innate immunity
Abstract:
Innate immunity is the most ancient form of immune defense in multicellular organisms. Because of the
evolutionary conservation of the innate defense mechanisms, the fruit fly, Drosophila melanogaster, has
emerged as a popular model for studying innate immunity. The immune defense of Drosophila that consists
solely of innate immunity, relies on both humoral and cellular mechanisms. The fruit fly has three types of
hemocytes, namely plasmatocytes, crystal cells, and lamellocytes that are responsible for cellular responses,
such as phagocytosis, melanization and the encapsulation of wasp eggs. The cellular immune response against
parasitoids involves the activation, mobilization, proliferation and differentiation of the different hemocyte
types. In this study, we have assessed the role of Edin (elevated during infection) in the immune response
against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin
was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that
Edin is an important determinant of the encapsulation response. Although edin expression in the fat body
was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes.
Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the
increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. Our study
shows that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase
of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.
Immunology
Author: Melina Ahonen ([email protected])
Co-author(s): Anna Grönholm, Marko Pesu
Affiliation(s): Immunoregulation, BioMediTech, University of Tampere
Studying the role of proprotein convertase furin in the regulation of T cell identity by
CRISPR/Cas9 technology
Keyword(s): furin, T cell identity, super-enhancer, CRISPR/Cas9
Abstract:
Proprotein convertases take part in many biological processes by cleaving immature proproteins to their
active form. One of these proprotein convertases is furin that plays an important role in immune system by
regulating the production of many cytokines and inducing T helper cell differentiation toward Th1 subset. The
regulation of furin s gene expression is still partly unknown, but recent studies have indicated that furin has
a putative distal super-enhancer in CD4+ Th1 cells. The main aim of this study was to delete furin s putative
super-enhancer from mouse T cell genome with CRISPR/Cas9 technology in order to see if super-enhancer
has a role in the regulation of T cell identity. We also clarified responsiveness and cytokine profiles of EL-4
and HT-2 T cells to be used in the screening the effect of deletion. CRISPR DNA constructs were transfected
to EL-4 cells using 4D-NucleofectorTM, cells were stimulated with plate bound anti-CD3/28 ± IL-12 and the
relative gene expression of furin and cytokines was measured by RT-qPCR. Both T cell lines responded to T
cell stimulations and expressed furin, thus providing good T cell model for the study. Our preliminary results
indicated that the deletion of furin s putative super-enhancer by CRISPR/Cas9 slightly reduced the expression
of furin and IL-2. The study will be continued with further experiments with the same CRISPR constructs and
also stable cell lines with super-enhancer deletion will be created, so that the role of furin s putative superenhancer in the regulation of T cell identity could be investigated more deeply.
Immunology
Author: Markus Ojanen (1) ([email protected])
Research group leader: Mika Rämet (1,3,4)
Co-author(s): Sanna-Kaisa Harjula (1), Kaisa Oksanen (1), Anna Grönholm (1),
Henna Myllymäki (1), Carina A. Bäuerlein (1), Mataleena Parikka (1),
Marko Pesu (1,2), Mika Rämet (1,3,4)
Affiliation(s): (1) BioMediTech, University of Tampere; (2) Department of Dermatology, Tampere University Hospital;
(3) Department of Children and Adolescents, Oulu University Hospital; (4) PEDEGO Research Unit, Medical Research
Center Oulu, University of Oulu
Genes affecting host response against mycobacteria in zebrafish (Danio rerio)
Keyword(s): Tuberculosis, Zebrafish, Mycobacterium marinum
Abstract:
Human tuberculosis (TB) is an epidemic disease caused by Mycobacterium tuberculosis (Mtb). Susceptibility
to TB depends on the Mtb strain and on a number of host factors; environmental conditions, personal health
status as well as genetic variation. Today, increasing amount of data are available on genetic polymorphisms
related to TB immunity, and to study the complex effects of host genetics in a mycobacterial infection, a
suitable animal model is required. In this study, we used Mycobacterium marinum infection in zebrafish to
model TB. Agilent microarray analysis was performed for wild-type fish after low-dose (~50 CFU) M. marinum
infection at 3 and 14 days post infection (dpi). 35 and 187 genes were induced at least 5-fold at 3 and 14 dpi,
respectively. Counts of down-regulated (<0.3-fold expression) genes in infections were 221 at 3 dpi and 130
at 14 dpi. Genes involved in several different processes, such as muscle contraction and immune response,
showed induction. From these genes, we found 11, which also associate with the risk of human TB, and chose
seven to investigate their relevance for mycobacterial resistance. Knock-out (KO) zebrafish for these genes
are being produced with a CRISPR-Cas9-based mutagenesis. Subsequently, the outcome of M. marinum
infection will be determined by measuring bacterial burden and investigating granuloma formation in both
KO embryos and adults. In addition, host response is monitored by analyzing cytokine expression profiles and
blood cell composition. We believe that our results will bring important insights into pathogenesis of TB and
into host genetics behind it. ”
Immunology
Author: Saara Aittomäki ([email protected])
Co-author(s): Tapio Lehtinen 1), Susanna Valanne 2), Mika Rämet 2,3,4,5), Marko Pesu 1,3)
Affiliation(s): 1) Immunoregulation, BioMediTech, University of Tampere 2) Experimental Immunology, BioMediTech,
University of Tampere 3)
Pirkanmaa Hospital District, Tampere 4)Department of Children and Adolescents,
Oulu University Hospital 5)Department of Pediat
The role of Furin1 in Drosophila immunity
Keyword(s): Innate immunity, Proprotein convertase, Drosophila melanogaster
Abstract:
Many important biological processes involve proteolysis of larger protein precursors by proprotein convertase
enzymes. We have investigated the role of the proprotein convertase family member Furin1 of Drosophila
melanogaster in innate immunity. Furin1 expression was knocked down in the fat body by crossing transgenic
UAS-Furin1-RNAi flies with C564-GAL4 or Fb-GAL4 flies. Adult flies from these crosses and control crosses were
infected with Gram negative Enterobacter cloacae or Gram positive Micrococcus luteus plus Enterococcus
faecalis and their survival was monitored. Our preliminary results indicate that flies with decreased expression
of Furin1 in the fat body are more sensitive to bacterial infection than control flies. This is possibly due to the
lack of induction of antimicrobial peptide expression via the Toll and Imd signalling pathways, as analysed
by quantitative RT-PCR. The fat body structure of Furin1-RNAi x Fb-GAL4;UAS-GFP larvae appeared normal
when compared to the w1118 x Fb-GAL4;UAS-GFP control cross larvae. Both RNAi-mediated knockdown of
Cactus and overexpression of IMD rescued the antimicrobial peptide expression from flies with decreased
levels of Furin1 in the fat body. This suggests that Furin1 may function upstream of the Toll pathway member
Cactus and the IMD pathway member IMD. Further studies will be carried out to elucidate the mechanism
for Furin1 mediated regulation of the immune signalling pathways.”
Immunology
Author: Mirja Niskanen (1) and Henna Myllymäki (1) ([email protected])
Co-author(s): Mika Rämet (1,2,3,4)
Affiliation(s): (1) BioMediTech, University of Tampere (2) Department of Pediatrics, Tampere Universtiy Hospital (3)
Department of Children and Adolescents, Oulu University Hospital (4) Department of Pediatrics, MRC Oulu
Reactivation of latent mycobacterial infection in the adult zebrafish
Keyword(s): Tuberculosis, Reactivation, Zebrafish, Mycobacterium marinum, Resuscitation promoting factors
Abstract:
Tuberculosis still causes 1.5 million deaths annually. Moreover, it has been estimated that 2 billion people
carry a latent Mycobacterium tuberculosis infection. 5-10% of the latent infections are reactivated even
decades after bacterial exposure. Often reactivation is caused by immunosuppression, but otherwise the
mechanisms of reactivation are poorly understood, hence reactivation cannot be prevented. Lack of adequate
animal models has hampered tuberculosis research. Mycobacterium marinum infection in zebrafish (Danio
rerio) leads to a disease that closely resembles human tuberculosis with natural latency and reactivation,
presenting a promising model for studying reactivation of tuberculosis. Our aim was to develop a reliable
method to reactivate latent mycobacterial infection in adult zebrafish. Fish were infected with a low dose of
mycobacteria, and the following latent infections were reactivated by dexamethasone feeding. Reactivation
of latent infection was seen as increased bacterial burdens: after six weeks the bacterial counts per fish had
increased 50-450-fold. We also wanted to develop a reproducible method to reactivate dormant M. marinum
in vitro. First, M. marinum cultures were exposed to hypoxia. As a result, the growth of bacteria ceased and
they fell into dormancy. Later, dormant cultures were reactivated by inoculating them into fresh medium in
oxygen rich environment. Reactivation was analyzed by measuring the expression of Resuscitation promoting
factors (Rpfs). We found that M. marinum Rpfs are induced during reactivation and each displays a distinctive
expression pattern in different phases of reactivation. These results suggest that the Rpfs could be used as
markers for mycobacterial reactivation.
Immunology
Author: Meri Uusi-Mäkelä ([email protected])
Co-author(s): Carina Bäuerlain, Markus Ojanen, Henna Myllymäki, Mika Rämet
Affiliation(s): MU, CB, MO, HM, MR=BioMediTech, University of Tampere, Tampere, MR lisäksi Finland 2Department of
Children and Adolescents, Oulu University Hospital, Oulu, Finland 3PEDEGO Research Unit, Medical Research Center
Oulu, University of Oulu, Oulu, Finland
CRISPR-Cas9 based Mutagenesis for Studying Tuberculosis in Zebrafish (Danio Rerio)
Keyword(s): Mycobacterium marinum, Zebrafish, Danio rerio, chemokines, cytokines, immunity, CRISPR-Cas9,
tuberculosis
Abstract:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based mutagenesis is an invaluable
method for generating targeted modifications in an organism s genome. The method is based on the activity of
Cas9, an RNA-guided endonuclease which catalyses site-specific cleavage of double-stranded DNA. Repairing
the double-stranded breaks with non-homologous end-joining will lead to frameshift mutations, rendering
the gene non-functional. Recently, CRISPR-Cas9 based mutagenesis has been established in zebrafish (Danio
rerio), an important model organism in immunological research. We have previously established this method
by successfully silencing eGFP in gfp-expressing mutant embryos. The efficiency of the silencing was evaluated
with the T7 endonuclease mutation detection assay and DNA polyacrylamide gel electrophoresis. We now
apply this method to knockout chemokines and their respective receptors to test their role in mycobacterial
infections in zebrafish. Our experiments involve microinjection of the guide RNA together with Cas9 mRNA
into a 1-cell stage embryo. The generated heterozygous mutants can be bred into homozygous knockout fish
lines. We will monitor the survival of mutant embryos, which have been infected at different time points after
fertilization, as well as the survival of adults, compared to wild-type fish. Furthermore, we will try to boost
the mutant immune response by vaccination with bacterial antigens and compare the effect to wild-type
vaccinated and unvaccinated fish. Overexpressing an interesting cytokine could serve as vaccine enhancement
and give us additional information about the role of that specific molecule. With these experiments, we are
aiming to provide important information for designing novel protective treatments against tuberculosis.
Measurement and stimulation systems and methods
Author: Risto-Pekka Pölönen ([email protected])
Research group leader: Katriina Aalto-Setälä
Co-author(s): Kimmo Kartasalo 1, Marisa Ojala 2, Joose Kreutzer 1, Marlitt Viehrig 1,
Feihu Zhao 1, Jyrki Rasku 3, Jukka Lekkala 1, Katriina Aalto-Setälä 2, Pasi Kallio 1
Affiliation(s): 1. Tampere University of Technology, Department of Automation Science and Engineering, BioMediTech,
Tampere, Finland. 2. University of Tampere, BioMediTech, Tampere, Finland. 3. University of Tampere, School of
Information Sciences, Tampere, Finland
Orientation analysis of hiPSC derived cardiomyocytes exposed to uniaxial mechanical
stretching using CytoSpectre software
Keyword(s): CytoSpectre, hiPSC, cardiomyocyte, uniaxial stretching, PDMS
Abstract:
hiPSC derived cardiomyocytes (CMs) cultured in vitro are considered structurally and functionally immature
compared to CMs in adult tissue. They lack stimuli which are present in human body, such as mechanical stress
of surrounding extracellular matrix. A custom made PDMS device was designed and manufactured at TUT to
mimic the mechanical stimulus of a beating heart. Structural analysis of CMs has been based on subjective
human assessed characterization of microscopy images of cells visualized by immunocytochemistry. An
automated software tool, CytoSpectre, was developed for this purpose in collaboration with TUT to carry out
objective analysis without biased human error. By exposing the hiPSC derived CMs to uniaxial mechanical
stretching, we managed to orientate the sarcomeres of CMs perpendicular to the axis of stretch, thus
providing a more mature shape of the CMs. Orientation of the sarcomeres in stretched as well as control
CMs was analyzed by CytoSpectre.
Author: Antti Ahola ([email protected])
Co-author(s): Paruthi Pradhapan, Eeva Laurila, Katriina Aalto-Setälä
Affiliation(s):
Analysis tool for human induced pluripotent stem cell derived cardiomyocyte beating
dynamics using video imaging
Keyword(s): Cardiomyocyte, biomechanics, motion analysis
Abstract:
Human induced pluripotent stem cells (hiPSCs) provide a platform for drug development and testing, due to
availability of cardiomyocytes (CMs) with various genetic profiles. Until recently, hiPSC derived CM analysis
has been limited to electrophysiologal methods. We have previously shown video analysis to be a feasible
addition to commonly used electrophysiological studies. It provides a detailed analysis of single dissociated
CM beating dynamics using digital image correlation (DIC). Here we present a semiautomatic analysis tool
designed for hiPSC derived CM biomechanics studies. The method used was presented by Ahola et al. in
2014. DIC is used for calculating velocity vector fields between video frames across hiPSCs. Directional
motion is quantified by dividing the cell to 8 sectors around a beating focus point. The vectors in each sector
are divided into directional components, creating orthogonal signals for each sector. Average waveforms are
calculated using cross-correlation templates, similarly as by Pradhapan et al. in 2013. The analysis tool detects
cardiomyocytes, and suggests analysis parameters to the user. It calculates one-beat average waveforms,
representing the cellular beating characteristics, and calculates parameters from them. The results of the
analysis can be uploaded to an online database. Our tool is capable of detecting differences in mechanical
beating characteristics between different areas of a single CM. The method is non-invasive and non-toxic,
allowing repeated analysis of the same cells, without introducing foreign agents. The tool, along with the
collected cell data, provides a valuable asset for different cardiomyocyte studies, including drug screening
applications.
Author: Marlitt Viehrig ([email protected])
Co-author(s): Sampo Tuukkanen, Pasi Kallio
Affiliation(s): Department of Automation Science and Engineering, Tampere University of Technology
Transparent Electromechanical Stimulation System for Stem Cell Applications
Keyword(s): Electromechanical Stimulation, Stem Cells, Conductive Polymers, Piezoelectricity, Crystalline Nanocellulose
Abstract:
The functionalities of cells and tissues in the human body depend greatly on their specific microenvironment
created by a wide range of biochemical, electrical and mechanical influences. Current standard cell cultivation
technologies fail to mimic this complex microenvironment, which makes them unsuitable as physical relevant
in vivo models. Therefore, the development of a controlled, biomimetic cell cultivation technology that
recreates the microenvironment of cells is of high importance especially for stem cell research. Based on the
pneumatic cell stretching system developed by Kreutzer et. al. (2014) a conceptual prototype for a transparent
electromechanical stimulation system for stem cell applications is introduced. The prototype should extend
the functionalities of the cell stretching system without affecting the functionality, benefits and capabilities
of the original device in a major way. This is realized through the incorporation of a stretchable, transparent,
electrical stimulative modular component in the existing system. Two optional approaches are introduced
for the system design: (1) Direct electrical stimulation (independent from mechanical stimulus) through
transparent electrodes fabricated from conductive polymers and (2) Indirect electrical stimulation coupled to
the mechanical stimulus created by an embedded piezoelectric layer of crystalline nanocellulose. Reference:
J. Kreutzer, L. Ikonen, J. Hirvonen, M. Pekkanen-Mattila, K. Aalto-Setälä, P. Kallio. Pneumatic cell stretching
system for cardiac differentiation and culture. Med. Eng. Phys., 36 (4), 2014.
Author: Hannu Välimäki ([email protected])
Co-author(s): Jarmo Verho, Joose Kreuzer, Jukka Lekkala
Optical oxygen sensing in cell cultures
Keyword(s): Oxygen sensing, cell cultures, thin film fluorescene
Abstract:
There is a need for continuous monitoring and control of chemical parameters in cell culturing processes.
One key parameter is the dissolved oxygen level in the medium, DO, which is known to be a powerful marker
of the metabolic status of the cells. Moreover, in specific stem cell cases, it has been observed that controlled
(typically deprived) oxygen conditions can promote growth and influence differentiation. As a part of overall
culturing condition control system development, we are currently developing novel optical technologies
for DO sensing. The technology is based on the oxygen dependent fluorescence lifetime of PtOEPK dyes
embedded in a dilute concentration (1%) in a thin (1 µm) polystyrene film deposited on culturing glass plates.
To enhance the fluorescence read-out and to reduce the optical power radiated into the culture, an optical
set-up based on parabolic lens is utilized. The design facilitates total internal reflection excitation and very
high collection efficiency of the emission. The low thickness of the sensing film leads to short response times,
and the current design has resulted in a cost-effective DO-sensor prototype with over 40 dB signal-to-noise
ratio at 10 Hz bandwidth. In addition, the low thickness and dye concentration facilitate inverted microscopy
and, very importantly, make it possible to utilize the film simultaneously as an electric insulation layer for the
microelectrode arrays and, last but not least, enhance the biocompatibility of the device. We plan to expand
the sensor for imaging set-ups, and apply it in specific cell model research cases.
Author: Joose Kreutzer ([email protected])
Research group leader: Prof. Pasi Kallio
Co-author(s): Pasi Kallio
Affiliation(s): Department of Automation Science and Engineering, BioMediTech, Tampere University of Technology,
Tampere, Finland
CELL CULTURE ENVIRONMENTS FOR IN VITRO CELL STUDIES
Keyword(s): cell culture; drug delivery; human pluripotent stem cells; hypoxia; PDMS; perfusion; pH; shear stress;
Temperature
Abstract:
Human pluripotent stem cell (hPSC) based in vitro cell models are currently actively developed. The models
can be used for studying various disease mechanisms, developing new diagnostics methods and assessing
safety of chemical compounds such as drugs, for example. To obtain reliable data for conclusive results,
disturbances and perturbations caused by the environment should be minimized also during the experiments
and measurements performed outside an incubator. This is unfortunately, however, still rarely the case. We
have studied and demonstrated a variety of unit functions for culture techniques, stimulations, and enhanced
environment control for human based stem cells for neuronal, cardiac, ophthalmic, and osteogenic in vitro
applications. These studies include among others: temperature, CO2 supply, hypoxic conditions, medium
perfusions (active and passive methods), drug delivery, mechanical stimulation of cells, and culture substrate
topographies, chemistries, and geometries to restrict the cell growth. For example, pH and temperature of
culture change relatively fast when the dish is taken out from the incubator. We have developed compact
gas exchange system around the culture to maintain the desired gas concentrations and temperature in
the culture environment during the long-term experiments, imaging and/or hypoxia studies outside the
incubator. In addition to environment control we have demonstrated that with different microstructures cells
and somas can be separated, guided, and growth restricted. The ultimate goal of the research is to integrate
the studied unit functions into a versatile system for laboratory use and research purposes for novel and
controlled cell culture environment.
Author: Ville Rantanen (1) ([email protected])
Co-author(s): Antti Vehkaoja (1), Jarmo Verho (1), Petr Veselý (2), Anton Kontunen (1),
Raisa Mattila (1), Jani Lylykangas (3), Mirja Ilves (3), Eeva Mäkelä (4), Markus Rautiainen (4),
Veikko Surakka (3), Jukka Lekkala (1)
Affiliation(s): (1) Department of Automation Science and Engineering, Tampere University of Technology, Tampere,
Finland; (2) International Clinical Research Center, St. Anne’s University Hospital Brno, Brno, Czech Republic; (3) Research
Group for Emotions, Sociality, and Computing, School of Information Sciences, University of Tampere, Tampere,
Finland.; (4) Department of Otorhinolaryngology, School of Medicine, University of Tampere, Tampere, Finland
Device for Studying Reanimation of Unilateral Facial Paralysis
Keyword(s): electromyography, facial pacing, functional electrical stimulation, prosthetic technology, unilateral facial
paralysis
Abstract:
A device for studying facial pacing for people who suffer from unilateral facial paralysis is presented. The device
is able to measure electromyographic (EMG) signals from the healthy side of the face and simultaneously
activate the paralysed side with electrical stimulation. Electronics designed for the device are integrated
to a single tabletop unit together with a myRIO embedded hardware device by National Instruments (NI).
The presented device is designed to fulfil the safety requirements of the Council Directive 93/42/EEC of the
European Union concerning medical devices and IEC 60601 standards. The device is powered by a commercial
medical grade power source. The custom electronics include isolated measurement and stimulation
amplifiers that feed the signals to and from the myRIO. Complex real-time processing can be carried out in
the myRIO’s FPGA circuit for detecting muscle activations and producing appropriate stimulation signals. The
functionality is programmed with NI LabVIEW. The device is controlled wirelessly via a computer interface,
and the measured data is logged to the computer. Clinical study with the device is about to be started at
the Tampere University Hospital. First trials will determine and compare the required stimulation signal
waveform parameters for transcutaneous activation of facial muscles of healthy participants and patients
suffering from unilateral facial paralysis. Four measurement and four stimulation channels of the device
enable studying different electrode configurations and stimulation patterns for recognizing and reanimating
symmetrical facial expressions in the future.
Author: Virpi Alarautalahti ([email protected])
Co-author(s): Virpi Alarautalahti1,2, Symantas Ragauskas3,4,Andrea Holme5,
Hannele Uusitalo-Järvinen6, Jari Hyttinen1,2, Giedrius Kalesnykas3,7, and
Soile Nymark1,2
Affiliation(s): 1 Department of Electronics and Communications Engineering, Tampere University of Technology,
Tampere, Finland; 2 BioMediTech, Tampere, Finland; 3 Department of Ophthalmology, University of Eastern Finland,
Kuopio, Finland; 4 Institute of Innovative Medicine, Vilnius, Lithuania; 5 Flow Cytometry Core, University of Alberta,
Edmonton, Canada; 6 Department of Ophthalmology, University of Tampere, Tampere, Finland; 7 Experimentica Ltd.,
Kuopio, Finland
Functional Characterization of Ex Vivo Retinal Explants
Keyword(s): electrophysiology, retinal culture
Abstract:
In vitro retina model systems are needed to investigate the effects and responses of the treatments to the
retinal tissue. In this study, we developed a culture system for ex vivo retinal explants from adult mice and
characterized it using electrophysiological and immunohistochemical methods. Retinae of young C57Bl/6J
mice were cultured in serum free medium supplemented with B27 and N2 under ex vivo conditions for up
to 14 days. We used perforated multielectrode arrays to record neuronal responses of retinal ganglion cells
(RGCs) from retinal explants. Spontaneous and light-evoked activity of RGCs was recorded during the 14-day
period. Morphology and structural organization of the tissue was also examined using immunohistochemistry.
Cultured retinal explants preserved their functionality remarkably well even though neuronal activity was
gradually lost during the culturing period. Spontaneous electrical activity of RGCs could be seen up to 14 days
in culture. Light-evoked activity was lost faster, during the first week in culture. Our immunohistochemical
analysis supported the results from electrophysiological characterization. Retinal morphology was retained
for 14 days, although a thinning of the outer nuclear layer was observed. Based on our results, retinal explants
from adult mice retain their functionality and morphology for a remarkably long time in culture conditions.
This culture system thus represents a valuable tool in biomedical research for studies on responses of retinal
tissue to pharmalogical compounds.
Author: Anne Skogberg ([email protected])
Co-author(s): Pasi Kallio
Affiliation(s):
Nanocellulose surfaces as cell culture substrates
Keyword(s): Nanocellulose, cell culture substrate, cytotoxicity analysis
Abstract:
Cellulose is the most abundant natural polymer on earth and economically feasible new high value products
from forest industry would be desired. Wood based nanostructured celluloses, cellulose nanofibers (CNF)
and cellulose nanocrystals (CNC) can take various forms such as gels and films, and have potential biomedical
applications. Nanocellulose is interesting for cell culturing and tissue engineering applications due to its nonanimal or non-human origin, its availability, biocompatibility, possibility for controlled enzymatic degradation,
and various surface modification possibilities. The primary objective is to study various nanocellulose surfaces
as cell culture substrates, using different nanocelluloses, manufacturing and coating methods. Existing model
surfaces and commercial products will be used as a reference. The prepared surfaces will be characterized
e.g. with AFM, SEM, surface plasmon resonance and microrobotic tools. The second objective is to examine
cell adhesion, growth, proliferation and viability of nanocellulose surfaces using specified cell types. Surface
plasmon resonance will be used to study interaction between cells and nanocellulose surfaces, and to
perform cytotoxicity analysis.
Author: Antti Vehkaoja ([email protected])
Research group leader: Professor Jukka Lekkala
Co-author(s): Vala Jeyhani, Jukka Lekkala
Affiliation(s): Tampere University of Technology, Department of Automation Science and Engineering
Evaluation of short distance chest area electrode locations for respiration rate monitoring
with impedance pneumography technique
Keyword(s): Respiration monitoring, Impedance pneumography
Abstract:
Respiration rate is one of the most important and the most sensitive physiological parameters used in
monitoring the development of the health status of hospitalized patients. It is one of the five parameters
in widely used Early Warning Score (EWS) patient status assessment scale. Traditionally, EWS has been
determined intermittently and manually by the nursing personnel but recently there has been increasing
interest for developing unobtrusive health monitoring devices that would continuously monitor the essential
vital signs and set an alarm if e.g. the EWS score shows that the status of the patient is deteriorating. Impedance
pneumography is one possible method for measuring the respiration rate. In impedance pneumography, a
small current is injected to the body through electrodes located at the torso and impedance between the
electrodes is measured. The impedance varies along with respiration when the volume of the lungs increases
and decreases. Unobtrusive continuous health monitoring could be performed e.g. with a small patch attached
to the chest of the patient. However, short inter electrode distance does not necessarily provide adequate
measurement sensitivity for reliable respiration monitoring with impedance pneumography technique.
We evaluated the signal quality and respiration measurement performance of ten short distance electrode
pairs in frontal thorax area. From the tested configurations, the electrodes located at upper sternum and at
location V5 of the 12-lead ECG system provided the most accurate respiration rate and were least affected
by movement artefacts.
Mitochondria
Author: Cagri Yalgin ([email protected])
Research group leader: Howard T. Jacobs
Co-author(s): Howard T. Jacobs
Affiliation(s): BioMediTech, University of Tampere, Tampere, Finland (CY, HTJ); Institute of Biotechnology, University of
Helsinki, Helsinki, Finland (HTJ); Tampere University Hospital, Tampere, Finland (HTJ)
Neurological function, dysfunction and rescue in an animal model of cytochrome oxidase
deficiency
Keyword(s): Cytochrome oxidase, alternative oxidase, mitochondrial disease, Drosophila melanogaster
Abstract:
Mitochondrial diseases affect one in 5000 individuals with varying degrees of disability. Treatment is limited
and there is no cure. Mutations affecting the cytochrome oxidase (COX) cause musculoskeletal disease,
presumably by disturbing the electron flow in the respiratory chain, which may lead to decreased ATP
synthesis and increased ROS production. In the light of our previous work in which an alternative oxidase
(AOX) saved a range of phenotypes due to COX deficiency in Drosophila melanogaster, we are now focusing
on the effect of AOX on neuronal COX deficiency. Pan-neuronal downregulation of a COX subunit caused
30-40% pupal lethality, and a severe locomotor deficit in newly eclosed adult flies. AOX co-expression in
the same cells partially rescued the locomotor deficit in a dose-dependent manner, and fully rescued the
pupal lethality. In order to find which specific types of neurons are involved in this deficit and its rescue, we
are running a Gal4 screen: Through this approach we will downregulate COX in cholinergic neurons but also
in adrenergic, serotonergic, dopaminergic neurons and in glia, and study animal s locomotion. We expect
COX downregulation to recapitulate the locomotor deficit in at least one of these cell subtypes. We are also
studying changes in cell number, mitochondrial organization, metabolism and muscle innervation in situ
and in vitro with histological and metabolic assays on the fly brain and muscle. These data will guide our
mammalian research on the therapeutic use of AOX in COX-related disease models.
Mitochondria
Author: Sina Saari ([email protected])
Research group leader: Howy Jacobs
Co-author(s): Marcos T. Oliveira, Eric Dufour, Howard T. Jacobs
Affiliation(s):
Dietary Restriction Affects the Development of Drosophila melanogaster Expressing the
Ciona intestinalis Alternative Oxidase
Keyword(s): mitochondria, alternative oxidase, drosophila
Abstract:
We have expressed the mitochondrial alternative oxidase (AOX), a respiratory chain alternative enzyme
from the sea squirt Ciona intestinalis, in the fruit fly Drosophila melanogaster. In this system, AOX is able
to counteract deleterious effects of dysfunction in complexes III and/or IV of the oxidative phosphorylation
(OXPHOS) system suggesting that AOX could potentially be used in gene therapies for human mitochondrial
diseases that currently have no treatment. However, the natural lack of alternative enzymes in the
mitochondria of arthropods and vertebrates suggests a possible impediment to this approach. The focus of
our work was to investigate possible disadvantages of AOX expression on the metabolism and development
of D. melanogaster, under conditions that mimic ecological scenarios in which resources are limited. The
flies were reared on restricted diets that, unlike the standard laboratory diet, only included glucose and yeast
extract, as a source of amino acids. The results show that whilst developing normally on the standard diet,
on restricted diets AOX-flies showed lower pupal viability compared to AOX-nonexpressing flies. The results
suggest that AOX may decrease maximal mitochondrial ATP production needed for the development of fastmoving metazoans, such as D. melanogaster, which in turn may account for the loss of the gene coding for
the enzyme during evolution, despite its ability to regulate mitochondrial redox homeostasis and reduce the
damaging effects of ROS production when OXPHOS is dysfunctional. Whether this developmental defect is
due exclusively to calorie restriction or lack of specific supplements in the diet is yet to be determined.
Mitochondria
Author: Katharina Bremer ([email protected])
Research group leader: Howard T. Jacobs
Co-author(s): Howard T. Jacobs, Paul V. Debes
The functional role of the alternative oxidase in the tunicate Ciona intestinalis
Keyword(s): Alternative oxidase, Ciona intestinalis, Metabolism
Abstract:
In many species, the alternative oxidase (AOX) allows for an alternative mitochondrial electron transport chain
(ETC) pathway compared to the classic ETC. The classic ETC, comprised of four enzyme complexes (CI-CIV), is
essential for aerobic energy production via oxidative phosphorylation (OXPHOS). During ETC impairments at
the level of CIII and/or CIV, AOX can maintain the electron flow, thereby alleviating metabolic and signalling
stress. Interestingly, vertebrates have lost the AOX gene, whereas the tunicate Ciona intestinalis, closely
related to vertebrates, still possesses AOX. However, information on the function of AOX in animals in general,
and in Ciona specifically, is scarce. Currently, Ciona-derived AOX is studied in mouse and fly models to explore
the potential of AOX as therapeutic tool for mitochondrial diseases. Together, understanding the physiological
role of AOX in Ciona not only advances the field of general mitochondrial physiology but also mitochondrial
disease mitigation. So far, I investigated the transcriptional response of AOX to extreme temperatures, high
trace metal, and low pH levels in different tissues (heart, neural complex, ovaries, stomach) in Ciona adults
and during development. During both life stages, no AOX-specific transcriptional response was observed to
any of the stressors. However, mRNA levels of AOX followed the same trend as two CIV subunits and were
more abundant in adult s ovaries compared to other tissues across treatments. These results suggest that
AOX is still actively transcriptionally regulated in adult Ciona and as maternally-derived protein may have an
important function during Ciona development under certain circumstances.
Mitochondria
Author: Priit Joers ([email protected])
Research group leader: Howard Jacobs
Co-author(s): Sampsa Järvinen, Jack George, Cagri Yalgin, Alba Naudi Farree
Affiliation(s): BioMediTech, University of Tampere, Finland; University of Lleida, Spain
mtDNA - more than just coding for genes
Keyword(s): mitochondrial genome, metabolic flexibility, diabetes
Abstract:
Mitochondria has its own genome, coding for 13 transmembrane subunits of ETC complexes in animals.
Defects in mt genome cause the rise of abnormal subunits, leading to dysfunctional ETC. In that case electrons
start missing their designated targets and react with oxygen, forming a group of highly reactive compounds
termed reactive oxygen species (ROS). Therefore malfunctions in mt genome coding capacity lead to decreased
OXPHOS and elevated ROS with numerous donwstream effects harmful for an organism. Therefore disorders
affecting coding and expression of mitochondrial genes has always been viewed through the prism of
decreased mitochondrial function and increased ROS production. My research in Drosophila has challenged
this paradigm as I have discovered a novel way how mtDNA stress affects cellular metabolism. When adult
flies are subjected to action by mitochondrially-targeted bacterial scanning endonuclease EcoBI, they die
rapidly. However, general mitochondrial biochemistry remains undisturbed very little damage to mtDNA, no
change in OXPHOS, unchanged mt transcript and ROS levels and no induction in other mitochondrial stress
conditions like unfolded protein response. Instead flies exhibit massive loss of storage fats, no alterations in
carbohydrate stores and heavily increased circulating glucose. Moreover, this is accompanied with complete,
but highly selective deactivation of Akt, a central kinase in several major pathways linking nutritional state to
cellular homeostasis. These results indicate that in addition to coding for genes, mtDNA can act as a sensor
that controls the metabolic flexibility and thus might hold the key to treatments of metabolic diseases.
Mitochondria
Author: Suvi Vartiainen ([email protected])
Research group leader: Howard Jacobs
Co-author(s): Ashwin Sriram, Filippo Scialo, Alberto Sanz, Ming Guo
Affiliation(s): University of Newcastle, UK, University of California, Los Angeles
Overexpression of Rheb mitigates mitochondrial pathologies arising from Pink1deficiency
in Drosophila
Keyword(s): Parkinson’s Disease, mitochondria, muscle, Drosophila
Abstract:
Certain mutations in Pink1 in human cause autosomal recessive Parkinson s disease. In the model organism
Drosophila melanogaster, functional deficiency of Pink1 leads to morphologically abnormal, enlarged
mitochondria in muscle. Their accumulation has been attributed to dysfunctional mitochondrial turnover,
with muscle being the most severely affected tissue due to its high energy requirements. Overexpression of
the G protein Rheb was able to restore almost normal mitochondrial morphology in the muscles of Pink1
knockdown or mutant flies. Rheb has been shown to localize to actively respiring mitochondria, and promote
mitochondrial turnover regardless of membrane potential. Its ability to rescue the effects of Pink1 knockdown
was dependent on the autophagy machinery, but not on Parkin or a protein MAPL/Mul1 implicated in the
turnover of mitochondria-derived vesicles. We propose that constitutive activation of autophagy by Rheb bypasses the requirement for the signaling function of Pink1 in the specific turnover of damaged mitochondria.
Mitochondria
Author: Jack George ([email protected])
Co-author(s):
Affiliation(s):
The PGC-1 Homologue Spargel in Drosophila melanogaster and its Role in Early Development
Keyword(s):
Abstract:
Background: dPGC1 (a.k.a Spargel) is the Drosophila melanogaster homologue of the mammalian PGC1
family of transcriptional coactivators. Whilst it is known that Spargel regulates similar functions to its
mammalian counterparts within the Drosophila fat body (mitochondrial biogenesis and mitochondrial
activity), it is otherwise understudied in this invertebrate model organism. Further elucidation of its roles
in D.Melanogaster could provide novel insights on this family of genes that may not have yet been fully
appreciated/understood in mammals. Here I present such an example, whereby dPGC1 expression is shown
to be essential in the female germline for embryo viability. Principal Findings: Spargel mRNA accumulates
during oogenesis and early embryogenesis. Spargel knockdown at these stages leads to a collapse in normal
transcriptional regulation during embryogenesis and the resultant embryos go into developmental crisis.
These embryos are unable to develop into larvae and therefore die during embryogenesis. Conclusions/
Significance: Spargel is an important determinant of D.Melanogaster embryo viability and its strict control is
essential for coordinated embryogenesis.
Mitochondria
Author: Marten Szibor ([email protected])
Co-author(s): Eric Dufour, Marion Wiesnet, Nadine Woitasky, Rolf Schreckenberg,
Praveen K. Dhandapani, Maarit Myöhänen, Luca Giordano, Klaus-Dieter Schlüter,
Thomas Braun, Howy Jacobs
Affiliation(s): BioMediTech University of Tampere, Max-Planck-Institute for Heart and Lung Research Bad Nauheim,
Institute of Physiology University of Giessen, BI University of Helsinki
Effect Of Alternative Oxidase (AOX) On Mitochondrial Function In The Post-Ischemic Mouse
Heart
Keyword(s): heart failure, ischemia-reperfusion, alternative oxidase, mouse
Abstract:
Ischemic cardiac events are a leading cause for heart failure in the Western world ultimately leading to
premature death. The involvement of mitochondria has recently come more into focus because in the
post-ischemic heart activities of respiratory chain complexes are impaired. Although renormalized after
organ remodeling, already a transient disturbance of the electron flow affects not only the ATP production
in cardiomyocytes but it also impacts on metabolic circuit activities, shifts the cellular phenotype towards
apoptosis by mitochondrial cytochrome c release, and results in superabundant levels of superoxide. Especially
the high superoxide levels are detrimental for the cellular viability since it further damages mitochondrial
and nuclear DNA as well as proteins of the respiratory complexes thereby starting a vicious cycle of cell
impairment. Many lower organisms express an alternative oxidase (AOX) to protect from insults that impair
electron flow through the respiratory complexes. Under such conditions, AOX can accept electrons from
ubiquinol to directly reduce oxygen thereby branching the classical respiratory chain. As a consequence, the
electron flow is maintained and with it metabolic circuit activities, and the superoxide production remains
low. We expressed AOX from Ciona intestinalis in mouse and studied its effects on mitochondrial function
and organ remodeling after cardiac ischemia-reperfusion. Approaches like our xenotopic expression of AOX
in disease models allow a novel view on development and progress of many diseases, and may allow radically
different therapeutic approaches to treat the actual causes of diseases instead of alleviating symptoms.
Mitochondria
Author: Ana Andjelkovic ([email protected])
Co-author(s): Kia K. Kemppainen, Howard Jacobs
Affiliation(s): PhD student
AOX and cleft thorax in Drosophila melanogaster
Keyword(s): alternative oxidase, Drosophila, cleft thorax, ROS
Abstract:
Misregulation of epithelial gap closure results in birth defects including spina bifida and cleft palate, and it can
lead to impaired wound healing. We are using a genetic analysis approach in fruit fly to dissect the signaling
pathways regulating epithelial morphogenesis. Dorsal and thorax closure are two distinct morphogenetic
movements during Drosophila development where epithelial sheets move to seal the embryo at the dorsal
midline or to join the parts of the wing imaginal discs to form thorax, respectively. Defects in JNK signaling
result in failure of the dorsal closure and produce a cleft thorax phenotype in flies. My reasoning is that the
identification of JNK effectors that rescue the cleft thorax phenotype in Drosophila may do so in humans
as well. There is a critical role for ROS in intracellular signaling, including activation of the JNK pathway.
We hypothesized that mitochondrial ROS is an effector of JNK signaling, and thus may control epithelial
morphogenesis. To test this hypothesis, we introduced an alternative respiratory pathway in Drosophila which
bypasses the normal ROS-producing respiratory complexes; a novel strategy to manipulate mitochondrial
ROS production. I expressed the Alternative Oxidase (AOX) in pvr, hep, bsk, d-jun or d-fos knockdown flies,
which exhibit cleft thorax. AOX expression significantly decreased the incidence of cleft thorax produced by
the knockdown of hep, bsk and pvr. In contrast, AOX had no effect when d-jun or d-fos were downregulated.
From this study, I identified D-fos and/or D-Jun (AP-1) as the target of the AOX-dependent regulation.
Regenerative Medicine
Author: Tanja Ilmarinen (1) ([email protected])
Research group leader: Heli Skottman (1)
Co-author(s): Hanna Hiidenmaa (1), Peeter Kööbi (2), Soile Nymark (3), Anni Sorkio (1),
Jing-Huan Wang (4), Päivi Alajuuma (5), Olli Oksala (5), Hannu Uusitalo (2)
Affiliation(s): 1) BMT, UTA; 2) Department of Ophthalmology, SILK, UTA and TAYS Eye Center; 3) TUT, Department of
Electronics and Communications Engineering, BMT; 4) Department of Ophthalmology, SILK, UTA; 5) Santen Oy
Subretinal Biocompatibility of Ultrathin Polyimide Membranes Seeded with Retinal Pigment
Epithelium Cells Differentiated from Human Embryonic Stem Cells
Keyword(s): Human pluripotent stem cells, retinal pigment epithelium, polyimide, transplantation
Abstract:
Retinal pigment epithelium (RPE) degeneration has a major role in pathogenesis of retinal diseases such
as age-related macular degeneration (AMD). Transplantation of RPE differentiated from human pluripotent
stem cells (hPSC) offers a promising treatment for AMD. In many transplantation studies, including our
own, survival and integration of suspended RPE cells are poor. A scaffold would facilitate implantation and
integration of the graft. Previously, we have shown that polyimide (PI) membrane is suitable for growth of
RPE differentiated from human embryonic stem cells (hESC). In this study, we investigated the suitability of PI
for subretinal implantation of hESC-RPE in rabbits. The surface properties of PI membranes were studied with
scanning electron microscopy (SEM) and atomic force microscopy (AFM). PI with and without hESC-RPE were
implanted in the subretinal space of New Zealand White rabbits. Animals received oral immunosuppression
the entire follow-up time. In vivo follow-up was performed with fundus photography, electroretinography
(ERG), and optical coherence topography (OCT). Histological examination was done three months after
implantation. In surface analysis, PI showed good porosity characteristics. After transplantation, a gradual loss
of pigmentation on hESC-RPE-PI was detected, potentially due to cell death of the implanted cells. In the eyes
with PI alone, no obvious signs of inflammation or retinal atrophy were observed. However, mononuclear cell
infiltration and retinal atrophy were observed around the hESC-RPE-PI implants. The ultrathin PI was welltolerated in the subretinal space. The oral immunosuppression was potentially insufficient for reduction of
xenograft induced inflammation and needs to be adjusted in the future.
Author: Iina Vainio1 ([email protected])
Research group leader: Jari Hyttinen1
Co-author(s): Britta Nommiste2, Lyndon da Cruz3, Andrew Webster2,3,
Tony Moore2,3, Pete Coffey2, Olaf Strauss4, Jari Hyttinen1, Kati Juuti-Uusitalo5,
Amanda-Jayne Carr2, and Soile Nymark1
Affiliation(s): 1Tampere University of Technology, Department of Electronics and Communications Engineering,
BioMediTech, Tampere, Finland, 2UCL Institute of Ophthalmology, London, UK, 3Moorfields Eye Hospital, London, UK
4Charité University Medicine Berlin, Ophthalmology, Berlin, Germany 5University of Tampere, BioMediTech, Tampere,
Finland
RPE protein bestrophin1 affects Ca2+ channel functionality in a patient with autosomal
recessive bestrophinopathy
Keyword(s): Retinal pigment epithelium, autosomal recessive bestrophinopathy, stem cells, patch clamp, confocal
microscopy, voltage-gated Ca2+ channels
Abstract:
Autosomal recessive bestrophinopathy (ARB) is a retinal dystrophy characterized by loss of central vision. ARB
is related to abnormalities in retinal pigment epithelium (RPE) protein Bestrophin 1 (BEST1) that is proposed
to act as a transmembrane anion channel. However, the exact function of BEST1 in the RPE is unknown. In
this study we aim to investigate the effect of BEST1 mutation on the L-type Ca2+ channel functionality. To
achieve this goal we produced induced pluripotent stem cells (iPSCs) from an ARB patient with a nonsense
p.R200X BEST1 mutation and differentiated iPSCs into RPE. The iPSC-RPE formed a pigmented monolayer
with cobblestone morphology expressing classic RPE markers. The p.R200X mutation was confirmed and the
full length BEST1 protein was undetectable. Perforated patch clamp method was used to analyse currents
through voltage-gated Ca2+ channels. We detected decreased currents through voltage-gated L-type Ca2+
channels in ARB iPSC-RPE compared to control fetal iPSC-RPE. In addition, the confocal images resulted in
lower L-type channel expression level in ARB iPSC-RPE compared to control fetal iPSC-RPE. Our results suggest
that p.R200X BEST1 mutation disrupts the interaction between BEST1 and L-type Ca2+ channels reducing the
currents through L-type Ca2+ channels. This is possibly caused by a decrease in the number of pore-forming
L-type Ca2+ channel subunits in the cell membrane due to disturbed interaction between BEST1 and the
regulatory L-type Ca2+ channel subunit.
Author: Paavilainen Tanja ([email protected])
Research group leader: Susanna Narkilahti
Co-author(s): Pelkonen Anssi, Fayuk Dmitriy, Mäkinen Meeri, Narkilahti Susanna
Role of astrocytes in formation of functional neuronal networks in vitro
Keyword(s):
Abstract:
Astrocytes are the most numerous cell type in the CNS involved in normal development as well as dysfunctions
of neuronal networks. Growing evidence indicates that astrocytes can affect the neurodevelopmental process
and maturation of hPSC derived neuronal networks. So far, astrocyte role in neuronal networks is mainly
studied in rodent in vivo models. Advances in the stem cell field have enabled differentiation of human
pluripotent stem cells (hPSCs) into neurons and astrocytes, and creation of functional neural networks in
vitro. hPSC-derived in vitro models may provide additional platforms for studying astrocyte role in neuronal
networks. Here, we differentiated human pluripotent stem cells into 1) neuron enriched and 2) astrocyte
enriched cultures. Properties of these cultures were compared with immunocytochemistry, microelectrode
array measurements (MEA) and calcium imaging. The results show that the amount of astrocytes affects the
functional development of neuronal networks. To get closer to generating functional neuronal networks that
behave as networks do in the brain, we must include astrocytes to better mimic the cellular components that
comprise the network.
Author: Sanni Virjula1,2 ([email protected])
Research group leader: Susanna Miettinen
Co-author(s): Feihu Zhao3, Joni Leivo2,4, Sari Vanhatupa1,2, Joose Kreutzer2,4,
Anna-Maija Honkala1,2, Pasi Kallio2,4, Susanna Miettinen1,2
Affiliation(s): 1 Adult Stem Cell Group, UTA 2 BioMediTech 3 Biomechanics Research Centre, Biomedical Engineering,
College of Engineering and InInformatics, National University of Ireland, Galway, Ireland 4 Department of Automation
Science and Engineering, TUT
Mechanical Stimulation of Human Adipose Stem Cells: The Effects on Osteogenic
Differentiation and Cell Mechanical Properties
Keyword(s): adipose stem cells, mechanical stimulation, cell stretching, atomic force microscopy indentation, osteogenic
differentiation
Abstract:
Tensile strain is known to affect stem cell behavior and differentiation, and stretching has emerged as a
promising differentiation method also for bone tissue engineering besides traditionally used chemical
supplements. However, the significance of this stimulus on the osteogenic differentiation of human adipose
stem cells (hASCS) remains unclear. In this study, we investigated the effect of long-term mechanical
stimulation on the attachment, osteogenic differentiation and mechanical properties of hASCS in vitro.
The hASCs were exposed to cyclic equiaxial stretching for 10 days in osteogenic culture conditions using
a tailor-made, pneumatic cell stretching device. The cells were followed by light microscopy imaging, and
the proliferation and osteogenic differentiation were analyzed quantitatively using CyQUANT® and alkaline
phosphatase activity assays on days 0, 6 and 10. Cell morphology, cytoskeletal orientation and focal adhesions
were visualized using immunocytochemical vinculin and phalloidin staining on days 3 and 6. Furthermore,
mechanical properties of hASCs in terms of apparent Young s modulus and normalized contractility were
obtained by atomic force microscopy based indentation and computational approaches. Our results indicated
that equiaxial stretching inhibited proliferation and enhanced osteogenic differentiation of hASCs when
combined with chemical stimulation. Stretching also reduced cell size, enhanced cytoskeletal orientation
and made focal adhesions stronger and localized wider through the whole cell-substrate interface. Moreover,
cell stiffening was observed during osteogenic differentiation and especially under mechanical stimulation.
These results suggest that stretching modifies cell morphology, focal adhesions and mechanical properties of
hASCs, and it could be exploited to enhance the osteogenic differentiation.
Author: Miina Ojansivu (1,2,3) ([email protected])
Co-author(s): Sari Vanhatupa (1,2,3), Xiaoju Wang (4), Minna Kellomäki (2,5),
Leena Hupa (4) and Susanna Miettinen (1,2,3)
Affiliation(s): 1 Adult stem cell group, UTA, 2 BioMediTech, 3 Science Center, Tampere University Hospital, 4 Johan
Gadolin Process Chemistry Centre, Åbo Akademi University, 5 Biomaterials and Tissue Engineering Group, TUT
Molecular mechanisms of bioactive glass induced osteogenic differentiation of human
adipose stem cells
Keyword(s): adipose stem cell, bioactive glass, osteogenic differentiation, cell attachment, cell signaling
Abstract:
Bioactive glasses (BaGs) have been widely exploited in bone tissue engineering due to their inherent ability
to induce osteogenic differentiation of stem cells without additional chemical supplements. However, the
molecular level response of cells to BaGs is currently poorly understood. In order to elucidate the mechanism
of BaG induced osteogenic differentiation we cultured human adipose stem cells (hASCs) on BaG discs S53P4
and 1-06 in the presence of inhibitors for focal adhesion kinase (FAK), extracellular signal-regulated kinase
(ERK1/2), p38 and c-Jun N-terminal kinase (JNK). The activation of FAK and mitogen-activated protein kinases
(MAPKs) on the glasses at 6h, 3d and 7d time points was studied by Western blot analysis. Cell attachment on
the BaGs was examined by vinculin immunocytochemistry as well as integrin²1 and vinculin immunoblotting.
The osteogenic differentiation was evaluated by quantitative alkaline phosphatase activity and the expression
of osteogenic marker genes RUNX2a and OSTERIX at 14 and 21 days. Based on our results FAK and ERK1/2
phosphorylation was upregulated by glass contact, and both BaGs stimulated hASC osteogenic differentiation
which was significantly reduced by inhibition of FAK, ERK1/2, p38 and JNK signals. Cells cultured on the
BaGs had enhanced integrin²1 and vinculin production, and focal adhesions were smaller but more densely
arranged when compared to the polystyrene control. Taken together, FAK and MAPKs seem to be important
players in cellular response to BaGs. The glass surface also had a distinct effect on cell attachment mode
which may be one underlying factor explaining the osteogenesis-inducing ability of BaGs.
Author: Heidi Hongisto ([email protected])
Research group leader: Heli Skottman
Co-author(s): Heidi Hongisto (1), Jochen Rieck (1), Antti Jylhä (2), Tanja Ilmarinen (1),
Zoltán Veréb (3), Ulla Aapola (2), Roger Beuerman (4), Hannu Uusitalo (2,5),
Goran Petrovski (3), Heli Skottman (1)
Affiliation(s): (1) BMT, UTA; (2) Department of Ophthalmology, SILK, UTA; (3) University of Szeged, Hungary; (4)
Singapore Eye Research Institute, Singapore; (5) TAYS Eye Center
Comparative proteomic analysis of human embryonic stem cell-derived retinal pigment
epithelial cells and primary human RPE
Keyword(s): Human embryonic stem cell, Retinal pigment epithelium, Proteomics
Abstract:
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the potential to
provide an infinite cell source for cellular replacement therapies and clinical trials with hESC-RPE to treat
retinal degenerative diseases are ongoing. Many groups, including ours, have demonstrated that hESC-RPE
show features similar to native RPE including morphology, relevant gene and protein expression, epithelial
barrier properties, and functionality demonstrated as key cytokine secretion and phagocytosis capacity. To
extensively characterize the proteomics of hESC-RPE we compared the protein expression profiles of two
hESC-RPE cell lines to primary human RPE from cadaver donors using isobaric tags for relative quantitation
(iTRAQ) technology. Total protein was extracted, digested with trypsin, and peptides were labeled with
iTRAQ labels. Analyses were done with Nano-RPLC- TripleTOF instrumentation (Ab Sciex). The comparative,
quantitative proteomic profiling showed the hESC-RPE to closely resemble their native counterparts with
over 1000 common proteins identified. Overall expression profiles between the hESC-RPE lines were very
similar, showing that there was little cell line specific variation. Most proteins were expressed in hESCRPE cells at levels similar to human RPE. The protein expression reflected both the origin and the cellular
microenvironment of the samples. For example, a large number of mitochondrial proteins were present and
expressed at lower level in the hESC-RPE cells compared to native RPE, reflecting reduced energy requirement
profile under cell culture conditions. Important proteins involved in specialized functions of the RPE were
identified such as proteins involved in phototransduction/visual cycle and transporter proteins.
Author: Tiina Joki ([email protected])
Co-author(s): Laura Ylä-Outinen, Susanna Narkilahti
Affiliation(s): NeuroGroup/BioMediTech, University of Tampere, Finland
Characterization of human neural networks cultured on hydrogel matrix
Keyword(s): Hydrogel, Neural cell, 3D culture,
Abstract:
Background: Central nervous system (CNS) deficits are difficult to treat and often cause severe loss of normal
functionality to patient. Regenerative medicine may in the future offer solutions to help these patients. Cell
transplantation therapies and sophisticated 3D tissue models, could offer clinically relevant treatments or
diagnostic tools, for currently incurable deficits. In 3D, biomaterials can be utilized as extra cellular matrix
(ECM) mimicking substance to support cells. In neural field, hydrogels are considered as potential biomaterial
group due to properties similar to native neural tissue. Aims: The aim of this work was to study human
neural cell behavior when cultured with synthetic hydrogels. Methods: Human pluripotent stem cell derived
neural cells were cultured on top of hydrogel, under the hydrogel or mixed into the hydrogel. Laminin protein
coating was used as positive 2D growth control and non-coated cell culture plastic as negative control. The
cells were characterized using viability analysis, time-lapse monitoring and immunocytochemical staining
against neural markers. Results: Hydrogels were non-cytotoxic and supported cell growth. Cells both under
and on top of the hydrogels (2D cultures) were positive for neural markers. Gelation did not cause negative
effect to cultures. Cells inside the hydrogel were viable and migrating in 3D culture. Conclusions: According
to our in vitro studies, the material was able to support the neural cell viability and maturation. No negative
effect due to gel was observed thus the material seems very promising for further studies.
Author: Laura Hyväri ([email protected])
Co-author(s): Miia Juntunen, Miina Ojansivu, Susanna Miettinen and Sari Vanhatupa
Affiliation(s): Adult Stem Cell Research Group, University of Tampere, Finland; BioMediTech, University of Tampere,
Finland; Science Center, Pirkanmaa Hospital District, Finland.
The role of FAK, ERK and ROCK Signaling in the Osteogenic and Adipogenic Differentiation
of Human Adipose Stem Cells
Keyword(s): adipose stem cell, cell adhesion, adipogenic differentiation, osteogenic differentiation, cell signaling, FAK,
ERK, ROCK
Abstract:
Cell adhesion, mediated primarily through integrins and focal adhesions (FA), has been reported to regulate
cell differentiation in mesenchymal stem cells (MSCs). Focal adhesion kinase (FAK) is a central mediator of
integrin-activated signaling which is interconnected with various cellular pathways including Extracellular
signal-regulated kinase (ERK) pathway and Rho-ROCK (Rho-associated protein kinase) pathway. However, the
detailed molecular mechanisms that regulate differentiation lineage commitment remain largely unknown.
The aim of the present study was to assess the significance of the FAK, ERK and ROCK pathways on the
differentiation of human adipose stem cells (hASCs) towards osteogenic and adipogenic lineages. Inhibitor
molecules targeted to these pathways were used to block the signal transduction. Osteogenic differentiation
was assessed by analyzing the alkaline phosphatase (ALP) activity, matrix mineralization and the expression of
osteogenic marker genes OSTERIX, DLX5 and RUNX2a. Adipogenic differentiation was analyzed by assessing
the fat vacuole formation with Oil red O staining, and the expression of adipogenic marker genes LEPTIN and
aP2. Our results indicated that the inhibition of FAK, ERK and ROCK function suppressed both cell proliferation
and osteogenic differentiation dose-dependently. Adipogenesis was increased as a result of ROCK inhibition
and also the inhibition of FAK and ERK cascades appeared to have a slightly increasing effect on adipogenesis.
The ROCK inhibition also affected cell morphology leading to rounded shape of the cells. As a conclusion,
these results suggest that the cell adhesion and morphology modulated by FAK, ERK and ROCK signaling are
relevant regulators of the osteogenic and adipogenic differentiation of hASCs.
Author: Musammir Khan ([email protected])
Research group leader: Minna Kellomaki
Co-author(s): Michael Zharnikov2
Affiliation(s): Tampere University of Technology, 2 Heidelberg University, Heidelberg Germany
Chemical Derivatization and Biofunctionalization of Hydrogel Nanomembranes for Potential
Biomedical and Biosensor Applications
Keyword(s): Hydrogel Nanomembranes, Swelling, Derivatization, Specific Protein Adsorption, Biocompatibility
Abstract:
Poly(ethylene glycol) based hydrogel nanomembranes (PHMs) haveare demonstrated to be able to host
protein-specific receptors, providing, at the same time, protein-repelling and humidity-responsive matrix
with a characteristic mesh size up to 8.4 nm. The membranes were prepared by crosslinking of amino- and
eboxyepoxy-terminated STAR-PEG precursors and maintained their hydrogel and protein-repelling properties
even at a deviation of the precursor composition from the equilibrium value (1:1). The grafting density of
the test avidin proteins, specifically attached to the biotin moieties coupled to the free amine groups in the
PHMs, varied from 0.45x10^12 to 1.3x10^12 proteins/cm2 within the sampling depth of the experiments
(~11.5 nm), depending on the precursor composition, whereas the analogous values for the non-specifically
adsorbed proteins were lower by a factor of 4-5. The engineering of PHMs with biomolecule-specific
receptors and their loading with biomolecules is of potential interest for sensor fabrication and biomedical
applications, including tissue engineering and regenerative therapy.
Other / Printable electronics and sensors
Author: Mitra Akbari (1) ([email protected])
Research group leader: Leena Ukkonen
Co-author(s): Jari Juuti(2), Lauri Sydänheimo(1), Leena Ukkonen(1)
Affiliation(s): (1) Department of Electronics and Communications Engineering, Tampere University of Technology,
Tampere, FI-33101, Finland (2) Microelectronics and Materials Physics laboratories, University of Oulu, Oulu, FI-90570,
Finland
Characterization of Flexible Graphene Oxide Film Reduced by Pulsed Xenon Flash Printable
electronics and sensors
Keyword(s): Graphene; Xenon flash reduction; inkjet printing.
Abstract:
Graphene based nanomaterials have an excellent potential to implement in the electronic devices due to its
unique and superior electrical, mechanical, and physical properties. The real challenge of using graphene is
related to deposition methods and processes which affect dramatically on the final properties. In this study,
we present a water-based graphene ink consisting of graphene oxide and polystyrene dissolved in deionized
water. The following ink was inkjet printed on oxygen plasma treated Polyimide substrate (Kapton, TM).
The fabricated samples were annealed, and then reduced by pulsed Xenon flash within the microsecond s
ranges and at room temperature. The advantages of replacing Xenon flash reduction instead of conventional
methods such as chemical and thermal reduction, are to save time and energy along with the possibility of
using various kinds of substrates. The energy, amplitude and width of each pulse were adjusted to achieve
the optimum flash settings for the deposited graphene oxide. The reduced samples were characterized to
observe the influence of different processing conditions of the reduced graphene oxide films which have the
potential to be used as a sensing part in mechanical, environmental or biological sensors.
Other / Stem Cells
Author: Mäkinen Meeri ([email protected])
Co-author(s): Hyysalo Anu, Narkilahti Susanna
Affiliation(s): NeuroGroup, BioMediTech, University of Tampere, Finland
Electrophysiological characterization of human embryonic stem cell derived oligodendrocyte
precursor cells Stem Cells
Keyword(s): oligodenrocyte precursors, human pluripotent stem cells, electrophysiology
Abstract:
Oligodendrocytes are cells which myelinate the axons of the brain. Oligodendrocyte precursor cells (OPCs)
exist in the adult brain as the major proliferative cell population. Due to their proliferative and precursory
nature, OPCs are interesting in the context of regeneration and remyelination. However, OPCs are not a
homogenous cell population and differ in their electrophysiological properties. It has been suggested that
the electrically excitable population of OPCs would preferentially myelinate functionally active axons. In this
study, our aim was to produce and characterize OPCs from human pluripotent stem cells. For this, human
embryonic stem cells were differentiated into OPCs using previously described protocol. The oligodendroglial
identity of the cells was confirmed with antibodies. The development of electrophysiological properties of
OPCs was studied with whole cell patch clamp. The differentiating cell population was found to consist of
electrically active and electrically passive cells. The electrically active cells responded to current injections
with abortive or full action potentials.
Other
Author: Muhammad Waqas Ahmad Khan ([email protected])
Research group leader: Leena Ukkonen*
Co-author(s): Elham Moradi*, Toni Björninen*, Lauri Sydänheimo*, G. Steven Bova^
Affiliation(s): * Tampere University of Technology, ^University of Tampere
Wireless Implantable Sensors for Intracranial and Intra-tissue Pressure Sensing
Keyword(s): wireless sensor, implantable sensor,Intracranial pressure sensor, Intra-tissue Pressure Sensing
Abstract:
The recent advancements in microelectronics have enabled integrated low-power microelectronic systems
as platforms for minimally invasive implantable biomedical micro sensor. However, today most implant
systems are still “wired” to the external units. This increases the discomfort of the patient and limits the
usage of the system to clinical environment. In the future, the reality will be “being wireless for therapy”
enabling continuous and in-all-conditions utilization of implantable biomedical systems. To make this reality,
our research focuses on wireless technology and communication techniques to monitor specific physiological
parameters, such as the intracranial pressure (ICP) related to hydrocephalus and intra-tissue pressure related
to monitoring and diagnostics of prostate cancer. An increase in the amount of cerebrospinal fluid (CSF)
or blood in the brain, due to an illness or an injury, can lead to an elevated ICP. ICP monitoring is used in
the diagnosis and treatment of life-threatening conditions, such as traumatic head injury and intracerebral
hemorrhage. There are several invasive and non-invasive methods for ICP monitoring. Invasive methods
are accurate, but not fit for long-term monitoring, whereas non-invasive methods lack in the accuracy. To
achieve a fit alternative, we are currently working on two approaches for wireless ICP monitoring based
on capacitive and piezoresistive implanted sensor. The former approach utilizes a pressure sensitive MEMS
capacitor and the latter a wirelessly powered piezoresistive microsensor. Our future work aims at utilizing the
wireless pressure sensing technology in the monitoring of prostate cancer through sensing of the intra-tissue
pressure.
Other / Mechanobiology, Cell biology
Author: Teemu Ihalainen ([email protected])
Co-author(s): Lina Aires, Jens Moeller, Vesa Hytönen, Viola Vogel
Affiliation(s): Aires, Moeller and Vogel: Laboratory of Applied Mechanobiology, Department of Health Sciences and
Technology, ETH Zurich, Zurich, Switzerland. Ihalainen and Hytönen: BioMediTech, UTA, Tampere
Intracellular tension regulates nuclear lamina structure
Keyword(s): Cell mechanics, nucleus, mechanosensing, cytoskeleton, biomaterials
Abstract:
Deciphering how physical properties of cellular microenvironments regulate gene expression is a «holy
grail» in mechanobiology. Nowadays its well established that cell nucleus is tightly connected to the cellular
cytoskeleton, which in turn, is linked to the extracellular matrix and/or neighbouring cells via different
kind of adhesions. On the nuclear membrane, lamin proteins play a central role at the crossroad between
cytoplasmic signalling and nuclear events. Recently we have shown that the major lamin protein, lamin A, is
more exposed in the apical than basal nuclear lamina (1). We quantified how the protein exposure is affected
by cell spreading, the physical nature of microengineered cellular niches, pharmaceutical inhibitions and
direct mechanical stress. Most significantly with respect to mechanotransduction mechanisms, the newly
identified epitope overlaps with key regions implicated in DNA and histone binding, and various laminopathy
mutations that impair the nuclear mechanical stability. Our findings should help decipher how the physical
properties of cellular microenvironments regulate nuclear events and gene expression. Ref. 1: Ihalainen,
Aires et al., Nature Materials, EPub August 24, 2015, doi:10.1038/nmat4389
Other / Molecular biology
Author: Rolle Rahikainen ([email protected])
Co-author(s): Magdaléna Von Essen (1), Conor Kelly (1), Teemu Ihalainen (1),
Bernhard Wehrle-Haller (2), Vesa Hytönen (1)
Affiliation(s): (1) BioMediTech, University of Tampere, Finland. (2) Department of Cellular Physiology and Metabolism,
University of Geneva, Switzerland.
Destabilized talin-1 mutants for studying cellular mechanosensing
Keyword(s): Mechanosensing, cell-matrix adhesions, talin, vinculin
Abstract:
Cellular mechanosensing is known to be essential for many cellular processes, e.g. cell migration, stem cell
differentiation and tissue remodeling, but the molecular mechanisms behind it are currently only poorly
understood. Focal adhesion protein talin directly participates in the transmission of mechanical forces
between the cellular cytoskeleton and the extracellular matrix. It has been hypothesized that a mechanically
activated interaction between talin and another focal adhesion protein, vinculin, may act as the primary
mechanosensory switch in many animal cells. In this study, we have modified the mechanical stability of
the talin-1 rod domain by rational mutagenesis to study the role of the talin-vinculin interaction in cellular
mechanosensing. Steered molecular dynamics simulations have been used to design mutations that affect
the mechanical stabilities of the individual rod domain bundles. The mutated talin-1 proteins have been
studied by expressing them in mouse embryonic fibroblast (MEF) cells. Mechanical destabilization of the
talin rod domain was found to induce talin localization into cell-matrix adhesions and to increase the total
adhesion area in MEF cells. Interestingly, the level of talin rod domain destabilization was also found to affect
the cellular localization of the expressed talin proteins. While a single destabilizing point mutation induced
talin localization to the leading edge of the cell, drastic rod domain destabilization induced localization to
the trailing edge of the cell. This polarization in the talin distribution was found to be dependent on the
presence of the full length talin rod domain and independent of the increased talin localization into cellmatrix adhesions. The research is in progress.

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