Summer Conference’2015

Forum Scientium

What else ?LecturesPosters

Team building activitiesSaunaYoga

Canoe/KayakBarbecueBilliards

Meditation

1

WELCOME TO FORUM SCIENTIUM SUMMER CONFERENCE 2015

A very hearty welcome to this year’s summer conference in Vårdnäs. Located in picturesque

lake south of Linköping, Stiftsgården Vårdnäs will be our year’s conference venue. We hope the

beautiful surrounding will not only relax your mind but also spark enthusiasm within the

group.

We have a diverse group of invited speakers who will be focusing on wide range of topics

including research, publishing papers as well as a more crucial topic of stress handling during

PhD. We will also hear from a former FORUM member about his viewpoint regarding success in

life.

During the stay you will have access to sauna, and ofcourse with good weather a nice swim in

the lake. We also have canoes/kayaks booked for those who would like to grab this opportunity

to drift with the calm waters of the lake.

We hope you will have a fruitful and enjoyable time in Vårdnäs.

/Meenu & Jacob

2

Invited speakers

Clas Malmström – Handling stress

Clas Malmström is a charismatic speaker and gifted entertainer who has inspired an

audience ranging from leaders, managers, scientists and doctors. He specialized in the

rehabilitation of patients with severe psychosomatic and stress-related disorders. He is

acclaimed for his work with the diagnosis of both negative and positive stress and assay

development for clinical applications of salutogenic behavioral medicine. Currently he is a

part-time doctor, a writer, lecturer and advisor to several company managers.

Per Jenson

Per Jenson is Professor of Ethology at IFM, Linköping University and scientific leader of

the AVIAN group, consisting of five principal investigators, and a total of about 35 people

including technicians and master students. The research of his lab within the AVIAN group

is focused on genomic aspects on welfare and behaviour, where domestication related

effects form a central framework. Chickens and dogs are the main models for this. They

cooperate with the Swedish Defence Forces to study their dog breeding program, and also use companion

dogs and the standardised behaviour tests common in Sweden. This is combined with molecular methods

such as QTL-analysis and microarray technique to find the genetic basis for various behaviour affected by

domestication, such as social behaviour, learning and stress reactions.

Tomas Lindahl

Tomas Lindahl is Professor in Clinical Chemistry and Group leader of the Hemostasis

Research Group at Department of Clinical Chemistry. He started in hemostasis

research as PhD student at Karolinska Institute in Stockholm 1985, received his PhD

degree in 1989 and has been doing research in Linköping since 1993. His research

revolves around investigating the role of platelets in haemostasis and cardiovascular

diseases and also coagulation. He is also member of board of the Swedish Society on Thrombosis and

Haemostasis and of the Swedish Association of Clinical Chemistry

3

Per Hammarström

Per Hammarström, received his MSc in 1995 and PhD in Biochemistry in 2000 at

Linköping University (LiU) with Prof. Uno Carlsson. He then did a post doctorate (2000-

2002) at The Scripps Research Institute, in La Jolla, USA with Prof. Jeffery W. Kelly. He

started his independent research group as Assistant professor at LiU in 2003 and was

2008 awarded full Professor of Protein Chemistry. His early research was centered on

the role of protein folding intermediates in the protein folding process, and the

mechanistic function of molecular chaperones during productive folding. Following his post-doctoral years

working on transthyretin amyloidogenesis, his research has shifted towards investigation of protein

misfolding and its key role in a variety of amyloid and prion diseases.

Per Aspenberg

Per Aspenberg is professor of Orthopaedic surgery in Linköping University. Per

Aspenberg took his MD in Uppsala in 1974, and did his ortopaedics training in Västerås.

He defended his PhD thesis entitled "Bone Induction in Rodents and Primates" in Lund

1988. He is fond of developing new but simpler methods, and to use these to study

possibilities for drug induced acceleration of healing. His research aims to gain deeper

understanding of the healing of injured bone and tendon. His research group pioneered

the use of bisphosphonates in orthopaedics. They were also the first to show accelerated fracture and

tendon healing by drug treatments in humans. In opposition to the prevailing view, they have also showed

that atypical fractures are a side effect of bisphosphonates, and that osteonecrosis of the jaw is caused by a

bisphosphonate-related inability to heal chronic infection (osteomyelitis). Besides academics and research,

he also is an amateur artist and cellist.

Joel Hedlund

Joel Hedlund received his MSc in Engineering Biology from LiU and did his PhD in

Bioinformatics in prof Bengt Persson’s group at IFM, working on the characterization

of protein families using bioinformatics tools. After his dissertation in 2010 he took on

a position as application expert in bioinformatics at the National Supercomputer

Centre (NSC) at LiU. His presently is part of the executive team of the Nordic e-

infrastructure collaboration (NeIC) working as coordinator for efforts in the strategic

area for bio- and medical sciences.

4

Torbjörn Lindblom

Torbjörn Lindblom is an organizational consultant with assignments at individual, group and

organizational level. He has 10 years’ experience as operations manager at Feelgood. He

and his colleagues have developed and implemented training programs for executives,

managers and employees in a number of different organizations in various industries. He

has experience and knowledge of personal/executive coaching, team development and

sports leadership. Together with his colleagues, he provides the support for the development program for

PhD students in Linköping University which aims at encouraging high performance as well as care of one’s

own health, during their graduate studies.

5

Poster presentations

During the conference, all members will have two occasions to present their posters. The first presentation

is to the Forum members which is divided into four sessions (poster sessions 1-4 in schedule) and the second

presentation is for the poster jury (poster competition 1-4). Please check in the table below which group you

have been assigned to and also your poster number.

Poster competition: The sessions will start with a 1-minute one slide presentation of all the posters in that

session after which the presenters of that session should be present by the poster. The members have the

opportunity to view the poster and ask questions during this time. At the end of the fourth session, the

members will have the opportunity to vote for the best poster. The best poster will receive an award. For the

competition, the members will present their poster to the jury in 2-minutes after which the jury will have

few minutes to ask questions. For the best poster presentation, the jury will vote for one winner and three

second place. The winners and runners-up will receive an award.

Speed dating: There will be speed dating session alongside the poster competition. The idea with speed

dating is that the members should be able to gain better insight into what other Forum members are

working on and thus help the members to find potential collaborations. During speed dating, the members

will have 2 minutes to present their work to other Forum member in two by two sessions. Try to keep it to

1.5 minutes to have a little bit time for questions.

Poster group I Poster group II Poster group III Poster group IV

1. Amélie Wallenhammar

10. Niclas Björn 19. Anna Södergren 28. Angelika Holm

2. Jesper Fogelholm 11. Anna Zimdahl Kahlin

20. Fredrick Bäcklund 29. Malin Hammarman

3. L. Fernández del Río 12. Peter Eriksson 21. Erica Zeglio 30. Alejandro Vicente-Carrillo

4. Olof sandberg 13. Parmis Sepanloo 22. Mohsen Golabi 31. Magnus Bernhardsson

5. Katarina Bengtsson 14. Camila Sandén 23. Anders Elfwing 32. Jacob Kuruvilla

6. Sofie Sundberg 15. Emina Vorkapic 24. Theresia A. Sjöström

33. Robert Pilstål

7. Anna Svedberg 16. Kjersti Claesson 25. Martin Johnsson 34. M Atikuzzaman

8. Meenu R. Rajan 17. Andrey Höglund 26. Susanna Lönnqvist 35. Ankit Macawan

9. Bela Nagy 18. Christopher Aronsson

27. Andreas Skallberg

6

Abstracts

7

Structural and functional study of the B-box domain of

human TRIM21/Ro52 and its molecular involvement in

disease

Amélie Wallenhammar1, Madhanagopal Anandapadamanaban1, Alexander

Espinosa2, Marie Wahrén-Herlenius2 and Maria Sunnerhagen1

1) Molecular Biotechnology, IFM, Linköping University

2) Karolinska Institute, Stockholm

Poster number: 1

TRIM21/Ro52 is an E3 ligase, which belongs to the TRIM (tripartite motif) family of proteins

that encodes multidomain proteins typically involved in cellular signaling events.

TRIM21/Ro52 consists of a N-terminal RING domain encoding ubiquitin ligase activity, a Zn2+

binding B-box, a coiled-coil (CC) domain, and a C-terminal SPRY/B30.2 domain. As an E3 ligase,

TRIM21/Ro52 mediates target specificity in the post-translational event of ubiquitination

allowing control of biological processes such as protein degradation and activation.

Our biological data on Ro52 as described by ubiquitination assays suggest that the RING and the

B-box jointly regulate both auto-ubiquitination and substrate-ubiquitination. Furthermore, we

will present the current status of our structural NMR evaluation of the RING-B-box motif, as

well as the on-going work of determining the high resolution NMR structure of the B-box as a

single domain. Specifically, I will describe the characterization of interactions between the E2

UbcH6 and the RING-B-box motif as assayed by SPR/Biacore. This will further enlighten the

interplay between the key actors in the ubiquitination pathway; ubiquitin, E1, E2 and E3.

Increased structural and functional understanding of the TRIM21/Ro52 enzymatic mechanism

will promote development of novel therapeutic approaches for the treatment of patients with

Sjögren’s syndrome. The results of this study will contribute to the understanding of the high

cancer risk that these patients display, possibly by autoantibody binding to the RING-B-box

motif interfering with ubiquitination.

8

Finding the causal gene for quantitative variation in

red colouration

Jesper Fogelholm, Martin Johnsson, Dominic Wright.

IFM Biology, Linköping University.

Poster number: 2

The genetic regulation of coat colour is one of the most studied gene systems to date, with ~150 genes known to be involved. Currently, however, all have Mendelian, rather than quantitative, effects. Using QTL mapping in two intercross chicken populations we identify a 2Mb region associated with the intensity of red colouration. This region contained a selective sweep (fixed in domestic birds), with several genes in close proximity. Microarray analysis of all genes in the QTL interval using multiple individuals from two generations of Red Junglefowl x White Leghorn intercross populations revealed a handful of correlating genes. Expression of Neurotrophic tyrosine kinase receptor type 3 (NTRK3) was found to have the highest correlation with the intensity of red colouration in feathers. Furthermore another of the correlated probesets has also been shown to be differentially expressed in the hypothalamus of another generation of the same intercross. To the best of our knowledge this is the first time that a gene regulating a quantitative colour trait has been identified. Strikingly NTRK3 has also been shown to influence fear type memories in a mice model, which potentially could indicate that there is a link between behaviour and colour.

9

Polarizing properties and structural characteristics in

the cuticle of the scarab beetle Chrysina gloriosa L. Fernández del Río, H. Arwin, K. Järrendahl

Dept. of Physics, Chemistry and Biology, Linköping University, Sweden

Poster number: 3

Some scarab beetles have an eye catching metallic coloration originating from the reflection of light on

the exocuticle. The reflected light is elliptically polarized and in some cases even circularly polarized.

The exocuticle is mainly composed of chitin as well as proteins and its structure is organized on layers

of helicoidal chiral structures made of fibrils of chitin crystals.

The scarab beetle Chrysina gloriosa has well differentiated areas, gold- and green-colored, which not

only differ in color but also in polarization properties. Mueller matrices are measured on these two

areas with a spectroscopic ellipsometer (RC2, J.A.Woollam Co., Inc). The results are presented as

contour plots where we represent different parameters as a function of incidence angle θϵ[20˚; 75˚] and

wavelength λϵ[240; 1000] nm of the incident beam.

Parameters of particular interest are the m41 element

of the Mueller matrix, which is related to the circular

polarization behavior, the degree of polarization, the

ellipticity and the absolute value of the azimuth angle.

The gold-colored area is a good specular reflector on

which reflected light is left-handed and near-

circularly polarized at certain θ and λ as seen in Fig.1.

The green areas, on the other hand, scatter light and

the polarization depends strongly on the scattering

angle. It is observed that the scattered light is

elliptically polarized and exhibits similar

characteristics as the specular reflected light from the

gold-colored areas.

The structure of the two areas has been studied to

understand the different behavior. SEM images of the cross sections are presented showing layered

structures. The gold-colored areas show layers parallel to the surface but on the green areas the layers

have a cusps-like shape. In an optical microscope, polygonal cells with a size of 10 µm can be seen on

the green areas. These cells correspond to each of the cusps seen in the SEM images. AFM images are

also presented to provide a better understanding of the shape of the cells which causes the scattering

from the green areas.

Figure 1 Contour plots (λ, θ) of beetle C. gloriosa measured on a gold-colored area.

10

Abstract

Olof Sandberg

Poster number: 4

Background

We investigated how ruptured Achilles tendons are loaded in a brace. There is an ongoing

discussion whether patients should be recommended to bear weight on the injured limb.

However, little is known about the effects of bracing on tensional loading of the healing Achilles

tendon: it is uncertain if load-bearing actually stresses the Achilles tendon inside a brace.

Methods

We measured plantar flexion moment inside the brace, in order to estimate tensional

loading of the tendon, by use of an insole with pressure transducers.

Results

After wearing the brace for 1 hour, young healthy individuals reduced their maximum

flexion moment during gait by half. Patients with Achilles tendon rupture showed no

measurable flexion moment during gait with the brace, 4 or 7 weeks after injury. Only when

specifically instructed, could they produce a considerable plantar flexion moment. We noted

that gait speed with the brace at 4 weeks correlated with a heel-raise functional test at 1 year:

the higher the spontaneous gait speed, the less the functional difference between the injured

and the uninjured leg (r2=0.68; p=0.002).

Conclusion

The correlation with gait speed suggests that the patients’ general physical aptness has an

impact on the end result.

11

Conducting polymer electrodes for

gel electrophoresis

Katarina Bengtsson, Sara Nilsson, Nathaniel D. Robinson

Transport and Separations Group, Dept. of Physics, Chemistry and

Biology, Linköping University, Sweden.

Doi:10.1371/journal.pone.0089416

Poster number: 5

The purpose of this study was to investigate whether the conductive polymer PEDOT:PSS could

replace platinum electrodes in gel electrophoresis to perform protein separations.

During gel electrophoresis, electrochemical reactions have to take place at the electrodes to

maintain an electric field in the gel. This reaction is mainly electrolysis of water which causes

pH changes and gas formation. Further, the reuse of metal electrodes introduces a risk of cross-

contamination between used in successive runs. We propose that these problems can be

eliminated by using polymer electrodes instead of metal electrodes. Many of the problems are

avoided since PEDOT:PSS is an electrochemically-active polymer that is oxidized/reduced at

lower voltages than those required to electrolyze water.

Electronic measurements with and without polymer electrodes were done to demonstrate the

compatibility between the polyacrylamide gel, SDS buffer strips and PEDOT:PSS. Here we saw a

clear difference between polymer and metal electrodes at 1V, while they behaved similary at

100V. The current through the gel with polymer electrodes was 6 times higher than with

platinum electrodes, showing compability and how easily the PEDOT:PSS is oxidized/reduced.

A multi-colored protein ladder was separated using the commercial PhastSystem, with and

without PEDOT:PSS.

We showed that PEDOT:PSS can replace platinum electrodes in gel electrophoresis and that it is

compatible with the commercial PhastSystem from GE Healthcare. We hope that this can

facilitate the development of smaller, cheaper and disposable gel electrophoresis systems.

12

Cholinergic modulation of layer 6 Corticothalamic and Fast-Spiking interneurons

Sofie Sundberg1, Gonzalo Sanchez1, Sarah Lindström1 and Björn Granseth1

1 Division of Cell Biology, Department of Clinical and Experimental Medicine, Linköping

University

Poster number: 6

Neuromodulators are essential for fine-tuning neuronal signaling and behavior. Especially,

transitioning between states of brain activity involve triggers, such as release of acetylcholine in

relevant brain areas. Cerebral cortex layer 6 has the ability to affect network excitability and

cortical output signaling, and pose an interesting target for the study of the effect of

neuromodulators. We wanted to study the responses of the layer 6 corticothalamic, CT,

pyramidal cell type and inhibitory fast-spiking, FS, interneurons upon cholinergic stimulation.

Patch clamp whole-cell recordings were made from CT neurons and FS interneurons in acute

coronal slices of cerebral cortex from mice while bath applying cholinergic agonists and

antagonists. CT neurons showed higher sensitivity to cholinergic neuromodulation than FS

interneurons. The general cholinergic agonist Carbachol depolarized CT neurons by 13.72 mV

(SEM ± 1.56 mV, n = 9) and FS interneurons by 4.37 mV (SEM ± 1.93 mV, n = 7). Both cell types

also got an increased input resistance, with higher responses of CT neurons than FS

interneurons. CT neurons showed both nicotinic and muscarinic responses, where FS

interneurons only showed muscarinic responses. Higher responsiveness of CT neurons,

compared with FS interneurons, to cholinergic drugs suggests that CT neurons could, upon

physiological acetylcholine release, provide excitation to the cortical network influencing the

activity state. cortical

13

A validated LC-MS/MS method for quantification of the

anticancer drug erlotinib and its metabolites

Anna Svedberga,*, Henrik Gréena,b,c, Anders Vikströmd, Joakim

Lundebergc, Svante Vikingssona

Poster number:7

a Clinical Pharmacology, Division of Drug Research, Department of Medical and Health Sciences, Linköping University, SE-

581 85 Linköping, Sweden, b Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, SE-587 58 Linköping,

Sweden c School of Biotechnology, Science for Life Laboratory, KTH Royal Institute of Technology, SE-171 21 Solna, Sweden d Department of Pulmonary Medicine, University Hospital, SE-581 85 Linköping, Sweden

Erlotinib is a tyrosine kinase inhibitor, approved as monotherapy in first, second and third line of non-small cell lung cancer (NSCLC). Large variations in response and toxicity are observed in patients treated with erlotinib. To study if drug concentration affect inter-patient variability, a liquid chromatography tandem mass spectrometry method was developed and validated according to international guidelines for quantification of erlotinib and its metabolites in human plasma [1]. The substances were extracted using protein precipitation, separated on a BEH XBridge C18 column (100 x 2.1 mm, 1.7 µm) by gradient elution of acetonitrile and 5 mM ammonium acetate in a total run time of 7 min. Erlotinib, OSI-420 and didesmethyl erlotinib were quantified in the concentration range 25-5,000 ng/mL, 0.5-500 ng/mL and 0.15-10 ng/mL respectively. Precision and accuracy was <14 % except for OSI-420 at LLOQ (17 %). Due to the lack of commercially available reference substances, human liver microsomes were used for metabolite generation. Fourteen metabolites were identified, three of them not previously reported. The metabolites were measured semi-quantitatively and validated with respect to selectivity, precision and stability.

Figure 1: Chromatogram representing a patient sample after 1 month of erlotinib treatment

[1] A. Svedberg, H. Gréen, A. Vikström, J. Lundeberg, S. Vikingsson, A validated liquid chromatography tandem mass spectrometry method for quantification of erlotinib, OSI-420 and didesmethyl erlotinib and semi-quantification of erlotinib metabolites in human plasma, Journal of Pharmaceutical and Biomedical Analysis, 107 (2015) 186-195.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

%

-1

99

Erlotinib_20140325_054 15: MRM of 2 Channels ES+ 586.3 > 410.1 (U2)

2.72e6100

0

Inte

nsi

ty(%

)

I = 2.7e6

OSI-420

OSI-597 (IS)Erlotinib-d6 (IS)

Erlotinib

M13

M3

M6M11

M16

Didesmethylerlotinib

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 (min)

14

Reduced FoxO1 in human adipocytes from patients with

Type 2 diabetes explains network wide impaired insulin

signaling

Meenu Rohini Rajan, Elin Nyman, Preben Kjölhede, Gunnar Cedersund and Peter Strålfors

Poster number: 8

Control of gene expression via Forkhead box O (FoxO) transcription factors may be the most

important way for insulin to control cellular concentrations of key rate controlling enzymes in

the cellular metabolism of mammals. Whereas insulin control of FoxO has been widely studied

in cell lines and experimental animals, very little is known about FoxO in primary human cells.

Likewise, virtually nothing is known about how FoxO contributes to the insulin resistant state

of type 2 diabetes (T2D) in human beings.

Herein we have in depth investigated insulin signaling to control of FoxO1 in primary human

adipocytes obtained from patients with T2D and in parallel from non-diabetic control subjects.

We have integrated insulin control of FoxO1 with the rest of the insulin-signaling network. We

show that insulin-regulated phosphorylation of FoxO1 is exclusively downstream mTORC2 and

phosphorylation of PKB at Ser473, and that the insulin-control of FoxO1 therefore is not

affected by the insulin resistance in T2D. Instead the cellular concentration of FoxO1 is reduced

in T2D, which attenuates insulin-independent effects of FoxO1, which, in turn, cause inhibition

of mTORC1 and attenuation of the feedback to IRS1. Hence, our findings demonstrate that a

major component of the mechanism of insulin resistance in T2D is a reduced abundance of the

FoxO1 protein, which attenuates the insulin signaling network-wide to cause insulin resistance

- the fundamental defect in T2D of obesity.

15

Pharmacogenetic biomarkers for chemotherapy induced

adverse drug reactions Niclas Björn

Division of Clinical Pharmacology, Department of Medical and Health Sciences,

Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden

Poster number: 10

Classical chemotherapy is a common category of cancer treatment that uses chemical

substances. It basically works by reducing the growth rate of and killing tumor cells.

Chemotherapeutic treatments have narrow therapeutic windows; even very small variations in

the dosage can result in a lack of efficacy or a toxic side effects. At the moment, most

chemotherapy dosages are based on the body surface area (BSA) of the patient. However, the

variation in BSA is far less than the variation seen in drug exposure, clinical effects and adverse

drug reactions (ADRs). The classical chemotherapies are associated with significant inter-

individual variability in both therapeutic effect and ADRs. The most important factor

responsible for these differences is now recognized to be the normal genetic variability, it is

estimated to account for 20-95% of variability in drug disposition and effects.

The overall aim of the project is to investigate the possibilities of predicting the therapeutic

response and ADRs in patients undergoing classical chemotherapy. This will be conducted for

the standard chemotherapy treatments against lung cancer, ovarian cancer, breast cancer and

acute myeloid leukemia (AML). We want to identify and validate a set of genetic biomarkers

that can be used to predict the outcome of the chemotherapy, minimizing toxicity, maximizing

positive anti-tumor effects and adjust dosage according to the patient’s individual genotype,

before treatment is even started.

The projects will primarily include genotyping and bioinformatics of both candidate gene/SNP

sequencing and whole exome/genome sequencing. The projects will see to validation of

previously found biomarkers as well as assessing novel biomarkers for further studies. In-

house laboratory experiments for confirmation of findings will also be conducted to strengthen

results.

16

Characterization of the drug metabolizing enzyme

thiopurine methyltransferase (TPMT) 497A>G

Anna Zimdahl Kahlin1, Sara Helander1, Lars-Göran Mårtensson2,

Malin Lindqvist Appell1

1Department of Medical and Health Sciences, Division of Drug Research,

Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden 2Department of Physics, Chemistry, and Biology, Linkoping University, 581 83

Linkoping, Sweden

Poster number: 11

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme that converts cytotoxic thiopurine drugs into both active and inactive metabolites. In Caucasians 10 % carries a single nucleotide polymorphisms (SNP) causing a less functional TPMT enzyme leading to an increased risk of severe adverse reactions during treatment with normal thiopurine doses. Recently, we discovered the SNP TPMT 497A>G in a young female patient with acute lymphoblastic leukemia. This SNP had earlier been reported from the genomic ClinSeq Project but had not been further characterized. In our study the TPMT 497A>G was characterized to describe the properties and function of the resulting protein in comparison to the wild type TPMT. We have determined the enzyme activity in RBC and WBC, analyzed the mRNA sequence and the heredity. To investigate the stability and structural changes of the protein structure we produced recombinant human TPMT 497A>G in E.Coli and analyzed it with circular dichroism. TPMT 497A>G causes an amino acid shift of Y166C. Our results show that 497A>G is a heritable variant of TPMT that decreases the patient´s enzyme activity dramatically. The overall structure of the enzyme is normal but the amino acid shift causes a pronounced less stable enzyme which influences the patient´s metabolism of thiopurine drugs.

17

Development of new promising multimodal nanoparticles for Biomedical Imaging

Development of lanthanide-based theranostic

nanoparticles

Peter Eriksson a, Andreas Skallberg a, Robert Boyd b, Caroline

Brommesson a, Xuanjun Zhang a & Kajsa Uvdal a

a Division of Molecular Surface Physics & Nanoscience, Department of Physics,

Chemistry and Biology, Linköping University

Poster number: 12 b Division of Plasma & Coatings Physics, Department of Physics, Chemistry

and Biology, Linköping University

Over the last 10 years has the interest of combining different modalities into one “all-in-one”

nanoprobe grew remarkably. In this project we mainly focus on combining therapeutic and

diagnostic properties into one theranostic particle. This is achieved by using two lanthanides,

cerium and gadolinium into a combined nanoparticle synthesis. Cerium is a fascinating material

in the content that it has been shown to have several possible therapeutic abilities, where the

multi-enzyme antioxidant ability is of major interest in my research. In addition, cerium has

great crystalline properties and allows gadolinium to homogenously be implemented in the

crystal structure of cerium. Gadolinium is a favorable element to implement into cerium

nanoparticles, since it has shown to enhance the antioxidant properties of cerium and it is the

most effective element in order to enhance positive contrast in magnetic resonance imaging.

18

A link between mechanics, inflammation and healing:

Changes in the inflammatory cell population during

tendon healing

Parmis Sepanloo1, Robert Blomgran2, Jan Ernerudh3, Per Aspenberg1

Orthopedics1, and Microbiology and Molecular Medicine2, Clinical immunology3,

Department of Clinical and Experimental Medicine, Faculty of Health Sciences,

Linköping University, Linköping, Sweden.

Poster number: 13

Introduction: Inflammation is important to start the healing process, but the composition of the

immune cell population during tendon healing is not comprehensively described, and it is

unknown if this is influenced by mechanical loading. We here used flow cytometry to describe

the distribution and kinetics of inflammatory cell population during Achilles tendon healing.

Methods: The right Achilles tendon of 24 rat were transected, the tendon left un-sutured to heal

spontaneously, and the wound closed with two stiches. At 1, 3, 5 and 10 days after tendon

transection, the healing part of the Achilles tendon was removed and digested using collagenase

prior to cell isolation. Cells were then stained with antibodies (CD45, CD11b, CD68, CCR7,

CD206, CD163, CD3, CD4, CD8a, CD25, Foxp3) to characterize different sub-categories of

macrophages and T-cells. Data was acquired using flow cytometry.

Results and conclusion: Investigating the participation of innate and adaptive immune cells

during tendon healing, a very small fraction of CD45+ cells were found to be T cells, while

majority of the CD45+ cells were CD11b+ cells of the innate immunity.

The trend in T cell activation mirrored that of the macrophages. An early activation with high

abundance of CD25+ T cells (corresponding to a high levels of CCR7+ macrophages) was

followed by a shift or increase in cells with more regulatory functions such as regulatory T cells

(corresponding to the high abundance of CD206+ macrophages) at day 10. The increasing

proportion of T cells over time, especially regulatory T cells, and high abundance of CD206 M2

macrophages suggests that these cells may play a role in the remodeling process.

19

Tuning liposome membrane permeability by insertion

and folding of de novo designed polypeptides

Seng Koon Lim1,*, Camilla Sandén2,*, Bo Liedberg1, Daniel Aili2

1 Centre for Biomimetic Sensor Science, Nanyang Technological University,

Singapore 2 Division of Molecular Physics, Department of Physics Chemistry and Biology,

Linköping University, Sweden * Equal contribution by authors

Poster number: 14

Membrane-active polypeptides, such as antimicrobial peptides (AMPs), are of large interest for

development of novel therapeutics, including peptide-mediated drug delivery- and release

systems, transfection agents and antibiotics. The initial association of AMPs to lipid membranes

is in general caused by electrostatic interactions and thus not a result of a specific molecular

recognition event. Finding routes to tune specificity and activity of membrane-active

polypeptides is hence of large importance to improve their efficacy and minimize harmful side

effects for therapeutic applications.

Here we describe a de novo designed antimicrobial-like polypeptide (JR2KC) that partition into

zwitterionic lipid membranes when specifically and covalently anchored to the membrane.

Anchoring and subsequent membrane partitioning triggers a structural transition of the

polypeptide from random coil to α-helical conformation and induces pore formation. The extent

of pore formation can be dynamically tuned by varying either the number of anchoring moieties

or the peptide concentration. Moreover, a dual and highly specific control system for regulating

the membrane activity of JR2KC is demonstrated. Addition of a complementary polypeptide

(JR2E), designed to heterodimerize with JR2KC, effectively inhibits pore formation. Peptide

dimerization competes with membrane partitioning, which prevents JR2KC from partitioning

into the membrane. JR2E contains two cleavage sites for the matrix metalloproteinase MMP-7,

which is an enzyme involved in tumour metastasis and inflammatory process and upregulated

in many malignant tumours. Digestion of JR2E by MMP-7 reactivates the membrane penetrating

capacity of JR2KC.

This system thus provides a precise and very specific route for tuning the permeability of lipid

membranes and a novel strategy for development of recognition based membrane active

polypeptide that also enables indirect enzymatically controlled release of liposomal cargo.

20

Effects of Imatinib in experimentally induced abdominal

aortic aneurysm

Emina Vorkapic1, Elma Dugic1, Mikko I. Mäyränpää3, 4, Joy Roy4, Per

Eriksson5 and Dick Wågsäter1

Poster number: 15

1 Division of Drug Research, Department of Medicine and Health Science, Faculty of Health Science, Linköping University, Linköping, Sweden. 2 Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland 3 HUSLAB, Division of Pathology, Meilahti Laboratories of Pathology, Helsinki University Central Hospital, Helsinki Finland. 4 Vascular Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. 5 Atherosclerosis Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden.

BACKGROUND: AAA is characterized by increased infiltration of inflammatory cells, increased

production and activation of chemoattractant and a phenotypic change of vascular smooth muscle cells

(SMCs) in response to factors such as platelet-derived growth factors (PDGFs) among others. Imatinib is

a selective inhibitor of several tyrosine kinases, including PDGF receptors. It is increasingly used in

patient with malignant diseases but recently it is becoming evident that Imatinib may have vascular

protective properties. We hypothesized that Imatinib might have protective properties on above

mentioned features.

METHODS: Male ApoE-/- mice were infused with AngII (1000 ng/kg/min) for 4 weeks to induce AAA. A

group of ApoE-/- mice were infused with saline as controls. Daily treatment with 10 mg/kg Imatinib or

tap water as control via gavage was provided for 4 weeks.

RESULTS: After infusion the adventitial aortic diameter was significantly reduced by 18% (P<0.05) in

AngII infused mice treated with Imatinib as compared with mice given tap water. Gene expression of

the SMC marker SM22α was significantly increased by 48% (P<0.01) in AngII infused mice after

Imatinib treatment compared with mice given tap water which was further confirmed with

immunohistochemical staining of SM22α. Treatment with Imatinib demonstrated a reduction by 86%

(P<0.05) of CD3ε expression compared with mice given tap water.

CONCLUSION: Our findings indicate that treatment with the tyrosine kinase inhibitor, Imatinib, inhibits

infiltration of inflammatory T-cells, and may also affect SMC proliferation and migration, which in turn

may have a protective effect on the development of AAA.

21

Quantification of real-time in-vitro thrombus formation:

moving form arbitrary units to platelet count

Kjersti Claesson, Tomas Lindahl, Lars Faxälv

Linköping University: Dept. of Clinical and Experimental Medicine

Poster number: 16

The diversity of flow systems and quantification methods obstruct result comparisons and validation. Several parameters can be used e.g. surface coverage, fluorescent intensity or thrombus volume. However, these parameters may not truly reflect the actual accumulation of platelets since thrombus volume and coverage measurements does not separate thrombus increment by platelet accumulation from the volume reducing parallel process of platelet contractility. Fluorescence intensity measured in a single focal plane at the surface may lead to underestimation of aggregating platelets further out compared to adhering platelets at the surface. These issues might generate a misrepresentative result and the need for standardization is high. We aimed to construct a robust method for analysing thrombus formation, providing information on thrombus volume and platelet density, thereby also information about platelet contractility. Hirudinized blood was drawn through a 200μm wide flow channel, over a collagen patch, at a wall shear rate of 1400s-1. The platelets in 5% of the blood volume were fluorescently labelled and micrographs were captured using time-lapse and z-stack. A Python-script was used for image analysis, extraction of platelet number and position. Labelled platelets were detected and the mean distance to closest neighbours was determined. From this we calculated the total number of platelets and estimated the thrombus volume. The density of platelets was calculated, yielding additional information about the platelet contractility. The method was compared to confocal microscopy. The combined information about platelet number, thrombus volume and platelet density gives important information regarding thrombus development and can lead towards better standardization.

Picture that

represents you!

22

methQTL analysis to identify genetic polymorphisms

underlying methylation differences between wild and

domestic chickens'

Andrey Höglund, Martin Johnsson, Per Jensen, Dominic Wright

IFM, Biology, Linköping University, Linköping Sweden

Poster number: 17

DNA methylation is an epigenetic modification that affects gene expression levels and is found

in all vertebrates. This enables the otherwise static genome to generate functionally

differentiated cells influenced by predetermined genetic programing and/or external cues from

the environment. Recent studies have shown that the underlying DNA sequence affects the DNA

methylation levels. Here, we seek to identify the mechanics of DNA methylation in the chicken

and locate regions on the genome that are associated with differential methylation levels. A

DNA methylation quantitative trait loci (methQTL) analysis was conducted using 130

hypothalamus samples collected from the eighth generation of an advanced intercross between

wild and domestic birds. Individual resequencing of MeDIP samples (30 million reads/ sample)

taken from each hypothalamus was compared with individual microarray data, whilst 768 SNP

markers/ sample were also genotyped. This allows a comparison between genotype, global and

local methylation status and gene expression to all be compared in the same cross as part of a

combined methQTL/ eQTL mapping experiment. Results are ongoing, but should aid in the

identification of genomic regions correlated both with genotype and the genetic regulation of

expression, as well as to further elucidate breed differences between wild and domestic

chickens.

23

Self-Sorting de Novo Designed Heterodimeric Coiled Coil Peptides with Defined and Tunable Self-assembly Properties

Christopher Aronsson,† Staffan Dånmark,† Feng Zhou,‡ Per Öberg,§ Haibin Su,‡ and Daniel Aili*†

Poster number: 18

†Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, 581 83 Linköping, Sweden.

‡School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798.

§Vehicular Systems, Department of Electrical Engineering (ISY), Linköping University, 581 83 Linköping, Sweden.

Coiled coil peptides with defined assembly properties and dissociation constants are highly

attractive components in synthetic biology and for fabrication of peptide-based hybrid

nanomaterials in particular. In this work we describe a set of four promiscuous 28-residue de

novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are

non-orthogonal in the sense that they can form four different heterodimers albeit with large

differences in affinities. The peptides dissociation constants spans in more than four orders of

magnitude, from the micromolar to the picomolar range, leading to large difference in melting

temperatures. Due to this, they are prone to thermodynamic social self-sorting when all are

mixed, as confirmed by thermal unfolding experiments of different peptide combinations, along

with simulations. The peptides self-sort with high fidelity into predominately two dimers

corresponding to the coiled coils with the highest and lowest affinities for heterodimerization.

24

Platelet responsiveness during storage – formation of subpopulations and LAMP-1 as new quality markers

Anna Södergren1, Nahreen Tynngård1-3, Gösta Berlin1,2, Sofia Ramström1

1Department of Clinical and Experimental Medicine, 2Department of Clinical Immunology and Transfusion Medicine, 3Department of Clinical Chemistry, Linköping University, Linköping, Sweden

Poster number: 19 Background: Storage of platelet concentrates (PCs) results in a platelet storage lesion which may reduce the hemostatic function of transfused platelets. Quality testing of PCs commonly involves analysis of blood gases, metabolic variables and spontaneous expression of platelet activation markers. However, response towards agonists is rarely studied. Aims: The aim of this study was to find new and potentially more sensitive flow cytometry markers to detect changes in platelet function during storage. Methods: PCs prepared by apheresis (n =6) were examined on days 1, 5, 7 and 12. Exposure of lysosomal associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) and formation of different platelet subpopulations was assessed by flow cytometry in resting state or following stimulation with platelet agonists (collagen-related peptide (CRP), PAR1-activating peptide (AP) and PAR4-AP). The protocol was also tested in six PCs with reduced swirling. Results: The ability to form subpopulations upon activation was significantly decreased already on day 5 for some agonist combinations, e.g. seen as a decrease in the fraction of small platelets (25±3% on day 1 and 15±3% on day 5 upon activation with CRP+PAR1-AP+PAR4-AP, mean±SEM). In PCs with reduced swirling the ability to form subpopulations was severely reduced or even abolished. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. Agonist-induced exposure of PS and LAMP-1 gradually decreased with time (from 38±5% and 77±3%, respectively, on day 1 to 11±1% and 50±9% on day 12), and agonist-induced LAMP-1 exposure was absent in PCs with reduced swirling. Conclusion: The platelet activation potential measured as agonist-induced expression of LAMP-1 and fragmentation into platelet subpopulations are potential sensitive markers that could be beneficial to include in evaluation of the platelet storage lesion.

Figure. Expression of P-selectin, PS and LAMP-1 on platelets in resting samples (spontaneous expression) and in response to activation by CRP+PAR1-AP+PAR4-AP. PCs were divided according to swirling grade; fully retained swirling (day 7-12, n=6), reduced swirling (day 12-22, n=2) and without swirling (day 15-17, n=4). (A) Platelet expression of P-selectin, PS and LAMP-1 in resting (spontaneous expression) and activated samples (striped bars). (B) Correlation of PS and LAMP-1 expression after agonist stimulation.

25

Preparation and application of functionalized protein fibrils Fredrik Bäcklund, Olle Inganäs, Niclas Solin

Biomolecular and Organic Electronics, Department of Physics, Chemistry, and

Biology, Linköping, Sweden

freba@ifm.liu.se

Poster number: 20

When the protein insulin is heated in aqueous acid, it self-assembles into fibrous structures

known as amyloid fibrils. By co-grinding fibril forming proteins with a hydrophobic

functionalizing guest molecule, a protein-guest molecule composite material can be created that

retains the proteins innate ability to form fibrils. Subsequently formed fibrils will thus have the

structural properties of the protein fibril as well as the properties of the incorporated guest

compound1-3. The grinding procedure is applicable for a wide range of chromophores

commonly used for organic electronics and photonics and has been shown to be useful for

applications within biomedicine1 as well as optoelectronics3.

As the composite material forms fibrils, incorporated small linear molecules can be forced into

highly specific alignment and structural organization1,2. The fibrils simultaneously act as

dispersive agents and as an organizing matrix fixing linear molecules along the fibril long axis.

This enables a solution processable alternative to vaccum depositioning for large scale

molecular orientation. As a proof of concept, we have used the grinding methodology to show

new routes for the spacial organization and processing of (small linear hydrophobic)

oligothiophenes, obtaining polarized emission from fibrils functionalized with

oligothiophenes2.

1. Bäcklund, F. G. Solin N. Development and Application of Methodology for Rapid Screening of Potential Amyloid Probes. ACS Combinatorial Science, 16, 721-729 (2014)

2. Bäcklund, F. G. Wigenius, J. Westerlund, F. Inganäs, O. Solin, N. Amyloid fibrils as dispersing agents for oligothiophenes: control of photophysical properties through nanoscale templating and flow induced fibril alignment. Journal Of Materials Chemistry C: Materials For Optical And Electronic Devices 2, 7811-7822 (2014)

3. Rizzo, A. Solin, N. Lindgren, L.J. Andersson, M.R. Inganäs, O. White Light with Phosphorescent Protein Fibrils in OLEDs. Nano Letters 10, 2225-2230 (2010)

26

Electronic Polymers in Lipid Membranes Patrik K. Johansson,*a David Jullesson,b,c Anders Elfwing,c Chiara

Musumeci,c Erica Zeglio,c Niclas Solin,c and Olle Inganäsc

a Department of Bioengineering, University of Washington, Seattle, WA, US-98105, United States. b Chalmers University of Technology, SE-41296, Gothenburg, Sweden. c Department of Physics Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden.

Poster number: 21

Cells use cellular organelles called vesicles for intra- and extracellular transport of substances. Those transportation ways can be exploited in order to insert materials in the membrane double layer using artificially made vesicles called liposomes. The new added compounds can provide the cells membrane with new functionalities that were not naturally present before. The aim of this work is to introduce an electrically conductive material into the cell membrane in order to modulate the activity of the ion channels in the membranes. Conductive polymers are suitable candidate for this purpose due to their flexibility and biocompatibility. However, there are two requisites that should be satisfied in order to have conductivity and to be able to insert the polymer in the lipid bilayer:[1] the polymer should carry a dopant and be hydrophobic. Self-doped[2] conductive polyelectrolytes satisfy the first requisite by having the dopant covalently attached to the backbone. The second one can be achieved by formation of a supramolecular hydrophobic complex of a conjugated polyelectrolyte with oppositely charged molecules. In this work a self-doped conducting polyelectrolyte (PEDOT-S[3]) has been introduced into the lipid bilayer of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) liposomes. In order to do so PEDOT-S has been rendered hydrophobic by supramolecular interaction with alkyl-ammonium salt. This permitted the insertion of the polyelectrolyte into the bilayer structure without a significant loss in conductivity. A fluorescent probe (Nile Red) has been used to confirm the presence of PEDOT-S close to the hydrophobic bilayer, while the conductivity has been monitored with conducting-AFM.

[1] Widge A.S.; Matsuoka Y.; Kurnikova M., Langmuir, 2007, 23 (21), 10672-10681.

[2] Patil O. A.; Ikenoue Y.; Basescu N.; Colaneri N.; Chen J.; Wudl F.; Heeger A. J., Synth. Met., 1987, 20, 151-159.

[3] Karlsson R. H.; Herland A.; Hamedi M.; Wigenius J. A.; Åslund A.; Liu X.; Fahlman M.; Inganäs O.; Konradsson P.,

Chem. Mater., 2009, 21 (9), 1815-1821.4.

Light Emission

Light

Quenching PEDOT-S

27

Use of Phenylboronic Acid Derivatives in Molecular Imprinting of Whole Bacterial Cells

Mohsen Golabi, Edwin W H Jager, Anthony P F Turner Biosensors and Bioelectronics Centre, IFM, Linköping University, Linköping,

Sweden

Poster number: 22

The development of novel, rapid and inexpensive methods for the detection of bacteria will be

beneficial in many fields including food and water safety, biosecurity, bioprocess control and

clinical diagnostics. Of the possible alternatives, biosensors offer great potential to replace or

complement traditional culture-based detection methods, which are time consuming, expensive

and need equipped laboratories and trained staff.

Molecularly-imprinted polymers (MIPs) are bio-inspired artificial receptors that are finding

increasing use in biosensors. Unlike bio-receptors, they are more stable, inexpensive and easy

to produce. Although imprinting of chemical and biological molecules has been very well

studied, there is limited work on the imprinting of whole bacterial cells.

Bacterial cells are well-known to present several sugar compounds on their outer surface. In

this paper, we explore the reversible interaction between boronic groups and diols for the

development of highly specific MIPs for intact bacterial cells. 3-aminophenylboronic acid-based

MIPs, for the detection of Staphylococcus epidermidis, were fabricated via chronoamperometric

methods and SEM images were used to verify the successful capturing and releasing of the

whole bacterial cells. Successful capture and easy release of the bacterial cells, via a competitive

approach, was demonstrated. Furthermore, the usefulness of this imprinting process for the

specific detection of Staphylococcus epidermidis versus non-target bacteria, Staphylococcus

aureus and Streptococcus pneumoniae, was also demonstrated by the use of impedance

spectroscopy measurements of bacterial binding to MIPs and NIPs (non-imprinted polymers)

electrodes.

28

Conducting protein nanowires using metallic polymer

Anders Elfwing,a Chiara Musumeci,a Fredrik Bäcklund,a Niclas Solin,a Olle Inganäsa a Biomolecular and Organic Electronics, Linköping University, Linköping, Sweden aelf@ifm.liu.se

Poster number: 23

As the size of electronic are shrinking to the molecular level, bottom-up approaches has been

suggested to enable the validity of Moore´s law for coming decades. The use of biomacromolecules

such as DNA, fibrous proteins and nanocellulose hold a promising route to act as scaffolds for

conducting material. These naturally occurring or easily synthesized objects can be produced or

harvested at low cost and have an intrinsic nanometer precision. Conjugated polymers hold

promising characteristics such as mechanical flexibility and structural similarity for interactions

with biological structures. The working horse of organic electronics, PEDOT (poly(3,4-

ethylenedioxythiophene), has increasingly found use in organic light emitting diodes and organic

photovoltaic due to high conductivity, high optical transmission and excellent stability. The water

soluble variant PEDOT-S show similar characteristics and has proven to render electrical

conductivity to a variety of biological scaffolds such as protein nanowires, silk fibers and single DNA

chains.1

In this study we report on the investigation of insulin amyloid fibers coated by the water soluble

conducting polymer. Using conducting AFM the electrical conductance of single fibers can be

mapped and novel “internal” probes have enabled us to study the self-assembly process.

AFM pictograms showing the height profile (left) and corresponding current mapping (right) of protein fibers

covered by PEDOT-S.

Hamedi M, Herland A, Karlsson RH, Inganäs,O. Electrochemical devices made from conducting nanowire

networks from amyloid fibrils and alkoxysulfonate PEDOT. Nano Lett 2008 jun 8 (6) 1736-40.

29

The search and evaluation of high performance ion

exchange membranes for iontronic applications

Theresia A. Sjöström, Amanda Jonsson, Erik O. Gabrielsson, Loïg

Kergoat, Daniel T. Simon, Klas Tybrandt, Magnus Berggren.

Linköping University, Department of Science and Technology, Laboratory of Organic Electronics, SE-601 74 Norrköping, Sweden.

Poster number: 24

Iontronics is a growing field where the main mission is to deliver charged biomolecules with

high spatiotemporal precision. To do this successfully, the fabricated devices depend on stable

and high performing materials. The requirements for a new material are that it has to be stable

during processing (including different temperatures and different solvents) and as importantly,

have a high transport number. The transport number is a measurement between 0 and 1 that

tells how selective the ionic transport through the ion exchange membrane is for either cations

or anions. In this project the cation selective material poly(4-styrenesulfonic acid-co-maleic

acid) (PSS-co-MA) cross-linked with polyethylene glycol (PEG) is successively patterned and the

transport number is evaluated with three different lateral evaluation techniques. In all

techniques, the organic electronic ion pump1 is used. Concentration measurements of collected

samples with transported Acetylcholine gave transport number ~0.84, a lateral EMF technique

using K+ gave apparent transport number ~0.9 and a final technique including a lot of different

ions gave a mean transport number of ~0.96. This indicates that the material is suitable for

iontronic applications, since it is stable during processing and all different evaluation

techniques shows high transport numbers.

30

Signatures of selection in an admixed feral chicken

population

Martin Johnsson1, Eben Gering2, Pamela Willis3, Thomas Getty2,

Dominic Wright1

1 IFM Biology, Linköping University, Linköping, Sweden. 2 Kellogg Biological

Station, Michigan State University, USA. 3 Department of Biology, University

of Victoria, Canada

Poster number: 25

Feral organisms are domesticates that live in a wild environment. This means exposure to

different natural and sexual selection pressures compared to a domestic environment. The

extent that such a population can adapt to a feral environment depends on the genetics of

domestication traits. A free-range chicken population has successfully invaded the Pacific island

of Kauai. There are at least two possible sources for this population. First, the chickens could

originate from Red Junglefowl brought over by the Polynesians. Second, they could be feral

domestic chickens. Many domestic chickens escaped when hurricanes hit the island in the

1980s and 1990s, and could have contributed. We collected phenotypes and DNA samples from

Kauai chickens. Based on plumage, vocalisations and mitochondrial DNA, they appear to be an

admixed population of domestic chickens and Red Junglefowl. We then investigated the

genomics of this population with whole-genome sequencing. We calculated pooled expected

heterozygosity in windows across the genome as a measure of genomic diversity. On average,

heterozygosity was high, consistent with admixture. Regions of low heterozygosity may be

targets of selection during feralisation. Most regions are different compared to those selected

during domestication. This suggests domestication and feralisation have different genetic

architectures.

31

Tracing of transplanted keratinocytes with CFSE

Susanna Lönnqvist1, Maria Karlsson1, Gunnar Kratz1,2

1. Division of Experimental Plastic Surgery, Department of Clinical and

Experimental Medicine, Linköping University, Linköping, Sweden

2. Department of Hand and Plastic Surgery, Linköping University,

Linköping, Sweden

Poster number: 26

Delivering keratinocytes with the aid of gelatin microcarriers to wounds has been investigated

but the contribution of the transplanted keratinocytes has been difficult to establish. We have

investigated the use of 5 (6) Carboxy-fluorescein N-hydroxysuccinimidyl ester (CFSE) as a

traceable stain for human primary keratinocytes. In this in vitro study different CFSE

concentrations and viability and migrational capacity of CFSE- stained keratinocytes have been

evaluated. The tested CFSE-concentrations did not significantly affect the viability of the

keratinocytes or their ability to migrate when compared to unstained control keratinocytes.

CFSE - stained keratinocytes were cultured in spinner flasks to adhere to gelatin microcarriers

followed by administration to in vitro wounds inflicted in viable human skin in culture. Tracing

of transplanted keratinocytes in tissue sections with the aid of fluorescence microscopy

revealed that keratinocytes retained the CFSE stain over three weeks of tissue culture and

migration of keratinocytes from the microcarriers to the wound bed. We propose a novel use of

CFSE as a passive, easy to use stain for tracing of keratinocytes in tissue sections.

32

Benzenesulfonamide-Terminated Thiol on Plain Gold

Surface; Molecular Orientation and Electron Structure

Andreas Skallberg, Peter Eriksson, Kajsa Uvdal

Division of Molecular Surface Physics and Nanoscience, Department of

Physics, Chemistry and Biology, Linköpings University, Linköping, Sweden

Poster number: 27

Benzenesulfonamide-terminated thiol monolayers on gold surfaces have been prepared to be

used as a model system for molecular recognition. The benzenesulfonamide-terminated thiol

adsorbed to gold have been characterized by means of molecular orientation and electron

structure, using X-ray photoelectron spectroscopy (XPS), Near-Edge X-ray Absorption Fine

Structure Spectroscopy (NEXAFS) and Infrared Spectroscopy (IR). The results shows that the

benzenesulfonamide-terminated thiols form a well-organized monolayer with an average tilt

angle of the aromatic ring structure towards the normal of the gold surface is approximately

63o.

33

Area and Volume in Infection and Inflammation-

Aquaporin 9 in Human Macrophages

Angelika Holm1, Karl-Eric Magnusson1, Elena Vikström1

1Medical Microbiology, Dept. of Clinical and Experimental Medicine, Faculty of

Medicine and Health Sciences, Linköping University, SE-58185, Linköping, Sweden,

angelika.holm@liu.se

Poster number: 28

The ability to regulate volume and shape is essential for cells in all tissues and in various

physiological processes. The water channels, aquaporins (AQPs) are critical in this regard. Cell

swelling is a common denominator in inflammation and immune cell activation and volume and

shape regulation is highly important in both cell damage recognition, motility and in

phagocytosis. We along with other groups have shown the importance of AQPs in cell migration.

AQP9 is the main water/glycerol-channel in human immune cells and is involved in cell

migration. We developed a model where AQP9 is proposed to accumulate in the plasma

membrane with water influx creating a pressure induced space between the actin cytoskeleton

and the plasma membrane. This allows the actin polymerization to take place, rendering the

formation of the protrusion.

Different inflammatory inducing agents affect the expression and the distribution of AQP9 in

macrophages and elevated AQP9 expression has been detected in patients with chronic

inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease (IBD) and

it was proposed to be a potential marker for chronic inflammation. But little is known about the

role and expression of AQPs in inflammatory processes.

We aim to investigate the role and expression of aquaporins (AQPs) and specifically

AQP9 in infection and in chronic inflammatory conditions.

We use Pseudomonas aeruginosa as an infection model to study inter-kingdom signaling, in

relation to volume- and area-regulation and AQP9 expression in macrophages. We are

specifically interested in the effects of the bacterial communication system called Quorum

sensing (QS) that elicit transcription of virulence genes. Infecting macrophages with P.

aeruginosa wild type and the QS- mutant reveal a difference in the ability of the bacteria to

induce phagocytosis and it changes the distribution of AQP9 in the macrophages.

34

Different Gene Response to Mechanical Loading during

Early and Late Phases of Rat Achilles Tendon Healing

M. Hammerman, P. Sepanloo, S. Ramstedt, P. Aspenberg

Orthopeadics, Departemnt of Clinical and Experimental Medicine, Faculty of

Health Sciences, Linköping University, Sweden

Poster number: 29

Introduction Mechanical loading stimulates tendon healing, resulting in a stronger tendon. The positive effect is seen both in the early inflammatory phase and in the later proliferative phase of healing (1-5 respectively 6-14 days after tendon injury).

Hypothesis Mechanical loading leads to different gene expression responses in the early and late phases of tendon healing, especially regarding genes related to inflammation. Methods The Achilles tendon was transected in rats and they were unloaded by tail-suspension the day after. Three or fourteen days after tendon transection, half of the rats were exposed to a 5 minutes episode of loading by treadmill running, and then re-suspended for 3 hours until they were euthanized for RNA collection. 4 rats in each group were used for microarray analysis and the remainder (5-6 rats) for confirmatory quantitative real-time PCR.

Results Day 3: Microarray analysis showed that mechanical loading regulated 137 genes. 9 up-regulated genes related to inflammation were confirmed by PCR, which seems to be important for invasion and activation of leukocytes. Day 14: Microarray analysis showed that mechanical loading regulated 91 genes and no genes related to inflammation was confirmed by PCR. Discussion Mechanical loading during early healing seems to increase the immune response by increasing the invasion and activation of leukocytes. In contrast, mechanical loading during late healing did not seem to have a strong impact on the immune response. This might be relevant regarding the frequent administration of anti-inflammatory drugs during tendon healing in the clinic, and needs to be investigated further.

35

Membrane receptor mapping in ejaculated ram spermatozoa

Vicente-Carrillo, A.1, Casao, A.2, Pérez-Pé, R.2, Cebrián-Pérez, J.A.2, Muiño-Blanco, T.2, Rodríguez-Martínez, H.

1 Developmental Biology, Department of Clinical and Experimental Medicine,

Linköping University, Linköping, Sweden. 2 Department of Biochemistry and Molecular and Cell Biology. Faculty of

Veterinary, University of Zaragoza, Spain.

Poster number: 30

Sperm receptors play a crucial role in male fertility. Specifically, the CatSper receptor family and

Opioid receptor family modulate sperm motility while the hyaluronan receptor HA-CD 44

participates in sperm-cumulus chemotaxis in several species. Their presence and distribution in

ram spermatozoa might, thus, be a relevant parameter for ovine andrology, as well as to

understand molecular interplays between ram spermatozoa and the pre-fertilization

environment. Hereby, the presence and distribution of CatSper 1, 2, 3 as well mu (µ), delta (δ)

and kappa (κ) opioid and the HA-CD44 receptors have been explored for their localization in

fresh spermatozoa collected from nine mature Rasa Aragonesa rams using an artificial vagina. A

swim-up selected sub-population of most robust spermatozoa was used for

Immunocytochemistry (ICC) and Western Blotting (WB). The ICC studies revealed that CatSper

1 was localized in the acrosome and post acrosome, CatSper 2 in the acrosome, CatSper 3 in the

post-acrosome domain (with a weaker immunolabelling in the tail principal piece) and CatSper

4 in the acrosome and post-acrosome domains. The µ and δ opioid receptors were distributed

uniformly in the acrosome while the κ opioid receptor was mainly localized in the sperm tail,

mainly its principal piece (some weaker staining was also observed in the acrosome). The HA-

CD44 was present in the acrosome. The WB confirmed the presence of all these receptors. In

conclusion, Ram spermatozoa possess a full battery of membrane receptors identifiable as

CatSper 1, 2, 3 and 4; the µ, δ and κ opioid receptors and the HA-receptor CD44. Supported by

Vetenskapsråde and Formas, Stockholm.

36

Anti-RANKL Treatment Improves Screw Fixation In

Cancellous Bone In Rats

Magnus Bernhardsson, Olof Sandberg, Per Aspenberg

Poster number: 31

Orthopedic implants may loosen because of increased bone resorption adjacent to the implant. Treatment with bisphosphonates has been shown to improve implant fixation by reducing osteoclast activity. With this study we wanted to test if a treatment against osteoclast differentiation (instead of activity) would be more efficacious than bisphosphonates. We used a rat model to compare osteoprotegerin-Fc (OPG-Fc) with a high or an extreme dose of bisphosphonate (alendronate). A stain-less screw was inserted in the right proximal metaphyseal tibia and a hole was drilled in the same region of the left tibia. The rats were randomized to four groups (total n = 42) and received subcutaneous injections of either OPG-Fc (8 mg/kg, twice weekly), a high dose of alendronate (20 μg/kg, six days per week), an extreme dose of alendronate (200 μg/kg, six days per week) or saline (twice weekly) for four weeks. OPG-Fc increased the pull-out force by 153 % (p < 0.001; Figure 1A) compared to saline controls. No significant difference could be seen between OPG-Fc and alendronate. The total bone volume per total volume (BV/TV) in the former drill hole was measured using microCT, and increased by 754 % (p < 0.0001; Figure 1B) with OPG-Fc compared to saline controls. OPG-Fc also resulted in a significantly higher BV/TV compared to both alendronate doses (p < 0.001; Figure 1B). The results suggest that a treatment with an anti-RANKL agent may be relevant for fixation of orthopedic implants and fracture healing in cancellous bone in rats.

Figure 2. Mechanical and microCT data. OPG-Fc increased pull-out force compared to controls (A). Bone formation was higher in the OPG-Fc group compared to controls and both alendronate groups (B).

37

Surface proteomics for tracking nanoparticle targeting

based on nanoparticle corona

Jacob Kuruvilla1, Susana Cristobal1

1Department of Clinical and Experimental Medicine, Linköping University,

Linköping, Sweden

Poster number XX

Poster number: 32

Nanoparticles are found to be coated with proteins once they are exposed to biological fluids forming the protein corona which can critically affect the behavior and this dictates its interaction with the biological system. Previous studies have focused in studying the whole corona instead of the exposed proteins. Here we introduce a surface proteomic approach where we tag the exposed protein part of the nanoparticle corona which would add the most important information regarding nanoparticle characterization helping in designing their targeting capabilities. Titanium dioxide (TiO2) nanoparticles of sizes 10 and 30nm were incubated in DMEM cell culture media for the formation of the nanoparticle protein corona. The outermost protein layer on the nanoparticles was tagged using biotin on which trypsin on-bead digestion was carried out. NeutrAvidin agarose resin was used to purify the tagged proteins. These proteins were analyzed using mass spectrometry and quantified using a label-free approach.

38

From threading to weaving; on the way towards a

paradigm shift. Robert Pilstål

Department of Physics, Chemistry and Biology, Linköping University

Poster number: 33 It all started with the discovery of the structure of DNA. From there on it was inevitable that we

would decipher the code of life, understand how it translates from coded sequence to

molecular structure and how important such structures are in understanding cell function,

explaining the causes of dire diseases and discovering new remedies. Yet, we have not fully

understood the sequence to structure to function relationship of our evolutionary code; it is

still in bits and pieces.

Today we perform massive [DR]NA and protein sequence comparisons by simply matching letters

with each other, minimizing the mutational distance between them. With this simple method we

can find relationships between divergently evolved structures, and link function from known

entities to newly discovered ones, thus determining their function in turn. This method works fine

if we have the time and resources to use other methods to first discover the function, and then

propagate those attributes to the structure family by sequence similarity. But it breaks down as

soon as we try to look at the great unknown; huge multidomain proteins, protein complexes and

interactions, cofactors and so forth. Then we need to extract this information directly from the

code!

Recent advances in theory has lent us tools to extract even more detailed data from

sequences; structural connections from sequence covariation. Just as we once started to build

sequence families from the code extracted from the genome, we now have to start build

structural clans from the relations that we can extract from those families.

I try to illustrate how such a development could empower us and what it would mean by

comparing threading, an over 20 year old method of structure prediction with weaving; a new,

yet only hypothesized method. Furthermore I discuss what major hurdles I believe we have to

overcome to really get there and what I'm doing to chip in.

39

Spermatozoa rather than seminal fluid drives immune-modulation in the sperm storage tubuli of Red Jungle Fowl hens M Atikuzzaman1, A Höglund2, M Johnsson2, M Alvarez- Rodríguez1, R Mehta1, D Wright2 and H Rodriguez- Martinez1

1Department of Clinical and Experimental Medicine (IKE),

2Department of Physics, Chemistry and Biology (IFM), Linköping University (LiU), Sweden

Poster Number: 34

Spermatozoa along with the seminal fluid (SF) they bathe in, being alien to the female,

negotiate with the oviduct an avoidance of rejection that can warrant fertilization of each

newly ovulated oocyte. Previous findings showed mating caused significant changes in the

expression of genes related to immunity in the sperm storage tubuli (SST) and adjacent

uterus of hens. Here we studied whether SF could be equally responsible for such shifts in

gene expression in pure Red Jungle fowl hens (RJF), the low-fertile ancestor of today´s hen

highly fertile breeds. The RJF were allotted to (i) control-unmated, n=4; (ii) treatment 1-

mated with fertile roosters, n=4 or (iii) treatment 2-inseminated with homologous SF-only,

n=4). Regional oviduct samples were removed 24 h thereafter. Gene expression was studied

using cDNA microarray analysis (12 x 135 k array, Roche NimbleGen) and declared significant

if different at FDR corrected P value (q ≤ 0.05). Mating induced up- and down-expression of

genes, 435 and 230 respectively, yet only in the SST while the SF-insemination failed to

establish any such differential changes. Gene ontology (GO) analysis of the “mated, T1-group”

SST, holding spermatozoa, showed that genes related to the ‘immune system process’ (GO:

0002376) were either down-regulated (10%) or up-regulated (5%). On the other hand, 9% of

the up-regulated SST genes were immune responsive (GO: 0006955) in contrast to

42% of down-regulated genes under the GO term category ‘response to stimulus’ (GO:

0050896). The findings suggest spermatozoa but not necessarily the SF are able to down-

modulate immune-reaction for their survival. Supported by FORMAS, Stockholm.

40

Opiates may attenuate platelet activation

Salumeh Bastami1, Nahreen Tynngård2,3, Ankit S. Macwan3, Tomas

L. Lindahl3 and Srinivas Uppugunduri3

1Department of Medical and Health Sciences, Linköping University,

Linköping, Sweden

2Department of Clinical Immunology and Transfusion Medicine, and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

Poster number: 35 3Department of Clinical Chemistry and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

Morphine and other opiates are widely used for pain treatment in a variety of conditions.

Opiates also affect respiratory function, sedation, diuresis and the functioning of the

gastrointestinal tract. The µ, δ and κ opioid receptors are involved in mediating the effects of

opiates through G-proteins. However, the effect of opiates on platelets is poorly studied. Since

most patients receiving pain relief also require a functional hemostatic system, it is important

to understand their impact on platelets and consequently coagulation. The aim of the study

was to characterize effects of five

commonly used opiates; tramadol, fentanyl, oxycodone, morphine and ketobemidone on

platelet aggregation, and activation response. The expression of the activation markers P-

selectin and fibrinogen was analyzed by flow cytometry. Platelet aggregometry was assessed

by impedance technology. Phosphorylation status of AKT was measured by Western blotting.

All the tested opiates showed inhibition of platelet activation albeit differentially. Tramadol,

ketobemidone and oxycodone significantly inhibited platelet aggregation and activation. The

study indicates that effect on coagulation system should be considered while using opiates for

treatment.

Venue: Stiftsgården Vårdnäs, SE-590 45 BROKIND

Ph. No: +46(0)13368500info@vardnas.com

Forum Scientium

Time Wed 26th Aug'15 Time Thu 27th Aug'15 Time Fri 28th Aug'15

8:00 AM 7:30 AM Breakfast 7:30 AM Breakfast

8:30 AM Poster session III

9:00 AM Coffee/put up posters 9:15 AM Poster session IV

9:30 AM 10:00 AM Coffee 9:15 AM Break

10:30 AM Poster session I

11:15 AM Poster session II 11:30 AM Voting for best poster 10:10 AM Coffee

12:00 PM Lunch 11:45 AM Lunch

Poster competition I & II

Speed dating III & IV 11:20 AM Break

3:15 PM Coffee 2:00 PM Coffee

3:30 PM Poster competitionIII & IV

Speed dating I & II 12:00 PM Lunch

4:30 PM Change 3:30 PM Change 1:00 PM Closure/evaluation

4:45 PM YOGA 3:45 PM Team building activities

6:00 PM Sauna/free activity 6:00 PM Free activity

7:30 PM Dinner (BBQ) 7:15 PM Photography

9:15 PM After dinner activity 7:30 PM Dinner/Prize

9:15 PM After dinner activity

1:45 PM

1:00 PM 1:00 PM

2:30 PM

Travel to VårdnäsNorrkoing-07:00 AMLinköping-08:00AM

Information/introduction of new

members

Travel back toLinköping & Norrköping

Per Jensen'The dog-the behaviour geneticist’s best friend'

Tomas Lindahl'Novel mechanisms of platelet

regulation-targets for new antithrombotic treatments'

8:30 AM

Per Hammarström'Misfolded proteins cause

neurodegenerative diseases'

Torbjörn LindblomFeelgood:Development Pgm. for PhD students

11:30 AM

9:25 AM

Per Aspenberg 'How to make enemies in

science'

10:35 AMJoel Hedlund

'How to succeed in life (caveat emptor)'Clas Malmström

'How to handle Stress'

10:30 AM

Summer Conference 2015 Schedule