6th Student retreat of the Microbiology and Immunology

PhD program  

 12-14 September 2013

Locarno, Switzerland    

Kontaktgruppe  für  Forschungsfragen  

 

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Table  of  Contents    

Table  of  Contents  .....................................................  2  Scientific  Program  ....................................................  3  Guests  ......................................................................  5  Abstracts  ..................................................................  9  Presentations:  .........................................................................................  9  Session  1  ...................................................................................................  9  Session  2  ................................................................................................  12  Session  3  ................................................................................................  15  

Posters:  ....................................................................................................  19  Session  1  ................................................................................................  19  Session  2  ................................................................................................  35  

Social  Event  -­‐  Winetasting  ......................................  51  General  Information  ...............................................  53  Contacts  .................................................................  54  Participants  ............................................................  54                

 

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Scientific  Program    Thursday,  12  September  2013    11:00  –  11:30                          Arrival  and  Welcome    17:30  –  18:30                        Guest  speaker:                                                                                    Prof.  Antonio  Lanzavecchia,  MD                                                                                    Institute  for  Research  in  Biomedicine,                                                                                    Bellinzona    20:00  –  22:00                          Poster  Session  1      Friday,  13  September  2013    8:00  –  10:00                                  Workshop  “Presentation  Skills”                                                                                        by  Janneke  van  Woerden      10:30  –  12:30                            Workshop  “Presentation  Skills”    14:00  –  15:00                          Guest  speaker:                                                                                        Prof.  Urs  Albrecht                                                                                      Department  of  Biology  &  Biochemistry                                                                                      University  of  Fribourg    15:30  –  16:30                        Guest  speaker:                                                                                    Dr.  Santiago  Gonzalez                                                                                    Institute  for  Research  in  Biomedicine,                                                                                    Bellinzona      16:30  –  18:00                            Student  presentations,  Session  1                                                                                        (3  students)    19:30  –  21:30                          Poster  Session  2    

 

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Saturday,  14  September  2013    9:00-­‐10:30                                        Student  presentations,  Session  2                                                                                        (3  students)    11:00  –  12:00                                Student  presentations,  Session  3                                                                                          (3  students)    14:45                                                                Departure                                                        

 

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Guests    Prof.  Antonio  Lanzavecchia,  MD   Immune Regulation Director Via Vela, 6 6500 Bellinzona, Switzerland lanzavecchia@irb.usi.ch +41 91 820 0310 +41 91 820 0305 Antonio Lanzavecchia earned a degree in Medicine at the University of Pavia where he specialized in Paediatrics and in Infectious Diseases. From 1983 to 1999, he worked at the Basel Institute for Immunology and since 1999 he is the Director of the Institute for Research in Biomedicine in Bellinzona. He taught immunology at the Universities of Genova and Siena and since 2009 is Professor of Human Immunology at the Swiss Federal Institute of Technology ETH Zurich. He is Member of the EMBO and Fellow of the Royal College of Physicians. Awarded the EMBO medal in 1988 and the Cloëtta prize in 1999, Antonio Lanzavecchia published more than 270 papers. His research has covered several aspects of immunology: from antigen processing and presentation to dendritic cell biology, from lymphocyte activation and trafficking to T and B cell memory.

         

 

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Dr.  Santiago  Gonzalez   Infection and Immunity Group Leader Via Vela, 6 6500 Bellinzona, Switzerland santiago.gonzalez@irb.usi.ch +41 91 820 0360 +41 91 820 0305 Santiago F. González holds two PhD degrees, one in microbiology from the University of Santiago de Compostela (Spain) and one in immunology from the University of Copenhagen (Denmark). From January 2007 to September 2011 he was a postdoc in the group of Michael Carroll at the Immune Disease Institute, Harvard Medical School, in Boston (USA). He has been awarded two EU Marie Curie Fellowships, one for his postgraduate studies in Denmark where he studied skin inflammation and the connection between innate and adaptive responses from a molecular perspective. The second fellowship was a Marie Curie International Outgoing Fellowship awarded in 2008 for a project shared between Harvard Medical School and the National Center for Biotechnology (Madrid). The project focused on the study of the defense mechanism against Influenza virus. He has published several papers related with antigen trafficking, memory B cell, and the regulation of the immune system in high impact journals. During his work at Harvard he studied the transport mechanism of an influenza vaccine in the lymph node. He found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. These results have important implications for the

 

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generation of durable humoral immunity to viral pathogens through vaccination and were published in Nature Immunology. In November 2012 he joined the Institute for Research in Biomedicine in Bellinzona as a group leader studying pathogen-host interaction. Prof.  Urs  Albrecht  Department of Biology & Biochemistry, University Fribourg Chemin du Musée 5 CH-1700 Fribourg urs.albrecht@unifr.ch +41 26 300 8636 +41 26 300 9735  Urs Albrecht studied at the University of Zurich and got his diploma in Biochemistry in 1988. He earned his PhD in 1993 under the supervision of Prof. D. Schümperli in the Department of Zoology at the University of Bern. Afterwards he did a postdoc at the Department of Biochemistry at the Baylor College of Medicine in Houston, USA, with Prof. G. Eichele. In 1998 he was got a position as assistant professor at the same institute and from 1999-2001 at the Max-Planck Institute for Experimental Endocrinology in Hannover, Germany. Since 2001 he is an associate professor at the department of biochemistry at the University of Fribourg in Switzerland where he studies circadian rhythms in mammals. From 2008-2011 he was head of biochemistry Unit of the department of medicine at the University of Fribourg.

     

 

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Dr.  Janneke  van  Woerden    j.vanwoerden@aim.uzh.ch

 Janneke van Woerden studied Biology at the University of Utrecht and did her master’s degree in animal biology at the same university. She obtained her PhD in Anthropology in 2011 from the University of Zurich. She trained with Pamela Alean, who offers courses in transferable skills (e.g. project management, oral and poster presentations, career development, supervision of students) for researchers and teachers at institutions of higher education. Outline of the workshop "Effective scientific presentations": Learning outcomes By the end of the course the participants will be able to…

- prepare and give an oral or a poster presentation about their research work in an effective manner for the specific target audience

- recognize the elements of best practice when they observe the presentations of others

Content

- Elements of professional research presentations: audience orientation; objectives of the presentation; structure; preparation and delivery; personal performance; use of body language and mimic

- Design of powerful posters; oral presentations of the posters

- Effective use of power point for oral presentations.

 

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Abstracts  

Presentations:  Session  1    Identification of new therapeutic targets to modulate lymphatic vessel function Erica Russo, Alvaro Sanchez Teijeira, Maximilian Nitschké, Maria Iolyeva, Ann-Helen Willrodt, David Aebischer, Simone Häner, and Cornelia Halin Institute of Pharmaceutical Sciences, ETH Zurich Wolfgang-Pauli Strasse 10, Zurich erusso@pharma.ethz.ch Afferent lymphatic vessels are present in most tissues, where they begin as blind-ended capillaries, which merge into larger collecting vessels that connect with draining lymph nodes (LNs). Afferent lymphatic vessels are essential for the maintenance of tissue fluid homeostasis, absorption of dietary fatty acids (in the intestine) and mediate the trafficking of immune cells. Dysfunction of the lymphatic vasculature may contribute to the pathogenesis of human diseases: For example, lymphedema is a condition caused by damaged lymphatics resulting in an accumulation of lymph fluid and tissue swelling. Lymphatic vessels may also serve as a conduit to LNs and thereby are involved in systemic metastasis of cancer cells. Moreover, recent evidence suggest that lymphatic vessels are also involved in the regulation of tissue inflammation. An important goal of our research is to identify and validate new molecules, which could serve as therapeutic targets to modulate lymphatic vessel function and immune responses. In particular, we are interested in investigating the molecular mechanisms that control the migration of leucocytes,

 

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particularly dendritic cells, into and within lymphatic vessels. Furthermore, we are currently studying molecular mediators that are involved in (lymph)angiogenesis and lymphatic drainage. For this, we use standard molecular and cell biology methods, mouse models of inflammation and leukocyte migration, as well as intravital microscopy. In addition, we frequently employ FACS analysis, FACS-sorting and transcriptional profiling. MCMV-specific T Cell Immunity in Glandular Tissue Jenny Thom, Annette Oxenius ETH Zürich - Institute of Microbiology Wolfgang-Pauli-Strasse 10 CH-8093 Zürich jenny.thom@micro.biol.ethz.ch Cytomegaloviruses (CMVs) are ubiquitous β-herpesviruses that establish lifelong infection and latency. Prolonged secretion of virus in saliva represents a major route of CMV transmission with acinar glandular epithelial cells propagating virus for months after virus replication is controlled in other organs. CD8 T cells have been shown required and sufficient to control lytic viral replication in visceral organs. The salivary gland (SG) is uniquely capable of establishing and maintaining an immunological setting where CD8 T cells fail to control viral replication and otherwise dispensable IFNg-producing CD4 T cellsbecome essential to cease viral spread. We aim to characterize tissue intrinsic and viral factors that render CMV resistant to CD8 T cell mediated clearance in this important organ of viral dissemination. Using murine CMV (MCMV) our group showed that the activation of MCMV-specific CD8 T cells is prevented in the SG due to antigen deprivation via MCMV mediated down-regulation of MHC I molecules, which is known to be exceptionally efficient in acinar glandular cells of the SG. Furthermore, we found that the SG has the potential to induce and maintain a tissue resident memory

 

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(TRM) phenotype in CD8 but not in CD4 T cells in an antigen-independent manner. Using CD4 and CD8 TCR transgenic mice with specificities for MCMV, we are further analyzing the inductive requirements and functional properties of TRM CD8 T cells in the SG. To gain deeper insight into tissue environmental influences on T cell migration, antigen recognition, and effector functions, we are investigating MCMV directed CD8 and CD4 T cell responses via intravital imaging. Virulence and public goods in Pseudomonas aeruginosa Elisa Granato Universität Zürich, Institute of Plant Biology, Y13-L05, Winterthurerstrasse 190, 8057 Zürich egranato@botinst.uzh.ch Explaining the evolution of cooperation is of great interdisciplinary interest. A special focus on microbial model systems has recently emerged, with great advances being made in understanding both theoretical and experimental aspects of cooperative traits in bacteria. Our group is especially interested in social phenomena associated with the secretion of “public goods” such as iron-scavenging molecules (siderophores). Besides their role in bacterial metabolism, these compounds are known to be key players in the establishment of virulence in pathogenic bacteria. Here, we aim to understand the relationship between population structure, siderophore production levels and virulence. Specifically, we test theoretical models predicting that in the absence of population structure (e.g. under well-shaken conditions), a population of siderophore-secreting cells can be invaded by non-producing, “cheating” cells, which no longer contribute but still benefit from the siderophores produced by others. As a consequence of this selection, siderophore availability and therefore virulence should diminish. In contrast, siderophore production and virulence should remain high in

 

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structured populations, where cheating mutants should not be able to invade. We want to address these questions by establishing a novel experimental evolution system. Specifically, we will investigate the interaction between the opportunistic human pathogen Pseudomonas aeruginosa and a model host organism, the roundworm Caenorhabditis elegans, where the secretion of siderophores is essential to establish infections. We aim to elucidate the changes in virulence and siderophore production associated with different levels of population viscosity and dispersal using experimental evolution in a variety of scenarios and selection regimes. Session  2   NADPH-dependent enzymes that react through a covalent ene-adduct as intermediate Raoul G. Rosenthal, Marc-Olivier Ebert, Patrick Kiefer, Dominik M. Peter, Julia A. Vorholt & Tobias J. Erb Swiss Federal Institute of Technology Zurich, Institute of Microbiology, Wolfgang-Pauli-Str. 10, HCI F430, 8093 Zürich, Switzerland rraoul@ethz.ch The pyridine nucleotides nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are ubiquitous redox coenzymes that are present in all living cells. Although about 16% of all characterized enzymes use pyridine nucleotides as hydride donors or acceptors during catalysis, a detailed understanding of how the hydride is transferred between NAD(P)H and the corresponding substrate is lacking up to date for many enzymes. Here we demonstrate the formation of a covalent ene-intermediate between NADPH and its substrate in the catalytic cycle of crotonyl-CoA carboxylase/reductase (Ccr), using nuclear

 

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magnetic resonance spectroscopy (NMR) in combination with high resolution mass spectrometry (MS). Our results allowed us to directly access the catalytic cycle of other NADPH-dependent enzymes in an unprecedented way, suggesting that formation of NADPH-ene intermediates is a more general principle in catalysis and also operates in type II fatty acid biosynthesis. Resolving Salmonella-triggered inflammation in time and space Boas Felmy, Wolf-Dietrich Hardt ETHZ HCI G409 Wolfgang-Pauli-Str. 10 8093 Zurich, Switzerland bfelmy@ethz.ch Salmonella enterica enterica serovar Typhimurium (S. Typhimurium) can be transmitted by contaminated food and is a major cause of foodborne diseases all over the world. After ingestion, a part of the Salmonella population invades the intestinal mucosa and activatecaspase-1 which leads to the maturation and release of pre-formed cytokines. Eventually, these mediate an inflammation of the gut which enables S. Typhimurium to outcompete the microbiota. Furthermore, this pathogen also induces proinflammatory gene expression controlled mainly by the nuclear factor (NF)-κB signaling pathway. We are interested in identifying the cell type initially responding to S. Typhimurium infection by expressing proinflammatory genes. There are two possibilities. Either epithelial cells, shortly after being invaded, or lamina propria cells are activated and subsequently express proinflammatory genes. To monitor this process we are using mice expressing a GFP labeled NF-κB variant and infect them with S. Typhimurium. The pathogen’s entry into intestinal epithelial cells and the intracellular localization of NF-κB is tracked by live microscopy.

 

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Translocation of NF-κB to the nucleus indicates activation of proinflammatory gene expression and thereby the quantification of the GFP signal in the nucleus will reveal activated cells. Additional information about this process will be provided by reverse transcription quantitative real-time PCR arrays, immunofluorescence stainings and in situ hybridization. Preliminary data suggests that S. Typhimurium activates proinflammatory gene expression in lamina propria cells which then probably activates epithelial cells in turn. Further research is needed to fully elucidate the mechanism behind this observation. Identifying a microglia specific transcription factor Anne Buttgereit, Iva Lelios, Melanie Greter and Burkhard Becher University of Zurich, Institute of Experimental Immunology, Winterthurerstrasse 190, CH-8057 Zürich, buttgereit@immunology.uzh.ch Microglia are the resident mononuclear phagocyte population of the CNS and are considered the first line defense against invading pathogens and in scavenging dying cells. In contrast to most other populations of the mononuclear phagocyte system (MPS), microglia are radiation resistant and maintain themselves locally and independently of circulating bone marrow derived precursors in the steady state. It was shown that adult microglia derive exclusively from primitive myeloid progenitors in the yolk sac prenatally before E8.5 and subsequently migrate into the brain through blood vessels around E9.5. To this day, there is no genetic tool to specifically manipulate microglia in vivo. To identify genes, that are specifically expressed by microglia but not by other cells or precursors of the MPS, we analyzed mRNA expression profiles of purified distinct ex vivo macrophage populations from lung, peritoneum,

 

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spleen and CNS. Based on these studies we selected one such potential ‘microglia-regulating’ candidate gene termed Sall1. Sall1 is a transcription factor highly expressed in progenitor cells during development and regulates kidney development and neurogenesis. Preliminary data generated from Sall1-GFP reporter mice confirm a specific expression of Sall1 by microglia but not by macrophages in other tissues. Validating these findings will subsequently allow us to (1) fate map microglia during embryogenesis, adult homeostasis and inflammation, (2) to examine the role of Sall1 in microglia in steady state and inflammation and most importantly (3) to achieve a microglia-specific deletion of any gene of interest using Sall1-Cre mice. Session  3   A molecular perspective on fungal defense and microbial interactions David F Plaza; Stefanie S Schmieder; Niels van der Velden; Silvia Bleuler-Martinez; Therese Wohlschlager; Nicola Haines; Markus Aebi; Markus Künzler Institute of Microbiology, ETH Zurich, Wolfgang-Pauli Strasse 10, HCI F420, Zurich david.plaza@micro.biol.ethz.ch Fungal life history is strongly influenced by a sum of abiotic and biotic environmental factors. Fungi participate in interaction networks involving macro and microorganisms in competitive, cooperative or exploitative forms and, as part of them, they are the objects of predation and foraging by nematodes and arthropods. The main research goal of our team is to obtain a comprehensive understanding on fungal defense against predation by identifying and characterizing effector defense molecules in the genome of model filamentous fungi. We had found that toxic lectins are a common theme in fungal defense

 

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showing a rich variety of carbohydrate specificities; therefore, we use different genetic tools to accurately identify target glycans recognized by these lectins in the model invertebrates Caenorhabditis elegans and Drosophila melanogaster as a first step to characterize their mechanisms of toxicity. In addition, we use RNA-seq as an instrument to study the genome wide regulation of the defense response against nematode predation in the model basidiomycetous fungus Coprinopsis cinerea and to identify novel effector proteins. Finally, we combine new microfluidic technologies with the use of an engineered dtomato reporter strain of C. cinerea to monitor in a confined and controlled area the spatial regulation of the defense response against nematodes in this fungus. Biosynthetic insights into a recently discovered family of natural products Maximilian J. Helf, Michael F. Freeman, Brandon I. Morinaka, Anna L. Vagstad and Jörn Piel ETH Zurich, Institute of Microbiology, HCI G 429, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland, mhelf@ethz.ch Marine sponges are a rich source of bioactive natural products, most of which are produced by bacterial symbionts that cannot be cultured by current methods. Many natural products cannot be easily chemically synthesized or isolated in large enough quantities from their original producers. To overcome this supply problem, our lab works on the identification, isolation and heterologous expression of biosynthetic gene clusters for natural products. We focus on compounds with intriguing structures and bioactivities, making the gene clusters accessible to analysis and genetic modification in the process. The highly cytotoxic polytheonamides from the marine sponge Theonella swinhoei belong to the largest known secondary metabolites. They are extensively modified peptides with 48

 

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residues, of which 22 are non-proteinogenic. Recent genetic work in our lab showed that polytheonamides are synthesized by an as-yet uncultivated bacterial symbiont via a remarkable pathway that involves numerous epimerizations, hydroxylations and methylations of a ribosomal precursor peptide. Polytheonamides are the first characterized members of a new family of natural products called "proteusins". Parts of the polytheonamide gene cluster have been expressed heterologously in E. coli. We are characterizing the function and substrate specificity of the modifying enzymes, paving the way for their use in tailored biosynthesis of non-natural compounds. There are dozens of similar, as yet undescribed proteusin gene clusters present in several bacterial genomes. Many proteusin gene clusters comprise putative enzymes of completely unknown function. Characterization of these enzymes may lead to the discovery of novel biochemical reactions and tools for biotechnology. Role of rhinovirus in asthma exacerbations and epithelial barrier integrity Can Altunbulakli, Cezmi A. Akdis SIAF, Swiss Institute for Allergy and Asthma Research, Obere Strasse 22, CH-7270, Davos Platz, Davos, Graubünden, Switzerland can.altunbulakli@siaf.uzh.ch Asthma exacerbations are commonly caused by upper respiratory infections by pathogens such as human rhinoviruses (HRV). Development of recurrent asthma exacerbations have been linked to a decrease in epithelial barrier function. In my PhD studies; the effects of individual and co-infections of different HRV strains on sinus and bronchial airway barrier function will be investigated. Epithelial barrier integrity will be investigated via measurements of transepithelial resistance (TER), paracellular flux, and localization of junctional proteins via confocal microscopy; performed on human primary airway

 

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epithelial cells cultured on air-liquid interfaces (ALI). Transcriptome analysis and quantitative PCR will be performed of RV infected epithelial cells to investigate differences in gene expression and identification of novel genes relevant to response against RV. Overexpression and inhibition studies by siRNA targeting potentially relevant novel genes will be performed on primary epithelial cells. Monoclonal Abs and transgenic mice will be developed to demonstrate the in vivo relevance of novel target genes. With this approach, our focus is on demonstrating human in vivo relevance and identification of novel genes that may be used as biomarkers for epithelial leakiness and rhinovirus infection.

 

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Posters:  Session  1   Microfluidic platforms for monitoring interactions between fungi and bacteria Martina Stöckli1, Claire E. Stanley2, Pauli T. Kallio1, Markus Künzler1, Andrew J. deMello2, Markus Aebi1 1: Institut für Mikrobiologie, Wolfgang-Paulistrasse 10, 8093 Zürich 2: Institut für Chemie- und Bioingenieurwissenschaften, Wolfgang-Paulistrasse 10, 8093 Zürich smartina@micro.biol.ethz.ch Bacteria and fungi coexist and interact in a variety of environments. These interactions have major implications on the biology of the interacting partners and on the environment that the microorganisms inhabit. Both organisms feed by absorption and thus antagonistic strategies have evolved due to trophic competition. The basis of these interactions, however, is in many cases not well understood, as their investigation is technically challenging. Herein, microfluidic platforms that address these questions are presented. These platforms are easy to handle as a filamentous fungus can grow into the microchannels from an agar plug that is placed next to a lateral opening. Subsequently, the platform can be co-inoculated with bacteria through a separate inlet, allowing changes in morphology, growth rate, and interaction patterns of the same hyphae, upon the addition of bacteria, to be investigated over time. In addition, a platform that allows rapid exchange of the fluid surrounding the hyphae is detailed. Using this platform the response of hyphae to different compounds can be studied at a single cell level. The interaction of the basidiomycetous model organism, Coprinopsis cinerea, with two different strains of the gram-

 

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positive bacterium, Bacillus subtilis, which differ in the secretion of lipopeptides, has been studied. Using these microfluidic platforms, it has been observed that both bacterial strains attach to hyphae in an end-on manner. However, in the presence of the lipopeptide producer strain the hyphae stop growing and exhibit leakage of the cytoplasm in certain cells. The role of the oral epithelium in protective immunity against C. albicans Simon Altmeier ETH Zürich Institut für Mikrobiologie HCI G 437 Wolfgang-Pauli-Str. 10 8093 Zuerich altsimon@ethz.ch The pathogenic fungus C. albicans is a major risk factor for immunosuppressed individuals such as those suffering from AIDS. During infection, C. albicans initially interacts with epithelial cells, which not only serve as a physical barrier, but are also involved in the activation of the immune response. This crosstalk of the epithelium with leukocytes results in neutrophil recruitment to the site of infection and the production of antimicrobial peptides, both of which are highly relevant for fungal clearance. This project aims at identifying factors that are produced by the non-hematopoietic compartment in response to the fungus and relevant for protective immunity. An example of such a factor is granulocyte colony stimulating factor (G-CSF), which is essential for neutrophil trafficking. Using a mouse model of oropharyngeal candidiasis, we will identify the cellular source of G-CSF and examine how G-CSF induction is regulated. Thus, different subsets of non-hematopoietic cells will be isolated from the oral cavity of mice. Host and pathogen-derived molecules mediating the induction of G-CSF will be examined in vitro using immortalized cells.

 

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Afterwards, the in vitro data will be confirmed in vivo by infecting knockout mice with wild type or mutant Candida. In addition to G-CSF, we aim to identify further factors produced by the non-hematopoietic compartment that are relevant for protective immunity. For this, we will employ a genome-wide transcriptional approach using RNAseq and mutant Candida strains displaying increased or decreased pathogenicity. Together, this will help to identify novel immune pathways relevant for protection from fungal infection. Phenotypic and functional characterization of mesenchymal lymphoid tissue organizer cells Hung-Wei Cheng, Qian Chai, Lucas Onder, Elke Scandella, and Burkhard Ludewig Institute of Immunobiology, Kantonal Hospital St. Gallen, Rorschacher Strasse 95 CH-9007 St. Gallen Hung-Wei.Cheng@kssg.ch Lymph nodes (LNs) are secondary lymphoid organs which develop already in the sterile environment of the fetus in particular areas of lymphatic drainage. Currently, lymph node organogenesis is seen as a programmed process involving the interaction between haematopoietic cells and non-haematopoietic cells. In the early stage of LN formation, RORgt-expressing CD45+CD3-CD4+IL7Ra+ lymphoid tissue inducer (LTi) cells are recruited to the site of LN anlagen by mesenchymal lymphoid tissue organizer (mLTo) cells. Clusters of LTi cells and mLTo cells will be formed, in which lymphoxin(LT)α1β2 from LTi cells ligates the LTβ receptor (LTbR) on mLTo cells. The activation of LTβR-signaling will further enhance the expression of homeostatic chemokines CCL19, CCL21 and CXCL13, which attracts more T and B cells to the inductive site. As T and B cells themselves express LTα1β2, a positive feedback loop will be generated that

 

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eventually leads to the complex microenvironment of T and B cells zones. Whereas the molecular details involved in LTi cell-dependent stimulation of LN development have been thoroughly described, the development and differentiation of mLTo cells remains enigmatic. In this project, we plan to generate an inducible Ccl19-tTA (tetracyclin transactivator) bacterial artificial chromosome transgenic mouse which permits inducible lineage tracing through genetic tagging of mLTo cells. This new transgenic mouse model will facilitate detailed tracking of these mLTodifferentiation pathways throughout secondary lymphoid organ development. Systems immunological analysis of lymph node stromal cell phenotype and function Mario Novkovic, Lucas Onder, Hung-Wei Cheng, Elke Scandella, Gennady Bocharov, Burkhard Ludewig Institute of Immunobiology, Kantonal Hospital St. Gallen, Rorschacher Strasse 95, CH-9007 St. Gallen Mario.Novkovic@kssg.ch The major function of the immune system is to protect the host from infection with pathogens by finding and eliminating malignant cells. Secondary lymphoid organs (SLOs) exist mainly to facilitate efficient interactions between the immune system and foreign antigens and they are strategically positioned at routes of pathogen invasion. Lymph nodes (LNs), for example, are found at convergence points of afferent lymph vessels and morphologically they can be divided into three regions: cortex, paracortex and medulla. Each region contains various subcompartments with different cell populations. Non-hematopoietic stromal cells, such as fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), blood endothelial cells (BECs), lymphatic endothelial cells (LECs) and marginal reticular cells (MRCs), contribute greatly to the induction of

 

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immune responses and general functionality of the immune system. Thus it is important to understand their structural organization into different subcompartments, interactions, differentiation patterns and plasticity on a cellular and molecular level, and how these affect the global systems level. Our goal will be to dissect and predict LN stromal cell behavior in health and disease by multi-scale integration of both lower and higher level biological data. Hence, two different systems immunology approaches will be applied. First, molecular signatures characterization of particular stromal cell subsets will be performed using a microarray-based comparative transcriptome analysis to determine differentially expressed genes. And second, a spatio-temporal (4D) mathematical model of the structural-functional organization of the popliteal LN will be developed to examine the importance of stromal cells in LN remodelling and the initiation of antiviral immunity. Pyoverdine diversity, cross-use, and exploitation among Pseudomonas strains in natural communities Elena Butaitė, Rolf Kümmerli UZH, Institute of Plant Biology, Winterthurerstrasse 190, Y13-L-52, CH-8057 Zurich elena.butaite@uzh.ch Fluorescent pseudomonads secrete the siderophore pyoverdine to scavenge iron from the local environment. While laboratory studies have shown that pyoverdine represents a public good that can be exploited by non-pyoverdine-producing mutants (cheats), survey studies on clinical and environmental isolates have reported an enormous variety of different pyoverdine types. Here, we predict that there is a causal link between these two findings. Specifically, we argue that the presence of exploitative cheats selects for changes in pyoverdine molecule structure and receptor affinity in the

 

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producer strain, in order to make pyoverdine more exclusive to producers and less accessible to cheats. This in turn should select for better adapted cheats that resume exploitation, or for ‘de novo’ cheats in the novel pyoverdine-type background. This continuous evolutionary antagonism is expected to drive local pyoverdine and strain diversity. In our study, we test this hypothesis by isolating strains belonging to the fluorescent Pseudomonas from soil and water. In both habitat types, we compare strains isolated from the same, close, or distant patches for their ability to produce pyoverdine, and to exploit the pyoverdine produced by other strains. If the hypothesis holds true, we would expect that: (a) non-producing strains co-exist with producers in local patches; (b) pyoverdine diversity occurs at the patch level; and (c) there is local adaptation. Furthermore, we predict that strain diversity and local adaptation should be more pronounced in soil than in water habitats, because the latter is less-structured allowing strains to mix more easily, thereby undermining local interactions. Design and implementation of synthetic CO2-fixation pathways Lennart Schada von Borzyskowski, Ramon Weishaupt, Thomas Schwander, Tobias J. Erb Institute of Microbiology HCI F430 ETH Zürich Wolfgang-Pauli-Strasse 10 8093 Zürich Vorholt Group Lennarts@ethz.ch The problem of steadily rising atmospheric CO2 concentrations requires society to find novel creative ideas to control this greenhouse gas. Previous efforts in this field have been focused on the optimization of existing natural pathways (e.g., the Calvin cycle in plants) that usually limit carbon flux.

 

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In contrast to such classical biotechnological efforts, the emerging field of synthetic biology provides an alternative approach to address this question. Here, completely novel pathways of improved functionality are intended to be created by the combination of different enzymatic reactions from different sources or organisms. This project aims at creating a novel “synthetic” CO2 fixation pathway in bacterial host organisms by combining enzymes from different metabolic contexts. In order to assemble the synthetic pathway, the corresponding genes and genetic elements are currently introduced with a specific plasmid assembler technique. An operating synthetic CO2-fixing pathway will be demonstrated by enzyme assays and 13C isotope labeling techniques combined with high resolution mass spectrometry metabolomics. The synthetic pathway will be optimized using experimentally-guided metabolome network modeling approaches and targeted knock-outs in the host organism. Subsequently, the CO2-fixing organism will be selected for growth with CO2 and the CO2-fixing strain will be compared to the wildtype, before the modified organisms will be forced to exclusively use this synthetic pathway for carbon assimilation. Such synthetic CO2-fixation pathways could find applications in biotechnology and “green” chemistry (e.g., the production of biomass or biopharmaceuticals) or in environmental protection (e.g., customized control of CO2 emissions). Structure-to-function characterization of Saccharomyces cerevisiae STT3 protein Elsy Ngwa, Kristina Poljak, Susanne Müller and Markus Aebi Institut für Mikrobiologie, Wolfgang-Pauli-Strasse 10, 8093 Zürich elsy.ngwa@micro.biol.ethz.ch Asparagine-linked protein glycosylation is a highly conserved and essential posttranslational modification in eukaryotes. The

 

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key step of this reaction is catalyzed by oligosaccharyltransferase (OST), an enzyme that transfers a pre-assembled lipid pyrophosphate linked oligosaccharide to selected asparagine residues on nascent proteins entering the endoplasmic reticulum. Most eukaryotes express this enzyme as a heterooligomeric complex of eight proteins in which the STT3 protein is the catalytic subunit. The structure and function of this essential STT3 protein is still not fully understood. The crystal structure of the bacterial homologue of STT3, PglB has recently been solved and a mechanism for the glycosylation reaction in bacteria has been proposed. However, such a mechanism cannot be generalized and accepted as universal. We take advantage of the PglB structure and mutagenize yeast STT3 at chromosomal level to study N-linked glycosylation of proteins and enzyme complex formation in vivo. We will use media containing 5FOA to analyze growth phenotypes of point mutants and thus enzyme activity. OST Subunit turnover rates, measured using Selected Reaction Monitoring (SRM) coupled with SILAC and Blue native electrophoresis will both be used to assess complex formation upon introducing point mutations. Glycosylation efficiency of OST will be determined by analyzing site occupancy for different substrate polypeptides using mass spectrometry and finally glycosylation phenotypes of point mutants will be analyzed by assessing the glycosylation status of the vacuolar carboxypeptidase Y. Bistable gene expression of the Salmonella Pathogenicity island 1 Ismeta Curkic, Alexander Sturm, Markus Arnoldini, Martin Ackermann, Wolf-Dietrich Hardt Institute of Microbiology, ETH Zürich, Wolfgang-Pauli Strasse 10 icurkic@ethz.ch

 

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Bistability of a bacterial system describes the existence of two stable phenotypes within an isogenic population. Furthermore, bistable gene expression in Salmonella Typhimurium (S. Tm) also guarantees survival of the bacterial population by establishing a cooperative bacterial system. While one fraction of cells propagates infection, the second population establishes the infection and triggers inflammation. In S. Tm, bistable expression of genes was already demonstrated for factors, including flagellar genes as well as the Salmonella Pathogenicity Island 1 (SPI-1) that encodes the type three secretion system 1 (TTSS-1). The expression of the TTSS-1 is governed by a complex regulatory cascade, including the core regulators hilC, hilD, hilA and the negative regulator hilE as well as a number of global regulators. However, the molecular mechanism of how bistability of the S. Tm TTSS-1 expression is achieved has remained unclear. We are interested in deciphering the key players triggering the phenotype switch from TTSS-1-to TTSS-1+. Therefore, we established a fluorescence microscopy technique using microfluidic devices, which allows observation of expression profiles of individual TTSS-1 regulators and growth of single cells. So far, we were able to confirm the previously observed growth retardation and reduced cell size of cells expressing the TTSS-1. Using a fluorescently labeled TTSS-1 reporter, we plan to characterize TTSS-1 expression under different experimental conditions and ultimately observe the expression of individual TTSS-1 core regulators. A chimeric Ricin B fold lectin involved in C. cinerea defense response Stefanie S. Schmieder, Kevin Bärlocher, David F. Plaza, Silvia Bleuler-Martinez, Markus Aebi, Markus Künzler Institute of Microbiology, ETH Zürich sschmied@ethz.ch

 

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In the fungal kingdom, a large number of lectins with different specificities have been isolated from fruiting and resting bodies of multicellular representatives of the phyla Basidiomycota and Ascomycota. These lectins are synthesized in the cytoplasm of the fungal cell and various lines of evidence suggest that these lectins play a function as recognition and effector molecules in the chemical defense of the host fungi against predators and grazers. Upon ingestion of the host cytoplasm, these lectins bind to specific glycoconjugates in the digestive tract of the predators which leads to inhibition of development and eventually killing of the predator by a yet unknown mechanism. Toxicity of several different fruiting body lectins is assayed using model organisms e.g. the bacterivorous nematode Caenorhabditis elegans. Here we present a novel chimeric lectin form Coprinopsis cinerea involved in defense against predatory nematodes. IL-36 cytokines in fungal infections Mareike Bindszus, Nabil Boulos, Jan Kisielow, Manfred Kopf Institute of Molecular Health Sciences, ETH, Schafmattstrasse 22, 8093 Zurich mareike.bindszus@biol.ethz.ch IL-36 cytokines are members of the Interleukin-1 family and their signaling shows many features already known from IL-1. Binding of IL-36α, IL-36β or IL-36γ to the IL-36 receptor (IL-36R) leads to activation of MAPKs and NF-κB, but only if the cytokine and its receptor are present as a ternary complex together with the IL-1 receptor accessory protein (IL1RAcP). Furthermore, the IL-36 receptor antagonist (IL-36Ra)can block the signaling via binding to the IL-36R. For all IL-36 cytokines (IL-36α, IL-36β, IL-36γ and IL-36R) specific N-terminal truncations were found to enhance their activity in vitro. Interestingly, the truncations followed an unconventional pattern. We are interested in understanding the processing of

 

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IL-36 cytokines and plan to study the role of their N-terminal leader sequences. Recent data from human as well as murine studies revealed that IL-36 plays a key role in the inflammatory skin disorder psoriasis. However, the physiological role of IL-36 cytokines and their beneficial effect in infections remains obscure. Preliminary experiments from our group showed that IL-36 receptor deficiency renders mice susceptible to systemic Candida albicans infection. In the future work we want to identify the cellular components and unravel the underlying molecular mechanisms that are responsible for the severely worsened phenotype in fungi infected mice. Search of putative autoantigens in multiple sclerosis by screening a brain-derived cDNA library Katharina Pallmer, Raquel Planas, Mireia Sospedra, Roland Martin Universitätsspital Zürich Klinik für Neurologie Sternwartstrasse 14 8091 Zürich In many T cell-mediated autoimmune diseases including Multiple Sclerosis (MS) there is still an incomplete understanding of which is/are the most important autoantigen(s).The search for possible autoantigens triggering MS has been so far too hypothesis-driven and focused on few myelin proteins. The generation of a brain-derived cDNA library offers therefore an unbiased opportunity to identify putative autoantigens. Brain-infiltrating clonally expanded CD4 and CD8 T cell clones (TCC) have been generated rom a MS patient who died after a very aggressive course. A brain-derived cDNA library will be generated using RNA from the brain lesions of the same patient. In order to screen the library, autologous EBV-immortalized B cells will serve as antigen-presenting cells (APC) after transfection with plasmids containing the brain

 

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cDNAs as inserts. The transfected APCs will process and present the antigens encoded by the brain cDNA inserts. When a TCC specifically recognizes an antigen it will be activated and will show an increase in Ca2+ influx. Using Fura-2 as Ca2+ indicator, activated TCCs can be detected applying ratiometric microscopy. APCs in direct contact with an activated TCC will be captured with a glass capillary and the identity of the antigen will be determined by sequencing the plasmid cDNA insert. With this unbiased approach possible new autoantigens in MS might be identified. The knowledge of the autoantigen spectrum in MS is essential to develop tolerization therapies. Integrating major genetic and environmental risk factors of an autoimmune disease: Influence of HLA-DR15 haplotype and EBV infection on the global repertoire of T cells in multiple sclerosis patients Fabienne Largey, Ilijas Jelcic, Mireia Sospedra Ramos, Roland Martin Neuroimmunology and Multiple Sclerosis Research Section Department of Neurology University Hospital Zurich fabienne.largey@usz.ch Multiple sclerosis (MS) is an autoimmune disease leading to central nervous system (CNS) damage with yet unknown etiology. Genetic and environmental factors contribute to disease risk. Epstein-Barr virus (EBV) stands out as the major environmental risk factor, whereas HLA-DR15 haplotype (HLA-DR15) is the major genetic risk factor.HLA-DR15 is assumed to increase the MS risk by preferential presentation of self-peptides from or similar to CNS-specific self-proteins to T cells selecting an autoreactive CD4+ T cell repertoire (TCR). Such autoreactive T cells may be activated and expanded by crossreactivity between self-antigens and foreign antigens, e.g. viral peptides, a mechanism termed molecular mimicry.

 

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Epstein-Barr Nuclear Antigen 1 (EBNA1)-specific CD4+ T cells are selectively expanded in MS patients and some of these cross-recognize MS-associated CNS myelin antigens. These findings indicate that molecular mimicry is a viable hypothesis to explain the link between EBV and MS. To elucidate the influence of HLA-DR15 and EBV infection on the T cell repertoires in MS patients, this project investigates (i) whether HLA-DR15 shapes an immune repertoire either in the naïve- or memory CD4+ T cell compartment predisposing to MS and (ii) whether the EBV-specific CD4+ TCR is biased in HLA-DR15+ donors compared to HLA-DR15– donors. Optionally, the influence of HLA-DR15 on the quality and/or quantity of cross-reactivity against CNS and other autoantigens within the EBV-specific TCR could be analyzed. The results obtained in this study are expected to lead to implications for clinically relevant novel biomarkers and for future treatments by autologous cell-based induction of antigenspecific immunotolerance. Pattern recognition requirements in vaccination versus immunity to infection Vural Yilmaz, Annette Oxenius and Roman Spörri ETH Zürich, Institute of Microbiology, HCI G 403 Wolfgang-Pauli-Str. 10, 8093 Zürich yilmazv@ethz.ch The induction of adaptive immunity is crucially dependent on innate activation signals that convert resting antigen-presenting cells (APCs) into immunogenic ones. APCs expressing the relevant innate pattern recognition receptors can be directly activated by pathogen-associated molecular patterns (PAMPs) to become competent to prime T-cell responses. Alternatively, APCs can be activated indirectly by proinflammatory mediators synthesized by PAMP-exposed cells. Yet such indirectly activated APCs fail to prime effector CD4+and CD8+ T cell responses against vaccines. This lead to the conclusion that

 

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direct pattern recognition is required for the successful priming of adaptive immunity. However, recent data from experiments with pulmonary influenza infection challenges this conclusion. There, IL-1R signalling on APCs replaces direct pattern recognition in promoting CD8+ T cell responses, but not CD4+ T cell responses. The present project aims to shed light on the differential requirement for direct pattern recognition in vaccination versus immunity to infection. We are particularly interested firstly, in the requirement for pattern recognition in the context of crosspresentation, secondly, in surrogate activation mechanisms where the presenting APCs do not express the appropriate pattern recognition receptors, and thirdly, the basis for the differential requirement for direct pattern recognition in the priming of CD4+and CD8+ T cell responses to influenza infection. New chain release mechanisms in polyketide biosynthesis Katja Jensen, Holger Niederkrüger, Anna L. Vagstad, Nancy Magnus, Alexander Brachmann, Jörn Piel ETH Zürich Institute of Microbiology HCI G429 Wolfgang-Pauli-Str. 10 8093 Zürich kjensen@ethz.ch Trans-AT-Polyketide synthases (trans-AT-PKSs) are huge enzyme complexes responsible for the biosynthesis of polyketides. Polyketides are secondary metabolites that harbor enormous structural variety and display potent activities. Their biosynthesis is carried out by the stepwise assembly of acyl building blocks in which intermediates are covalently bound to the PKS. The assembled polyketide chain is then released from the PKS by cleavage. Numerous polyketides are good drug candidates, but their industrial exploitation is challenging in many cases due to low production yields. To overcome this supply bottleneck, we seek to better understand their

 

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biosynthetic pathways. The aforementioned chain liberation is the first necessary step for pathway characterization. We have identified a new enzyme class named the acyl hydrolases (AH) found in many trans-AT-PKS pathways. They catalyze release of misassembled intermediates from the enzyme complex, acting as PKS housekeepers. The role of AHs as a tool for improving studies with trans-AT systems is being explored. We are also interested in characterizing modifications of the core polyketide structure after chain release. One interesting modification, a chlorination, occurs within the oocydin A pathway (a trans-AT-PKS model system). We are trying to shed light onto this unprecedented halogenation mechanism. On first glance, no enzyme homologous to known halogenases could be found in the gene cluster. Characterization of these pathways may facilitate the rational design of polyketide analogues. Furthermore these enzymes may serve as general tools in the field of secondary metabolite research. A novel in vitro/vivo selection selection strategy for lentiviral vectors Sandra Ivic1, Renier Myburgh1, Karl-Heinz Krause2, Roberto F. Speck1 1: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland 2: Dept. of Pathology and Immunology, Geneva Medical Faculty, Dept. of Genetic and Laboratory Medicine, Geneva University Hospitals, Centre Medical Universitaire, Geneva, Switzerland sandra.ivic@usz.ch Genetic engineering of hematopoietic progenitor cells (HPC) by lentiviral-based vectors suffers from their low transduction rate with ~20% when aiming a single integration event. This amount

 

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of transgene expression may not be sufficient for the correction of a number of diseases. We propose a novel in vitro/in vivo selection/suicide system based on the expression of the human reduced folate carrier 1 (hRFC1) and the human ribonucleoside-diphosphate reductase subunit M2 (RRM2). hRFC1 is the main transport protein for the uptake of folates which are indispensable for purine synthesis and therefore for DNA synthesis. A greater expression level of hRFC1 should render cell more sensitive to killing by methotrexate (suicide) but resistant against trimetrexate (selection) due to their pharmacological properties - untransduced cells are expected to show the opposite effects with higher resistance to methotrexate but lower threshold killing to trimetrexate. RRM2 catalyzes the reduction of ribonucleotides into desoxyribonucleotides and its overexpression should lead to resistance to its inhibitor hydroxyurea (selection of transduced cells). We expect an additive or even synergistic effect when trimetrexate and hydroxyurea are given together on selection/killing of transduced vs. untransduced cells. All compounds are FDA approved and thus potentially permitted for use in human. We are currently examining the concept proposed in vitro using various cell lines. Provided promising results, we then move forward to gene engineering HPC and their transplantation into humanized mice. Transduced cells would be monitored via GFP in vivo for their proliferation or disappearance depending upon the compounds administered.        

 

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Session  2   Differential dendritic cell activation in peripheral T cell tolerance Daniela Jaramillo, Annette Oxenius and Roman Spörri ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland. jdaniela@ethz.ch Dendritic cells (DCs) are the main antigen-presenting cells and play a crucial role in inducing both immunity and tolerance. To elicit functional T cell responses, DCs must provide three signals to naïve T cells: (1) peptide presented on a major histocompatibility complex molecules, (2) costimulation and (3) immunomodulatory cytokines such as IL-12. Although it is well-known that those three signals are required to mount immune responses, it is unclear whether they could also play a role in T cell tolerance. The current model suggests that while antigen presentation by activated DCs induces immunity, antigen presentation by immature DCs results in T cell tolerance. However, immature DCs frequently fail to tolerise low affinity selfspecific T cells. Importantly, pathogen recognition triggers the DCs’ transition from an immature to an activated functional state. This can either occur through direct pathogen interaction or indirectly via inflammatory mediators produced by other cells that have been exposed to pathogens. Such indirectly activated DCs are characterised by high expression of co-stimulatory molecules. However, they lack production of immunomodulatory cytokines (signal 3) and consequently are unable to mediate immunity. Therefore, the hypothesis arises that indirectly activated DCs are not immunogenic, but tolerogenic. This study aims to determine and characterise the tolerogenic potency of indirectly activated DCs in vivo. In particular, we will test whether antibody-mediated targeting of antigen to indirectly activated DC can be exploited to induce antigen-specific T cell tolerance.

 

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This study aims to gain new insights into DC-mediated T cell tolerance and its potential for clinical application. How chronic viral infections shape the adaptive immune response Ute Greczmiel, Kirsten Richter and Annette Oxenius Institute of Microbiology, ETH Zürich Wolfgang-Pauli-Strasse 10, 8093 Zürich ute.greczmiel@micro.biol.ethz.ch The murine lymphocytic choriomeningitis virus (LCMV) is a commonly used model for persistent human infections like HIV or hepatitis C. In general an acute LCMV infection (low dose) and a chronic persistent infection (high dose) can be distinguished depending on the strain and the dose that has been used. During acute infections, viral clearance is mainly dependent on the CD8+ cytotoxic T cells (CTL). However, the CTLs are functionally impaired during persistent infection preventing clearance of the virus. Also the differentiation of CD4 T cells is affected by a persistent LCMV infection. During acute infections these cells differentiate mainly into Th1 helper cells whereas they preferably differentiate into follicular helper T cells (TFH) at late stages during chronic infections. TFH cells are mainly responsible for the induction of the germinal center reaction where B cell memory forms and affinity maturation of antibodies and class-switch take place. Antibodies play an important role in the late control of the persistent LCMV infection when neutralizing antibodies emerge which help control the viral spread. This project addresses the questions how long TFH cells need to provide B cell help in persistent LCMV infection and how pronounced their help is at different time points of the infection. It is also of interest whether and when the types of antibodies

 

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produced change during persistent infection and whether there are changes in their specificity towards LCMV. Role of tight junctions in Chronic Rhinosinusitis (CRS) Rita A. Costa, Cezmi A. Akdis University of Zurich, Swiss Institute of Allergy and Asthma Research, Obere Strasse 22, 7270 Davos, Switzerland rita.costa@siaf.uzh.ch CRS is a heterogeneous group of mucosal inflammatory diseases of the nasal cavity and paranasal sinuses with distinct potential causes, but its exact pathogenesis stillremains unclear. Persistent mucosal inflammation and microbial colonization and sometimes infection are characteristics of CRS. The microbiome of chronic rhinosinusitis is composed mainly by Haemophilus spp., Moraxella spp., Pseudomonas spp., Streptococcus spp., Corynebacterium tuberculostearicum and Staphylococcus aureus. S aureus is a major pathogen responsible for a variety of diseases. Certain strains are more virulent than others, but all colonizing strains are potentially infectious. The pathogenicity of S. aureus is due to its capacity to produce a wide repertoire of toxins, exoenzymes, adhesins, and immune-modulating proteins. Mucosal epithelial cells play an integral role in innate immune defense by sensing signals from the external environment, generating various molecules to affect growth, development, and function of other cells, and maintaining the balance between health and disease. Tight Junctions (TJs) are the seals between neighboring epithelial cells and thus essential for the epithelium integrity and its barrier function. This project aims at clarifying the contribution of Staphylococcus aureus to the inflammatory cascade in patients with Chronic Rhinosinusitis and the role of defects in mucosal barrier and barrier function. We will define the regulatory mechanisms and expression profiles of TJs and their signaling components that play a role in barrier integrity, which

 

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may help to elucidate factors in the pathogenesis of CRS and aid in the development of new therapeutic modalities. Investigating the Biological Role of the Secreted Chorismate Mutases from Microbial Pathogens Susanne Mailand Laboratory of organic chemistry ETH Hönggerberg, HCI F338 Wolfang-Pauli-Strasse 10 8093 Zürich The enzyme chorismate mutase (CM) catalyzes the Claisen rearrangement of chorismate to prephenate. This reaction is the first committed step in the biosynthesis of phenylalanine and tyrosine in the shikimate pathway. The shikimate pathway is essential in bacteria, fungi, algae and plants. Some bacteria were found to produce, besides the housekeeping chorismate mutase, another chorismate mutase, which possesses an N-terminal signal sequence that directs the secretion of the enzyme from the bacterial cytoplasm. This is very puzzling as the entire shikimate pathway is localized intracellularly. Some evidence suggests that the secreted CM, denoted *AroQ, is a virulence factor. However, its exact mode of action is still unknown. In my Ph.D. thesis I will investigate the biological role of secreted CMs from pathogenic bacteria. To this end, I will use Mycobacterium marinum, Salmonella typhimurium and Pseudomonas aeruginosa as model organisms and construct *AroQ knockout strains, if not already available. Under infection conditions, I will compare wild-type and knockout strains with regard to phenotypic changes in the pathogens themselves as well as changes in the response of host cells upon infection. For instance, I am interested in the intracellular replication of the pathogen in the host. Employing immunological tests and metabolomics and proteomics studies, the host immune

 

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response and the interference with metabolic and signaling pathways of the host will be investigated. Molecular Biology of an Attachment Factor in Giardia lamblia Jacqueline Ebneter, Adrian Hehl UZH, Institute of Parasitology Winterthurerstrasse 266a CH‐8057 Zürich jacqueline.ebneter@uzh.ch Infection with Giardia lamblia, a protozoan parasite, leads to severe diarrhea in humans and animals and significantly increases morbidity in third world countries. Infection is caused via oral uptake of cysts in contaminated water or food. After excystation in the small intestine, the resulting flagellated trophozoites attach to the intestinal wall where they reproduce. The attachment is mediated by the adhesive disk, a large rigid structure that is composed of a concave microtubule spiral which is directly underlaid by the plasma membrane. The detailed composition of the adhesive disk and how the plasma membrane is connected to it is still under investigation. We propose that a giardial Epsin homologue mediates curvature of the plasma membrane and, by serving as a linker between the plasma membrane and the adhesive disk induces the concave shape of the adhesive disc. Our hypothesis is based on mammalian Epsin that was shown to tubulate liposomes and induce curvature of clathrin-coated pits. While its C-terminus was suggested to bind different transport-associated proteins, the N-terminal ENTH domain was shown to induce membrane curvature byits partial insertion into one layer of the membrane only. We show that the giardial Epsin localizes to the adhesive disk and provide evidence that correct targeting is mediated by the protein’s C-terminus. Further, we demonstrate that the conserved ENTH-domain mediates binding of the protein to membrane-associated structures. Taken together, our data

 

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support the idea of giardial Epsin as a curvature-inducing linker between the plasma membrane and the adhesive disk. Development of a biosensor for the detection of Cell-to-cell communication molecules A. Suppiger, C. Aguilar and L. Eberl Department of Microbiology, University of Zürich, Zollikerstr. 107, Switzerland a.suppiger@access.uzh.ch Many bacteria are able to coordinate their gene expression in a cell density-dependent manner, a phenomenon called Quorum sensing (QS). In Burkholderia cenocepacia, a Gram-negative opportunistic human pathogen, two QS systems are known. The better characterized QS system employs N-acyl-homoserine lactones (AHLs) that are synthesized by CepI and sensed by CepR. Recent work identified an additional system that relies on a fatty acid signaling molecule, cis-2-dodecenoic acid (BDSF). Synthesis of BDSF is catalyzed by RpfFBc, an enoyl CoA hydratase. Genomic sequence analysis revealed that homologs of rpfFBc are conserved in a range of bacterial species. However, data showing that these species produce BDSF-like molecules is scarce. In B. cenocepacia, BDSF and AHL QS systems regulate a specific but also an overlapping set of genes. For example, while the lectin operon bclACB is primarily regulated by BDSF, the expression of the large surface protein BapA is regulated by both AHL and BDSF. Here we show the construction and characterization of strains that are used to monitor the production of AHL or BDSF signal molecules in different bacteria. A sensor plasmid was engineered with the promoter region of bclACB in front of promoterless gfp or luxAB reporter genes. To maximize the versatility of this sensor plasmid, it was transferred to different genetic backgrounds of B. cenocepacia. The ability of the newly generated sensor strains to detect

 

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BDSF or AHL was evaluated in liquid media and in cross-streak experiments. Salmonella growth rates in the mouse gut Sandra Wotzka, Wolf-Dietrich Hardt, Emma Slack ETH Zürich, Institute of Microbiology Wolfgang-Pauli-Str. 10, 8093 Zürich wotzkas@ethz.ch Salmonella spp. are one of the main causes for food-borne disease as the pathogenic bacteria can colonize the intestine of animals and humans. The colonization of the gastrointestinal tract is a complex interaction between the bacterium, the microbiota, the intestinal epithelium and the host immune system. To gain insights into these interactions, it is necessary to understand what kind of virulence factors contribute to the infection and which evolutionary developed differences within the Salmonella population enable the colonization of the gut. Recent studies indicated that not only the immune system but rather a fully developed and complex microbiota seems to be essential for colonization resistance and pathogen clearance. The microbiota composition is strongly dependent on the host's genetics, environment, diet and immune status, determining the host´s susceptibility to a wide range of disease conditions, ranging from intestinal infection to autoimmunity. Environmental stresses or changes in diet can lead to an altered microbiota composition, which in turn may have an influence on the host's health status. To gain a better understanding of how variations in the microbiota contribute to a functional immune system and how directed shifts in the microbiota, associated with particular genetic abnormalities, may have an effect on the host phenotype is really important. We are therefore using a genomic and proteomic approach to analyze different mutant mouse strains all of which are initially colonized with an identical microbiota, to elucidate potential shifts depending on the genetic background of the mouse and

 

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the impact on the gut associated immune system during health and disease. Salmonella typhimurium virulence factors in mice Rebekka Bauer, Lisa Maier, Wolf-Dietrich Hardt Institut f. Mikrobiologie Wolfgang-Pauli-Str. 10, 8093 Zürich rbauer@ethz.ch Salmonella enterica serovar Typhimurium (S.Tm) is a major cause of food-related gastrointestinal disease in humans worldwide. In mice, infection with S.Tm causes an illness that more closely resembles human typhoid fever and only a small fraction of infected mice develop diarrhea. The inability of Salmonella to colonize the gastro-intestinal tract is called colonization resistance. The mechanisms that permit Salmonella to overcome gut microbiota-mediated colonization resistance and to induce and survive inflammation are governed by a host of virulence factors, many of which have been discovered through pointed analysis of single genes or operons. However, a holistic genome-wide screen has yet to be conducted in an in vivo model. This approach carries the advantage of unveiling complex networks of interactions at the genomic scale, many of which may not be discovered using more traditional, directed methods of gene analysis. Due to colonization resistance and interactions with the immune system, large pools of mutants cannot be screened in conventional mouse models without encountering severe bottlenecks. Mice with less complex microbiota (LCM) show a lower colonization resistance and no bottlenecks occur. LCM mice are therefore an ideal background for genome-wide screening of Salmonella mutants. We are using LCM mice to carry out a transposon-tagged mutagenesis screen to discover virulence factors that affect S. Typhimurium colonization and inflammation.

 

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Understanding the phyllosphere lifestyle: Metaproteomics of a synthetic bacterial community Daniel B. Müller1, Hannes Röst2, Olga Schubert2, Ruedi Aebersold2 and Julia A. Vorholt1 1: Institute of Microbiology, Wolfgang-Pauli-Str. 10, 8093 Zurich 2: Institute of Molecular Systems Biology, Wolfgang-Pauli-Str. 16, 8093 Zurich damuelle@ethz.ch The phyllosphere comprises the above ground parts of terrestrial plants, which are dominated by the leaves and constitute a huge habitat for microorganisms. In recent years several cultivation-independent studies revealed insights into the composition of the natural phyllosphere community of different host plants. Besides Actinobacteria and Bacteroidetes, Alpha- and Gammaproteobacteria were shown to predominate the phyllosphere with Methylobacterium, Sphingomonas and Pseudomonas being three of the most abundant and frequently detected genera. Besides genomic and transcriptomic approaches, two metaproteogenomic experiments gave insights into the most abundant proteins in natural communities and allowed to infer the physiology of different phyla colonizing the phyllosphere. However, protein coverage per species is relatively low in complex communities and a higher depth of protein identification as well as precise quantification would be desirable. It is timely to bring community analyses to a reductionist level and analyze a synthetic community under controlled conditions in a gnotobiotic model system. The two ubiquitous plant commensals Methylobacterium extorquens PA1, Sphingomonas sp. Fr1 as well as the opportunistic plant pathogen Pseudomonas syringae pv. tomato DC3000 were chosen to be part of a reductionist community and will be analyzed under single strain and co-cultivation conditions using the recently developed proteomics approach called SWATH-MS. This metaproteomic approach will gain

 

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deeper insights into general and species specific traits which are important for successful adaptation and survival in the phyllosphere. The role of innate immunity in control of Legionella pneumophila lung infection Pascal Ziltener, Stefan Weber and Annette Oxenius Institute for Microbiology, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland. pascal.ziltener@micro.biol.ethz.ch Though amoeba are the environmental niche for Legionella pneumophila, the bacteria can also replicate in alveolar macrophages upon transmission to the lung via aerosols, and have the potential to cause a severe form of pneumonia known as Legionnaires’ Disease. The intracellular replication of L. pneumophila depends on the injection of multiple effector molecules into the host cell cytoplasm via a type IV secretion system (T4SS). These effectors promote phagocytosis, inhibit fusion of the phagosome with lysosomes and thus allow the bacteria to establish a replication permissive compartment. Alveolar macrophages are able to detect the activity of the T4SS with cytosolic pattern recognition receptors such as NLRC4, which in combination with MyD88-dependent signals leads to inflammatory secretion of IL-1β. This innate immune response is believed to be important for the control of primary L. pneumophila infection, resulting in the recruitment of neutrophils and subsequent clearance of bacteria. Though reduced L. pneumophila clearance has been shown in mice lacking various innate immune effector molecules, including NLRC4-/-, Caspase-1-/-and IFNγR-/- mice, we show here that the bacterial load in the lung five days post intranasal infection is increased 10-foldmore in mice lacking TNF production in macrophages, monocytes and neutrophils (mTNF mice), and in MyD88-/- mice. Furthermore, macrophages

 

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lacking TNF production are as permissive for intracellular L. pneumophila replication as NLRC4 deficient macrophages. Further investigation of the in vivo immune effector mechanisms mediated by TNF will enhance our understanding of how L. pneumophila infection is controlled by the innate immune system. Functional characterization of yeast oligosaccharyl-transferase complex Kristina Poljak, Elsy Ngwa, Susanne Müller, Markus Aebi ETH Zurich Institute of Microbiology HCI F412 (Aebi group) Wolfgang-Pauli-Strasse 10 poljakk@ethz.ch N-linked protein glycosylation is the most frequent post-translational modification of proteins. It is a highly conserved process and occurs in pro- and eukaryotic cells. Oligosaccharyltransferase (OST) is the enzyme that covalently links a preassembled oligosaccharide to the amide of asparagine residues located in the acceptor sequon AsN-X-Ser/Thr of a large number of proteins. In prokaryotes and protozoan eukaryotes, this enzyme is composed of a single polypeptide and this protein is conserved among all domains of life. In multicellular eukaryotes, such as yeast, this complex consists of 8 subunits present in two isoforms: Stt3, Ost1, Ost2, Swp1, Wbp1, Ost4, Ost5 and Ost3 or Ost6, where the first five are essential proteins for survival of yeast cells. STT3 gene encodes catalytically active OST subunit. Except Ost1p, which acts as a chaperon to promote glycosylation of selected protein clients and Ost3/6p, which exhibit oxidoreductase activity and bind specific polypeptides, not much has been known about the other subunits. Deletions and specific mutations in oligosaccharyltransferase complex subunits Wbp1p, Swp1p, Ost4p, Ost2p and Ost5p will be generated in order to test their effect on the growth, process

 

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of N-linked protein glycosylation and assembly of the OST complex in yeast. N-glycosylation efficiency will be evaluated by an MS-based quantitative determination of N-glycosylation site occupancy and by analyzing glycosylation of a model glycoprotein CPY. Complex formation will be assessed by performing Blue Native Gel Electrophoresis, where the proteins are kept in their native state and by measuring protein turnover by mass spectrometry. In vivo enrichment of regulatory T cells by Bcl-2/Bcl-xL inhibition promotes tolerance in allotransplantation Sarah S. Gabriel, Thomas Fehr, Pietro E. Cippà University of Zürich, Physiology Winterthurerstr.190, 8057 Zürich sarah.gabriel@uzh.ch Background/Aim: Regulatory T cells (Tregs) are the guardians of immunological tolerance. Since Tregs and effector T cells (Teffs) express a different pattern of pro- and anti-apoptotic factors, the apoptosis pathway might represent a pharmacological target to shift the balance from alloreactive Teffs towards graft-protecting Tregs. In this study, we explored the effect of the pro-apoptotic small molecule Bcl-2/Bcl-xL inhibitor ABT-737 on Tregs and its relevance in transplantation. Methods/Results: CD4+CD25+FoxP3+ natural and induced Tregs displayed a relative resistance to ABT-737 in vitro and in vivo, when compared to Teffs in a FoxP3-GFP transgenic mouse model. A short treatment with ABT-737 in vivo induced a significant enrichment of Tregs among CD4 T cells. This substantially potentiated the immunomodulatory effect of an induction therapy protocol including donor-specific transfusion and costimulation blockade (anti-CD154), leading to long-term survival of donortype fully MHC-mismatched skin grafts without immunosuppression. Moreover, Tregs enrichment by ABT-737 was a critical component of a novel protocol to induce mixed chimerism and tolerance without myelosuppression.

 

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Conclusion: Treg enrichment can be achieved in vivo by exploiting the relative resistance of Tregs to Bcl-2/Bcl-xL inhibition. This approach might find future clinical application to promote graft survival and tolerance after solid organ transplantation. Monitoring the effects of the HLA haplotype on NK cell KIR repertoire development Vanessa Landtwing, Vivien Béziat, Kalle Malmberg, Christian Münz Institute of Experimental Immunology Y44 J88 Irchel Campus Winterthurerstrasse 190, CH-8057 Zürich vanessa.landtwing@uzh.ch The killer-cell immunoglobulin-like receptor (KIR) phenotype on natural killer (NK) cells of an individual is shaped by their expression of human leukocyte antigen (HLA) ligands. Because the genes encoding for KIR and HLA are unlinked, the ligands and receptors are segregated independently of each other, leading to an extensive polymorphism in HLA and KIR genotypes. In leukemia patients undergoing HLA-matched but KIR-mismatched transplantation it was shown that the patients KIR repertoire would either take on the phenotype of the donor or remain mismatched leading to severe clinical complications. Furthermore, several publications have indicated a higher susceptibility to certain diseases, autoimmune disorders and hereditary sicknesses depending on the combination of KIRs and HLA ligands. This demonstrates the significance of the crosstalk between KIRs and their HLA ligands in the human immune system. Therefore, we further characterized this interaction between the HLA-ligands and their respective KIRs. To do so we are working with a humanized mouse model using NOD/SCID/γc chain-null mice. These mice are irradiated and reconstituted with CD34+human stem cells that were isolated from human fetal livers. In order to study the effect of specific HLA ligands on the skewing of the KIR repertoire we

 

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determined the genotypes of the fetal livers. With this information we were able to monitor the development of the KIR phenotype on NK cells in mice that had been reconstituted homozygously with just one donor or heterozygously with a mixture from two donors. Qualitative and Quantitative Requirements for Vaccination-mediated Protection from Salmonella Typhimurium Kathrin Moor, Emma Slack Institut für Mikrobiologie ETH Zürich, HCI G413 Wolfgang-Pauli-Strasse 10, 8093 Zürich kathrin.moor@micro.biol.ethz.ch The mucosal epithelia in the intestine, lungs and genital tracts of mammals are the major sites of non-vector-transmitted pathogen entry. Multiple layers of protection allow the host to live peacefully with the dense commensal microbiota whilst maintaining vigilance to invading pathogens. Intense study of mucosal immunity in recent years indicates that mucosal homeostasis is a highly dynamic process involving active innate immune recognition of the microbiota and active suppression of T cell-mediated immunity. However, the exact mechanisms by which each component of mucosal immunity restricts infection without excessive pathology remain unclear. By using a model of vaccination-mediated protection from Salmonella enterica serovar Typhimurium (S. Typhimurium) we try to identify such mechanisms. We make use of Markov-chain modeling to determine the size of the bacterial population initially invading the intestine and tissues and the rates of bacterial replication and clearance within tissues. This provides insights in mechanisms of mucosal immunity and allows us to determine the quantitative relationship between activation of the innate and adaptive immune system and inhibition of each stage of S. Typhimurium infection. We could show that induction of Salmonella-specific intestinal IgA correlates with a decreased

 

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rate of bacterial invasion from the intestine into the mesenteric lymph nodes whereas the bacterial replication within the lymph node remains unaffected. Further we examine the role of the intestinal microbiota in modulating the response to mucosal vaccination. In a subproject we investigate the safety and efficiency of mucosal vaccination in the murine model of x-linked chronic granulomatous disease. VI MIM Retreat, 12- 14 September 2013 Memory T cell inflation during murine cytomegalovirus (MCMV) infection Nicolas Baumann, Nicole Torti, Annette Oxenius ETH Zürich - Institute of Microbiology Wolfgang-Pauli-Strasse 10 CH - 8093 Zürich baumanni@ethz.ch Cytomegaloviruses (CMVs) are β-herpesviruses and establish latency and lifelong chronic infection. CD8+ T cells play an important role for the control of acute infection and for the surveillance and suppression of reactivation events during viral latency. Two distinct kinetics of MCMV-specific CD8+ T cell responses are observed: 'Conventional T cells' undergo expansion followed by contraction after virus control. 'Inflationary T cells' however continueto proliferate and expand even after control of lytic replication. They accumulate to high numbers in peripheral organs where they exhibit an effector-memory phenotype. We recently found that latently infected non-hematopoietic cells in lymph nodes activate MCMV-specific central-memory CD8+ T cells. This leads to their proliferation and differentiation into effector cells, and they subsequently migrate to peripheral tissues and contribute to early control of viral reactivation events. Our aim is to investigate the mechanisms of maintenance of MCMV-specific CD8+ T cells in peripheral tissues. Therefore we will determine the half-life of MCMV-specific TEM in infected

 

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hosts and whether their maintenance is dependent on antigen persistence or cytokines. Additionally, potential clonal selection processes within MCMV-specific CD8+ T cells during MCMV latency will be analyzed, as it is unclear whether the most abundant MCMV-specific T cell clones during the acute phase will also be dominant during latency. Also, we aim to understand the determinants of the 'size' of memory inflation, mainly whether stabilization at high frequency is constrained by “space limitation” encountered in the periphery or whether the number of memory precursor cells or TCM in the lymph nodes determines the 'size' of memory inflation.

 

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Social  Event  -­‐  Winetasting  

Via Zandone, 11 6616 Losone Canton Ticino – Switzerland Tel. +41 91 7910817 Fax +41 91 7915908  vini@delea.ch www.delea.ch The company Angelo Delea was founded in 1983, inspired by the „Bordeaux“ wine-making with the refinement in Carati (barriuqes) made of excellent French oak-wood from Allier and Nevers. The company’s most important products are the Merlot Carato, Carato Riverva, the Chardonnay, the Apocalisse, the crus like Merlot from Losone “saleggi”, the Merlot from Locarno “San Carlo”, the Merlot from Gudo “Progero e Caneggio”, the “Montedato”, the Castello di Cantone in Rancate and the sparkling wine “Charme”. The new and spectacular Cellar includes the wine-shop, the most modern cellar of wine-making and the distillery from 1932 where various grappas and fruit distillates are produced. The Vineyards of own production are 20 acres among them is the agriturismo la Fattoria dell’Amorosa in Gudo-Sementina.

 

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For years Angelo Delea chose vineyards in different locations in the Ticino, where the most suitable climatic conditions contribute to the ripening of the precious grapes. Vineyards of our property include the Amorosa in Gudo with 3 acres of Merlot, Cabernet Franc and Sauvignon, 20000 stumps with particular clone selections, the Castello di Cantone in Rancate (Mendrisiotto) with 6 acres and 30000 stumps of the red Merlot, Cabernet Franc, Syrah and the white Chardonnay, Sauvignon and Sémillon. In Cavigliano, 1 acre is planted with Chardonnay, approximately 5000 stumps.

 

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General  Information   Youth Hostel Locarno Palagiovani Via B. Varenna 18, 6600 Locarno (TI)

Tel: +41 91 756 15 00 Fax: +41 91 756 15 01 locarno@youthhostel.ch

 

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Contacts   Erica Russo erusso@pharma.ethz.ch 0774473729

Lennart Schada lennarts@ethz.ch 0786053190

Kristina Poljak poljakk@ethz.ch 0788832857

Martina Stöckli smartina@ethz.ch 0787243162

Participants    1. Altmeier Simon………………………….Poster 1(S1) 2. Altunbulakli Can…………………..…Presentation S3 3. Bauer Rebekka …………………………Poster 2(S2) 4. Baumann Nicolas ………………………Poster 3(S2) 5. Bindszus Mareike ………………………Poster 4(S1) 6. Butaite Elena …………………………...Poster 5(S1) 7. Buttgereit Anne ……………………..Presentation S2 8. Cheng Hung-Wei ………………………Poster 6(S1) 9. Costa Rita ………………………………Poster 7(S2) 10. Curkic Ismeta …………………………..Poster 8(S1) 11. Ebneter Jacqueline Antoinette …….…Poster 9(S2) 12. Felmy Boas……………………….…Presentation S2 13. Gabriel Sarah …………………………Poster 10(S2) 14. Granato Elisa …………………….…Presentation S1 15. Greczmiel Ute …………………………Poster 11(S2) 16. Helf Maximilian………………………Presentation S3 17. Ivic Sandra …………………………….Poster 12(S1) 18. Jaramillo Daniela ……………………..Poster 13(S1) 19. Jensen Katja …………………………..Poster 14(S1) 20. Landtwing Vanessa …………………..Poster 15(S2)

 

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21. Largey Fabienne ……………………...Poster 16(S1) 22. Mailand Susanne ……………………..Poster 17(S2) 23. Moor Kathrin …………………………..Poster 18(S2) 24. Müller Daniel ………………………….Poster 29(S2) 25. Ngwa Elsy ……………………………..Poster 20(S2) 26. Novkovic Mario ……………………….Poster 21(S1) 27. Pallmer Katharina …………………….Poster 22(S1) 28. Plaza David …………………………Presentation S3 29. Poljak Kristina …………………………Poster 23(S1) 30. Rosenthal Raoul…………….………Presentation S2 31. Russo Erica…………………….……Presentation S1 32. Schada von Borzyskowski Lennart.…Poster 24(S1) 33. Schmieder Stefanie ……………...…..Poster 25 (S1) 34. Stöckli Martina ………………………..Poster 26(S1) 35. Suppiger Angela ……………….……..Poster 27(S2) 36. Thom Jenny………………………….Presentation S1 37. Wotzka Sandra ………………….……Poster 28(S2) 38. Yilmaz Vural ……………………….….Poster 29(S1) 39. Ziltener Pascal………………………...Poster 30(S2)

 

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