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    Swammerdam Institute for Life Sciences Annual Report 2006 Faculty of Science

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    Swammerdam Institute for Life Science Annual Report 2006 Faculty of Science

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    Annual Report 2006 04 Chapter 1 Preface by the director Contents 06 Chapter 2 Scientific Program 08 Research Cluster - The Living Cell 08 Molecular Microbial Physiology 10 Molecular Biology and Microbial Food Safety 12 Cellular Microbiology 13 Structure and Functional Organisation of the Cell Nucleus 15 Epigenetic Regulation of Gene Expression 16 Molecular Cytology 18 Research Cluster - Plants and Health 18 Plant Physiology 20 Plant-pathogen Interaction 22 Research Cluster - SILS Center for NeuroScience 22 Animal Physiology and Cognitive Neuroscience 25 Cellular and Systems Neurobiology 29 Hormonal Regulation of Signal Transduction in the Brain 31 Research Cluster - Life Science Technologies 31 Mass Spectrometry of Biomacromolecules 33 Biosystems Data Analysis 34 Micro Array Department and Integrative Bioinformatics Unit Swammerdam Institute for Life Science 2

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    Annual Report 2006 36 Chapter 3 Societal Activities 38 Chapter 4 Management 41 Chapter 5 Scientific Advisory Board 43 Chapter 6 Assessment of Research Quality 46 Appendix 1 Research Results 46 Appendix 1a: Molecular Microbial Physiology 49 Appendix 1b: Molecular Biology and Microbial Food Safety 51 Appendix 1c: Cellular Microbiology 52 Appendix 1d: Structure and Functional Organisation of the Cell Nucleus 54 Appendix 1e: Epigenetic Regulation of Gene Expression 56 Appendix 1f: Molecular Cytology 59 Appendix 1g: Plant Physiology 62 Appendix 1h: Plant-pathogen Interaction 63 Appendix 1i: Animal Physiology and Cognitive Neuroscience 65 Appendix 1j: Cellular and Systems Neurobiology 69 Appendix 1k: Hormonal Regulation of Signal Transduction in the Brain 72 Appendix 1l: Mass Spectrometry of Biomacromolecules 73 Appendix 1m: Biosystems Data Analysis 75 Appendix 1n: Micro Array Department and Integrative Bioinformatics Unit 77 Appendix 2 Contact Details Swammerdam Institute for Life Science 3

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    Annual Report 2006 The Swammerdam Institute in 2006 1. Preface by the director Introduction The Swammerdam Institute for Life Sciences (SILS) is one of the ten research institutes of the Faculty of Science of the Universiteit van Amsterdam. For the first time since its creation in 2000, the institute has been evaluated by an external (international) evaluation committee. In the eyes of the committee, the prospects of SILS are very good. It foresees a bright outlook for life sciences in Amsterdam, and it sees SILS playing an instrumental role in it. This is supported by the bibliometric study of SILS over the period 1995 – 2005, indicating an above average research potential of the current SILS researchers. The committee also indicated some points for improvement. Most of the research groupings are relatively small, which can make it hard to be internationally competitive. Collaborations are therefore seen as an important part of SILS portfolio for the future. It also pointed out the delicate balance between research and teaching; for SILS the teaching load is heavy and could be impacting the productivity of the various groups. In order to further strengthen SILS’ position in research, these and other recommendations from the committee are currently being discussed within the institute, and will lead to the creation of a revised strategic plan for the years 2007-2011. New and exciting developments have been taking place in 2006 that further solidify the position of SILS. The ongoing activities regarding the collaboration of the UvA (SILS) with neighbouring institutes AMOLF (Institute for Atomic and Molecular Physics), CWI (Centrum voor Wiskunde en Informatica) and with the Vrije Universiteit in the area of Systems Biology, have resulted in the signing of an agreement to form the Netherlands Institute for Systems Biology (NISB). The NISB focuses on the integrated functioning of metabolic, signal-transduction and genetic networks in combination with systems that drive the generation of shape and force in living cells. Regarding the research facilities, a new, state-of-the-art green house complex has been completed in the first half of 2006. Final plans for a new building for the entire Faculty of Science have been completed, and we are awaiting to finally co- locate all SILS research groups in the same building in the next few years. Swammerdam Institute for Life Science 4

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    Annual Report 2006 In 2006 our financial position appears to have stabilised. We are still awaiting the • Prof.dr A. Smilde was the Eastern Analytical Symposium 2006 Award final figures, due to changes within the financial systems within the university, Recipient for Achievements in Chemometrics. and the changing allocation of funds for research and education. The institute • The group of dr. Timo Breit was involved in the installation of an e-BioScience will continue its’ extremely careful financial management and will make clear Laboratory (e-BioLab) designed for the interpretation of the results of choices on what we will and will not undertake. The apparent lack of flexibility (computational) -omics experiments using a.o. visualisation on a high has also been identified by the external evaluation committee. In line with its resolution video wall. The e-BioLab will enable multidisciplinary co-operation recommendation we will also in the coming years strive to fund new initiatives. and is an initiative of SILS, IvI, SARA, NBIC and NIKHEF in the framework of the Virtual Lab for e-science project. Highlights With respect to education and training, we have observed yet another increase • Prof.dr J. Hugenholtz, affiliated to NIZO Food Research, the Wageningen in Bachelor students: Psychobiology attracted 136 students (108 in 2005), Centre for Food Science (WCSF) and the Kluyver Centre for Genomics of Biomedical Sciences attracted 140 students (123 in 2005). The institute has trained Industrial Fermentations (KCGIF), was appointed as honorary professor in and educated students to a total of around 1700 in the Life Sciences Bachelors & Industrial Microbial Physiology in May 2006. Masters. Moreover, we have seen 17 PhD students successfully defend their thesis • Both dr J. Teixeira de Mattos and prof.dr J. Hugenholtz have obtained a in 2006. The further increase in education-related activities is welcomed, but transnational SysMo grant. This allows the Molecular Microbial Physiology also forces the institute to carefully balance the needs (and related finances) for group of prof.dr K. Hellingwerf to initiate two new systems biology projects research AND education. (1) on regulation of respiration in Escherichia coli and (2) on growth and product formation in lactic acid bacteria. Future • At the end of 2006, prof.dr J. Wells was appointed as a Senior Scientist within the Wageningen Center for Food Sciences (WCFS) and he will participate in a In 2007 the recommendations of the external evaluation committee will be new project concerning intestinal homeostasis. translated into a new strategic plan 2007-2011. We are entering a period in which • dr P.J. Verschure received the Feulgen Prize of the Society for Histochemistry. considerable investments have been announced for areas in which our researchers • An EU TMR-grant was awarded to prof.dr Th. Gadella in collaboration with 7 are involved (e.g. cognition, neuro-informatics, systems biology). This creates other European groups working on Nod factor signalling. new opportunities to further strengthen our research position. • Dr C. Testerink was awarded a VIDI for the project titled: Modulation of Overall, we are facing an exciting, yet challenging future for the Life Sciences, in protein kinase function by the lipid second messenger phosphatidic acid. which SILS can and will play an important role. • Dr R. Mirabella was awarded a VENI for the project titled: C6 volatiles: a new class of signal molecules in plants. • A grant from the Foundation for Technology & Science (STW) was awarded to the group of prof.dr C. Pennartz, for the development and application of miniaturized equipment for ensemble recordings in mouse models of brain disease. • The group of prof.dr W. Wadman will participate in a large research program (TTI Pharma) on the role of the endocannabinoid receptor. This program will continue our collaboration with Solvay Pharmaceuticals, the leading industrial partner in the program. • Prof.dr M. Joëls was the main applicant of a EUROCORES on “Stress and Mental Health”, which was approved by the ESF. In addition, prof.dr M. Joëls received an HFSP programme grant. Swammerdam Institute for Life Science 5

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    Annual Report 2006 Life Sciences Research: Build on our Strengths 2. Scientific Program Introduction The Swammerdam Institute is bringing together more than 200 researchers with knowledge from many areas of science, amongst others: biology, (bio)chemistry, (bio)physics, medicine and data analysis- and information technology. In these areas research is carried out at the molecular, cellular, and organismal level. Integrating the knowledge from different areas is essential to reach our final goal: understanding life processes in organisms and cells. Bioinformatics and data analysis are essential in this process and, Systems Biology is increasingly becoming a focal point of attention. In the institute research is carried out within four clusters: ‘The Living Cell’, ‘Plants and Health’, ‘SILS Center for NeuroScience’ and ‘Life Science Technologies’. The first three clusters study processes that take place within and between cells, such as signal transduction, growth and cell division and the structure of chromatin. Between these research themes many interactions take place ranging from the use of common methods to research collaborations. The fourth cluster, Life Science Technologies, applies advanced technologies to fundamental biological research and carries out technology development, keeping the SILS researchers at the forefront of Science. Life Science Technologies has extensive collaborations with other research groups within and outside SILS. A great amount of technologies and relating expertise has been build up, and genomics and advanced microscopy facilities have been created for the benefit of all research groups at the Swammerdam Institute. The institute studies a number of central themes on an institute wide level. These are for instance gene expression, the structure of DNA molecules and chromatin, protein - protein interactions, protein structure – function relations and signal transduction processes, as these take place in man, animal, plant and micro-organism. Within the institute this leads to exchange of information and extension of research over the borders of different disciplines. Crossing the borders between the different research topics within the institute and via interactions with strategic research partners, important and unique discoveries are made that lead to the discovery of life’s secrets and to applications that improve the quality of life. Swammerdam Institute for Life Science 6

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    Annual Report 2006 Research Output Research groups within the Swammerdam Institute for Life Sciences Clusters PhD Academic Patents Theses Publications The Living Cell 5 69 3 The Living Cell Plants and Health 5 20 0 SILS Center for NeuroScience 5 60 0 Molecular Microbial Physiology Prof.dr K.J. Hellingwerf Life Science Technologies 2 28 0 Molecular Biology and Microbial Food Safety Prof.dr S. Brul Total 17 177 3 Cellular Microbiology Prof.dr J.M. Wells Structure and Functional Organisation Prof.dr R. van Driel Research Input of the Cell Nucleus Epigenetic Regulation of Gene Expression Prof.dr A.P. Otte Clusters FS1* FS2** FS3º Total Molecular Cytology Prof.dr Th.W.J. Gadella The Living Cell 19.9 13.2 14.9 48.0 Plants and Health 10.0 5.3 5.5 20.8 Plants and Health SILS Center for NeuroScience 14.5 4.2 6.3 25.0 Life Science Technologies 7.6 2.3 16.4 26.3 Plant Physiology Prof.dr M.A. Haring Total 52.0 25.0 43.1 120.1 Plant-Pathogen Interaction Prof.dr B.J.C. Cornelissen * FS1 = University Funding, ** FS2 = External funding, governmental grants SILS Center for NeuroScience º FS3 = External funding, e.g. EU grants, commercial funding Animal Physiology and Cognitive Neuroscience Prof.dr C.M.A. Pennartz Cellular and Systems Neurobiology Prof.dr W.J. Wadman Hormonal Regulation of Signal Prof.dr M. Joëls Research input in full time equivalent Transduction in the Brain The research input of the academic staff per full time equivalent has been Life Science Technologies calculated making use of the following parameters: Mass Spectrometry of Biomacromolecules Prof.dr C.G. de Koster BioSystems Data Analysis Prof.dr A.K. Smilde Full Professors: 0.5 fte research Micro Array Department and dr T.M. Breit Assistant and Associate Professors: 0.5 fte research Integrated Bioinformatics Unit Postdoctoral Fellows: 0.9 fte research Centre for Advanced Microscopy dr E.M.M. Manders, prof.dr Th.W.J. Gadella PhD Students: 0.75 fte research Research Technicians: 1.0 fte research Swammerdam Institute for Life Science 7

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    Annual Report 2006 Research Clusters Molecular Microbial Physiology Chairholder: Prof.dr K.J. Hellingwerf The Living Cell Jeroen Hugenholtz Professor Joost Teixeira de Mattos Associate Professor Cells, the basic units of higher organisms and the competing individuals in microbiology, are the central topic of study of ‘The Living Cell’. This cluster focuses on the chain of events all the way from modulation of gene expression, Introduction e.g. by signals coming from the environment, up to the resulting phenotype elicited by such signals. This process is studied in (cells of) micro-organisms, The general aim of our research team is to discover the properties that allow animals and plants, most often to resolve basic scientific issues, but also in a more living (microbial) cells to catalyze a large array of concurrent chemical applied context, in particular aiming to improve food and health. The mission of fluxes and information flows. From these processes and in particular their the cluster ‘The Living Cell’ is to understand life, in particular in its simplest form mutual interactions ‘life’, with its typical characteristics as: adaptation to the of a living cell, all the way from the molecular level, upwards via the complex environment, reproduction and evolution, has emerged. Micro-organisms are biochemical and genetic networks that it encompasses, to the individual organism particularly successful in this respect as can be concluded from the fact that they that can be successful in the struggle for survival. The unique information inhabit even the most extreme and variable ecosystems known to exist on this that this approach provides, the possibility of linking this information with earth (and possibly even beyond), they can grow at very high rates, and can even metabolomics and proteomics data, combined with the computational simulation adapt/evolve genetically. of the processes under study, is leading to a shift in paradigm, often referred to as ‘Systems Biology’. ‘The Living Cell’ is uniquely positioned to play a key role in Our work focuses on various aspects of this process, like (i) the details of this development. intra-molecular signal-generation in (photo) receptor proteins, (ii) signal transfer between subsequent components in a signal transduction chain, (iii) Six chairs contribute to the research cluster ‘The Living Cell’: ‘Molecular the regulatory function of modulated gene expression, and (iv) the functional Microbial Physiology’, ‘Molecular Biology and Microbial Food Safety’, integration of these processes in the physiology of a range of micro-organisms, ‘Cellular Microbiology’, ‘Molecular Cytology’, ‘Epigenetic Regulation of Gene relevant for food and health, etc. By combining theoretical (i.e. computational) Expression’, and ‘Structural and Functional Organization of the Cell Nucleus’. and experimental approaches, insight is obtained into basic principles that underlie functional interactions in (information) flux-carrying macromolecular networks, and accordingly into a biochemical system that sustains microbial (i.e. cellular) life. Research Highlights Until recently, light-sensing in micro-organisms was only studied in phototrophic organisms. We have now clearly demonstrated light-sensing responses also in the chemotrophs Escherichia coli, Bacillus subtilis and Idiomarina loihiensis. These organisms have been shown to contain the blue-light photoreceptor proteins YcgF, YtvA and PYP, respectively. Especially the findings in E. coli and B. subtilis are of interest, since these are generally considered to be model organisms for Gram-negative and Gram-positive bacteria, respectively. Swammerdam Institute for Life Science 8

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    Annual Report 2006 It was shown that the general stress response in B. subtilis is modulated by blue Other Highlights light via the LOV domain of YtvA. Besides this, a completely new light-effect, elicited by red light, was found in this organism. The presence of light was • Prof.dr. J. Hugenholtz, affiliated to NIZO Food Research, the Wageningen shown to inhibit biofilm formation in E. coli (with the involvement of YcgF) and Centre for Food Science (WCSF) and the Kluyver Centre for Genomics of in I. loihiensis. The involvement of light-receptors in the formation of biofilms Industrial Fermentations (KCGIF), was appointed as honorary professor in is of specific interest, because it has become clear that in Nature, cells often live Industrial Microbial Physiology in May 2006. in these “multi-cellular” structures, which increases their antibiotic resistance considerably. Whereas a lot of progress has been made in understanding the • Prof.dr K. Hellingwerf organized the ESF/EMBO sponsored meeting on cellular changes that occur when an organism switches from a planktonic life “Microbial systems- and synthetic biology” in October 2006, in San Feliu, style to a biofilm, not much is known about perception of the signals that can Spain. trigger this switch. The new photoreceptors discovered in our group may turn out to be a very convenient tool to further characterize these latter processes. • Dr. J. Teixeira de Mattos was elected as secretary of the scientific committee of the 13th European Congress on Biotechnology 2007 (European Federation of Biotechnology, Barcelona, Spain) and as member of the organizing committee of this meeting. Future Prospects • Work in the photobiology and molecular physiology projects will be continued to resolve the molecular mechanisms of the new processes discovered (i.e. photoreception in RsbP and uncoupling in yeast mitochondria). • The photobiology project will be extended to include more explicitly aspects of systems biology, through mathematical modelling in which photocycle characteristics of YtvA and AppA will be integrated into a model describing the cellular (stress) response. • Both dr Teixeira de Mattos and prof.dr Hugenholtz have obtained a Yeast cells have been grown under conditions of defined and controlled low transnational SysMo grant. This allows the group to initiate two new systems oxygen availability. Quantitative analysis revealed that there is a micro-aerobic biology projects on (1) regulation of respiration in Escherichia coli and (2) on range of growth conditions that results in yeast cells that are vastly different from growth and product formation in lactic acid bacteria. both fully aerobically and anaerobically grown cells, with respect to: their the transcriptome and proteome profile, the functioning of their energy conservation machinery and their general physiological state. The notable differences in efficiency of energy conservation suggest the existence of an efficient uncoupling mechanism in Saccharomyces cerevisiae unknown hitherto. In E. coli we have developed a method to measure all quinone components (i.e. the oxidized and reduced form of UQ, DMQ and MQ) under physiologically well-defined conditions. Regulation of the redox state of these components is more complex than predicted by current theories. Swammerdam Institute for Life Science 9

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    Annual Report 2006 Molecular Biology and Microbial Food Safety spores against weak-organic acid preservatives. The data provide targets for Chairholder: Prof.dr S. Brul inhibition of unwanted microbial growth. (3) Finally, we focus on models for host physiology and host-microbe interactions. In collaboration with the virology department of AMC, C. elegans and eukaryotic cell cultures are used to Frans Klis Associate Professor (Senior Scientist) study the adverse effects of antiretroviral compounds on host cells. Furthermore, Hans van der Spek Assistant Professor we focus on the study of pathogenic yeast (see also 1) and, together with the Gertien Smits Assistant Professor Cellular Microbiology group of prof.dr Jerry Wells, on bacterial vegetative pathogens. Our prime area of research is the modulation by these micro- organisms of epithelial cell barrier functions. Introduction Research Highlights Our group aims at understanding the behaviour of (micro)organisms in relation to their food or pharma related environment using approaches ranging from We continued development of a tool to predict spore survival upon thermal classical molecular biology and biochemistry to state of the art functional treatment (see Keijser, 2006). A full description of the germination-specific genomics and systems-biology. The tool kit we apply includes genome-wide gene-expression profile was generated (Keijser et al. submitted; see picture). micro-array analysis, proteomics, various advanced microscopy techniques and Inactivation of germination specific genes showed that the process is most likely microbial physiology using controlled cell culturing systems such as fermentors governed by redundant systems that get specific functions during germination of and chemostats. We have major contacts and contracts with the food and pharma thermally damaged spores or upon germination under stress. Finally, our studies industry focusing on the application of our research in practical settings. Results of stress response in B. subtilis towards the classical preservative sorbic acid are as much as possible quantified and analysed using a number of modelling identified 3 genes that upon inactivation led to sorbic acid hypersensitivity. This tools. The areas of interest cover: (1) The interaction of fungal cells, in particular discovery was patented with Unilever (ter Beek et al., 2006). Close inspection of bakers’ yeast (Saccharomyces cerevisiae) with environments often encountered the gene-expression profile of germinating spores showed that two of three genes in the food manufacturing industry and Candida species with medically relevant are expressed (transiently) during spore germination under optimal conditions. biotic and abiotic surfaces. The prime focus is on both thermal and weak-organic acid stress response and resistance development as well as on the analysis of the behaviour of cells in biofilms. In our experimental set-up we focus on analysing and subsequently being able to interfere with stress cross-tolerance mechanisms. Specifically in Candida we focus on the cell wall proteome and cell wall assembly enzymes and their regulation in the context of trying to identify novel antifungal targets. (2) The second area of interest covers the behaviour of bacterial spore- formers (mainly Bacillus subtilis and Bacillus cereus, a close relative of Bacillus anthracis). These are organisms of prime-importance to the food industry. Their occurrence necessitates the application of harsh food preservation processes such as high thermal treatments. The mechanistic basis of sporulation initiation, the occurrence of extremely high thermal resistance of bacterial spores (e.g. resistance to various minutes at 121˚C), as well as molecular mechanisms involved in the early phases of spore germination are still far from understood. We focus, in a long-standing collaborative effort with the Microbiology group, TNO Quality of Life and the food industry, on all three areas of research. Additionally we study the mechanisms involved in stress resistance of vegetative cells and germinating A systems overview of gene expression during spore-germination. Swammerdam Institute for Life Science 10

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    Annual Report 2006 In our studies on (spoilage and medically relevant) yeasts we developed new • Finally, Dr. Frans Klis was a member of the scientific advisory board of the analytical tools based on t-statistics for the analysis of genomic expression and Priority Program of the Deutsche Forschungs Gemeinschaft ‘Colonisation and fitness profiles. In S. cerevisiae we identified that membrane remodelling is most infection by human pathogenic fungi’. importantly involved in resistance against the natural antimicrobial compound chitosan (Zakrzewska, Euk. Cell, in press). Studies on the cell wall of C. albicans Future Prospects have shown the importance of changes in its cell wall protein structure for virulence (de Groot et al., 2006). We leveraged this tool in collaborative studies • In Bacillus we will expand our studies of weak-acid stress response both in of various other fungal cell wall proteomes such as Schizosaccharomyces pombe, vegetative cells and germinating spores beyond sorbic acid and also we will Aspergillus niger and Phytophthora ramorum. Also, we have made significant start assessing stress cross-resistance and sensitivity of strains in which the progress in in-vivo measurement of intracellular pH in yeasts. We are now levels of expression of putative mediator genes are modulated. Our studies on able to assess this parameter in the cytosol and the mitochondrial matrix. The spore germination will include a molecular physiology assessment of effects of data will be incorporated in cellular (mathematical) models of biochemical thermal stress on germination frequency and lag-time. events during stress with weak acids, heat and oxidative agents. Finally, we have performed genome-wide deletion studies on yeast stress tolerance acquisition • For the yeast work we aim at linking cellular cytosol and pH reference data to in collaboration with prof.dr Steve Oliver of the University of Manchester. (3) molecular data on genome-wide expression during stress exposure. For both We succeeded in quantifying the toxic effects of anti-retroviral drugs with a acid and thermal stress, biochemical data will be put in mathematical models of mitochondria specific qPCR assay in C. elegans. cellular stress physiology. Phenotypic profiling data in S. cerevisiae on cellular cross-tolerance mechanisms will be analysed to uncouple growth-rate related Other Highlights phenomena to specific stress related phenomena. • S. Brul was awarded a Distinguished Research Fellowship of the University of • In C. albicans we will continue our work together with the department of Tasmania in Australia. In that framework he will visit the University in June Klinical Proteomics of prof.dr Chris de Koster and ACTA. We will study 2007, lecture and organise a Workshop with local Faculty. biofilm formation on substrates mimicking mucosal surfaces, biofilm formation under polymicrobial conditions and we aim at the identification of • Furthermore, he received a Unilever grant and authors award. cell wall assembly enzymes. • The joint fungal expertise of our department (Dr. Frans Klis, senior researcher) • The study of retroviral drug toxicity in C. elegans and cultured human cells and the department for Mass Spectrometry of Biomacromolecules with will be reinforced with a starting PhD student. He will extend the observations post-doctoral fellow Dr. Piet de Groot attracted the attention of the dental on the effects of such compounds on mitochondrial numbers to assessing their faculty (ACTA). They consulted us in the framework of their study on the impact on mitochondrial function. We will perform studies both in C.elegans involvement of Candida in oral disease during the ageing process. In particular and cultured epithelial cells. the research aims at studying mixed oral bacteria Candida biofilms. A (joint ACTA-SILS) VIDI-grant proposal has been submitted on this topic by Piet de Groot. • At the major international biannual Food Microbiology meeting FoodMicro 2006 in Bologna with near to 1000 participants three of the abstract submissions of the group were selected for an oral presentation. Swammerdam Institute for Life Science 11

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    Annual Report 2006 Cellular Microbiology humans and to broaden this approach to combat other major pathogens. Chairholder: Prof.dr J.M. Wells Other Highlights Introduction At the end of 2006, Prof.dr Wells was appointed as a Senior Scientist within the Wageningen Center for Food Sciences (WCFS) and he will participate in a new The aim of the Cellular Microbiology research group headed by Prof.dr Wells is project concerning intestinal homeostasis. to investigate the interactions between microbes and humans and to ultimately translate this research into new strategies for combating or preventing diseases. Prof.dr Wells is a member of the Scientific Organizing Committee of the In previous collaborations the group has identified two-component signalling forthcoming Campylobacter and Helicobacter Research Organisation (CHRO systems that are essential for Streptococcus pneumoniae to survive and that cause International Congress to be held in The Netherlands Sept 2007), and he infection in the host. We are now aiming to explore their role and mechanism was a Workshop Panel Member and Chairperson at the European Mucosal of action. Of particular interest are gene products that are potentially useful as Immunology Group Conference, Prague October, 2006. targets for novel antibacterials, anti-infectives or vaccines. In collaboration with TNO we are investigating interactions of commensal and probiotic bacteria with Prof.dr Wells was invited speaker and keynote speaker at several meetings, for the innate immune system of the host. Dendritic cells are of major interest as instance the ESF-EMBO Joint Symposium on Bacterial Networks and Systems they are abundant at mucosal sites where they perform a crucial role in regulating Biology in Spain, and the Food Micro 2006 Congress in Italy. tolerance and inflammation. Research on these topics could have important implications for the therapy or prevention of infectious diseases, inflammatory Future Prospects bowel disease, allergy and autoimmune disorders. This year research will continue on elucidating the function and mechanism of Research Highlights the essential signalling system YycFG in the major human pathogen Streptococcus pneumoniae. Additionally a new TI-Pharma-funded project involving a large Research on a Senter/CDTI -funded project involving industrial and academic national consortium will start. This project is concerned with the exploiting Toll- partners from Spain and The Netherlands began this year. The research is aimed like receptors for drug discovery. at understanding the role of an essential signalling system and its mechanism of action in Streptococcus pneumoniae and other drug-resistant Gram-positive pathogens. Collaborative research on the structure of these proteins is also underway in order to facilitate the discovery and development of chemical inhibitors of this vital regulatory pathway. Research initiated in the group leaders former laboratory on the development of an intranasally administered vaccine against Streptococcus pneumoniae (pneumococcus) was published. Here, the harmless milk bacterium Lactococcus lactis, intracellularly producing the pneumococcal surface protein A (PspA) was evaluated as a mucosally delivered vaccine. Economical and effective vaccines against pneumococcus are needed for implementation in poorer countries where this pathogen is reported to cause around 1 million infant deaths p.a. Given the success obtained with this experimental vaccine and the safety profile of L. lactis there is considerable potential to develop a pneumococcal vaccine for use in Swammerdam Institute for Life Science 12

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    Annual Report 2006 Structure and Functional Organisation of the Cell Nucleus developmental choreography of histone modifications and histone positioning Chairholder: Prof.dr R. van Driel is observed at these regulatory elements. This integrated approach may now be extended to unravel in trans gene regulatory interactions, i.e. between regulatory elements located on different chromosomes. Paul Fransz Assistant Professor Maike Stam Assistant Professor • Microscopical investigations on heterochromatic chromocenters in Arabidopsis Pernette Verschure Assistant Professor cells revealed a fascinating organization of subdomains of epigenetically modified chromatin and of individual repeat classes. Results support our view of a sequential process of chromatin compaction. Introduction • In the EU-EpiVector project we have successfully set up a genetically stable The one-dimensional structure of the genome of an increasing number of episomal system to visualise episomes in the nucleus of living cells and eukaryotes is being fully sequenced. A major challenge is to understand how it manipulate its activity at the epigenetic level. This system is being used to functions in terms of orchestration of expression of the many thousands of genes explore structure-function relationships in the interphase nucleus and develop it encodes. Gene control in eukaryotes is conducted at three hierarchical levels. novel vectors for gene therapy. One is that of the individual gene, involving cis- and trans-acting factors, such as enhancers, promoters and transcription factors. Information at the second • Based on the VIDI-2003 project, which is focussed on controlled changes in level is stored as posttranslational modifications of histones, incorporation epigenetic gene regulation and chromatin structure, we are developing a patent of histone variants and DNA methylation patterns. The third level is the application on the newly developed system in mammalian cells. compartmentalisation of the interphase cell nucleus. Gene regulation at this last level immediately relates to the folding of the chromatin fibre in the nucleus. • In the Huntington (Htt) project we explore the causal relationships between expression of the mutated Htt protein, formation of aggregates in the nucleus, Our aim is to unravel epigenetic mechanisms that employ the functional cell death-survival, changes in histone modifications and gene expression in a compartmentalization of the interphase nucleus and related higher order cell model system. chromatin structure. Epigenetic systems are essential for among others stable cell differentiation and have been found deregulated in various diseases. We •In cooperation with our group, prof.dr Reinhart Heinrich (Berlin; who concentrate on the dynamic structure of chromatin and the behaviour of the tragically died October 2006) and Dr Frank Bruggeman (Manchester, nuclear machineries involved in gene transcription, gene silencing and DNA Amsterdam) have explored the behaviour of gene networks that are controlled repair. We combine structural studies, often on living cells, with molecular at the single gene and the epigenetic level. Based on these preliminary studies biological, biochemical and other molecular approaches. It is our ambition to we started to develop a new research line combining measurements on natural be among the first to develop quantitative and predictive models of regulatory and engineered gene networks in cultured cells in combination with predictive epigenetic networks that can guide experimental approaches. mathematical modelling. Research Highlights Other Highlights • In the past year we obtained new insights into the epigenetic mechanism Dr P.J. Verschure received the Feulgen Prize of the Society for Histochemistry. that controls paramutation in maize. Application of the 3C-technology, in combination with chromatin immunoprecipitation (ChIP), has shown that Dr P.F. Fransz is a member of the organising committee of the 16th International physical interactions occur between specific genomic regulatory elements Chromosome Conference, Amsterdam, 2007. that are 100 kb apart. In part correlated with these interactions, a striking Swammerdam Institute for Life Science 13

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    Annual Report 2006 R. van Driel is member of the advisory and site visiting board of various systems biology programs in Europe: the systems biology initiative of the European Science Foundation, the EU EraNet program ERASysBio, the German ForSys and HepatoSys programs, and the Irish national SFI CSET Systems Biology program. Future Prospects In the forthcoming year we will make the next steps in starting up a new research line focusing on a systems biology approach of analysing multi-level regulated gene networks. This will involve one PhD student. In addition two collaborative grant applications will be submitted. The work on the dynamic behaviour of heterochromatin in Arabidopsis (thesis Dr Tessadori) will be continued by using this system for analysis of the molecular mechanisms that control reversible heterochromatin disassembly. Here we will take full advantage of the genetic, cell biological and biochemical potential of this plant system. Attempts to transfer the paramutation system from maize to Arabidopsis will be continued. At the same time we intend to broaden the paramutation research line towards in trans interactions in general between loci in plants and in cultured mammalian cells. The epivector system developed in 2006 will be exploited to analyse in detail the chromatin state dependant behaviour of epivector chromatin in vivo and analyse the mechanism of the highly reliable equal distribution of the episomes during mitosis. Finally, an extra effort will be made to develop the in vivo chromatin structure/gene expression system designed by Dr Verschure. Closely related to these in vivo approaches is the ongoing work on DNA repair and the derived system on the analysis of the assembly of the transcription initiation complex. The newly developed research on Huntington’s disease will be continued in cooperation with the Breit lab. Swammerdam Institute for Life Science 14

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    Annual Report 2006 Epigenetic Regulation of Gene Expression reporter gene when flanked by STAR elements has a much higher histone Chairholder: Prof.dr A.P. Otte acetylation status than a non-protected reporter gene, nicely correlating with higher gene expression levels. Furthermore, around the promoter of the reporter gene that is non-protected by STAR elements, higher levels of Polycomb group David Satijn Assistant Professor proteins are found in comparison with STAR-protected promoters. These results provide molecular mechanistic insight in the action of STAR elements. Introduction Other Highlights It is our long term aim to understand epigenetic regulation of gene expression Through Crucell, four novel STAR technology licenses were sold to leading in terms of cell differentiation and human disease. Research is focused on biotechnology companies, such as Genzyme and Novartis. In 2006 more than 10 unravelling the function of multiprotein complexes, such as the repressing patents were awarded concerning STAR technology. Polycomb group (PcG) proteins. PcG proteins form multimeric protein complexes that are part of a cellular memory system that is responsible for Future Prospects the inheritance of gene activity to progeny cells. The action of PcG complexes is delimited by the presence of genomic elements, STAR elements, which In the coming year we focus on further understanding the relation between counteract PcG mediated repression. The dynamic interplay between these two, PcG proteins and STAR elements. Therefore, we will extend our analysis of the functionally antagonistic, systems is studied. We further study the implications of chromatin structure of STAR elements and STAR element-protected reporter de-regulated expression of components of these gene regulation systems on both genes. In addition, we will analyze and implement novel, cellular promoters that the activity of genes and on directing cells towards pathological states. convey even higher protein expression levels than currently used viral promoters. Our main expertise lies in: Finally, we want to utilize epigenetic regulatory tools to develop novel expression (i) The utilization of a unique anti-PcG antibody panel that we developed over systems in which protein expression, growth rates of the cells and secretion of the years. The panel is used by us to unravel the composition and dynamic the proteins can be coordinately modulated to achieve an optimal expression behaviour of PcG complexes, particularly in the context of human cancers, as platform for therapeutic proteins. As such we aim to investigate and modulate in well the analyzing genetic target loci of PcG complexes. a comprehensive way (ii) The employment of STAR elements to increase the predictability, expression (i) The role of STAR elements in expression and stability of protein expression; levels and stability of therapeutic proteins in mammalian cells. Our knowledge (ii) An inverse relationship between cell growth and protein expression levels; of epigenetic gene regulation has important biotechnological and commercial (iii) Secretion of proteins. implications. Research Highlights We developed a novel, very stringent selection system for the establishment of mammalian cell lines that produce therapeutic proteins. This allows us to screen ~ ten-fold fewer colonies than usual to obtain a high-producer cell line. In addition average protein expression levels are at least ten-fold higher than when traditional selection systems are employed. We have started to analyze the chromatin structure of STAR elements, using CHromatin ImmunoPrecipitation assays. We found that a stably incorporated Swammerdam Institute for Life Science 15

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    Annual Report 2006 Molecular Cytology diabetes and atherosclerosis. To this end we apply and develop quantitative Chairholder: Prof.dr Th.W.J. Gadella microscopy reporting on the chemical composition and physical state.These research themes heavily depend on advanced microscopy technology organized within the Centre for Advanced Microscopy (CAM, 2004). The goal of CAM Michiel Müller Associate Professor is to boost Life Sciences research using and developing (optical) microscopy Tanneke den Blaauwen Assistant Professor techniques. Current most prominent developments are Coherent Anti-Stokes Joachim Goedhart Assistant Professor Raman (CARS) Microscopy (dr Müller), Third-Harmonic Generation (THG) Erik Manders Assistant Professor Microscopy (dr Müller), Controlled Light Exposure Microscopy (CLEM) (dr Manders) and Spinning disk & Total Internal Reflection (TIR)- Fluorescence Lifetime Imaging Microscopy (FLIM) (prof.dr Gadella). Introduction Molecular Cytology is the study of the dynamic architecture of living cells. Our central theme is ‘Self-organization and signalling in living cells’. Self- organization is the intrinsic property of matter to organize itself in a (dynamic) structure, whereas signalling implies the activity of gene-products to control a local activity which can alter the local cellular architecture (e.g. driving morphogenesis). In order to achieve a certain 3D architecture in cells, these two important mechanisms work in concert. At Molecular Cytology both mechanisms are studied with emphasis on membrane-related architecture of living cells using advanced microscopy tools. The main research areas are: Figure 2. Prototypes of the CLEM unit, the essential 1) Spatial organization of sub-cellular signalling (group leader prof.dr Th.W.J. part of the CLEM microscope. Gadella and dr J. Goedhart). We want to understand how cells can achieve Left: three experimental CLEM units; right: the and maintain a local signal in order to drive morphogenesis, to define new commercial CLEM unit built by Nikon cytoskeletal anchorage or vesicle-docking sites. We focus on signal flow across and in the plane of the membrane of living mammalian cells starting from Figure 1. Endocytosis of cell surface material mediates cell plate formation during histamine/P2Y receptors, G-alfaQ to PLC activation triggering downstream plant cell cytokinesis. In contrast to a 40 year old dogma and text book knowledge calcium and kinase signalling. stating that the new cell wall separating two daughter cells during plant cell division is 2) Molecular mechanism of bacterial proliferation (group leader dr T. den synthesized from Golgi vesicles, we show that cell surface material is used to form this Blaauwen). Two dynamic self-assembling membrane-bound protein complexes structure. The picture shows the endocytic cell surface marker FM4-64 (red) labeling the (hyperstructures) are involved in the elongation and division of the bacterial cell. cell plate from initiation until completion. Microtubules (green) (MAP-GFP) are forming The complexes extend the lateral cell envelope and produce complete new cell the phragmoplast around it, time is given in minutes. poles. The actin homologue MreB and the tubulin homologue FtsZ recruit these complexes, respectively. The identity, function and dynamics of the proteins in Research Highlights these complexes are studied. 3) Self organisation in complex (biological) systems (group leader dr M. Müller). • We demonstrated that for plant cell division, cell surface material is (re-)used We aim to understand the self-organizational properties of biomembranes in to build the cell plate separating the daughter cells (Dhonukshe et al (2006), functional domains crucial in cellular signalling. We also study the mechanism Dev. Cell 10, 137-150). These results highlight a novel endocytosis pathway of lipid droplet formation and breakdown, and aim to find cell morphological contributing to cell plate growth and break the 40 year old (textbook) parameters linked to the (mal)function of lipid droplets, with implications for dogma of Golgi delivery and complete de novo synthesis of this new plasma membrane. Swammerdam Institute for Life Science 16

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    Annual Report 2006 • We have developed a new microscope technology (CLEM; Controlled Light 7 membrane-spanning receptors in mammalian cells. Exposure Microscopy) that strongly reduces phototoxicity and photobleaching • We will further improve the CLEM technology and further reduce (Hoebe et al (2007), Nature Biotechnology). This technology is now applied phototoxicity, a limiting factor for many of live-cell imaging experiments. in several collaborative research projects. The CLEM project is a collaboration • We aim to realize portable endoscopic THG microscopy of lipid droplets, as between the Centre for Advanced Microscopy (SILS) and the AMC. well as to report for the first time on cell type and cell location dependent lipid • We have developed the experimental and numerical procedures that permit for droplet chemical composition and morphology. the first time to obtain quantitative compositional and physical state specific • We aim to publish the first paper that describes a reliable FRET-system to study information from multiplex CARS microscopy images, without a priori the interaction of rare proteins in bacteria. knowledge of the vibrational spectrum (E.M. Vartiainen et al. (2006), Opt. • We aim to publish that MreB is not involved in DNA segregation in contrast Expr. 14(8), 3622-3630; H.A. Rinia et al. (2006), J. Biomed. Opt 11(5), 050502- to numerous publications that state the reverse. We also aim to publish that 1-050502-3). MreBCD/MraY and MurG are part of the elongation protein complex. Other Highlights • An EU TMR-grant was awarded to prof.dr Gadella in collaboration with 7 other European groups working on Nod factor signalling. • The patented CLEM technology, invented by dr Manders, has been licenced out to Nikon Japan and is commercially available from August 2006. • Best poster of the conference award: Svetlana Alexeeva, Gert-Jan Kremers, Dorus Gadella, and Tanneke den Blaauwen. Title: To FRET or not to FRET? Measuring protein-protein interactions during cell division in E. coli. Gordon Research Conference on Single molecule approaches in Biology (June 18-23) USA. Figure 3. (a) THG optical section of LDs in a HeLa cell, incubated overnight with oleic acid complexed to Bovine Serum Albumin. (b) High resolution THG image of the area indicated Figure 4. Co-labeling of oriC-region and DnaB. Left image shows the phase contrast in (a). Future Prospects image of the cells, their length and diameter as determined by the Object Image program (cf. Experimental Procedures). The purple dots show the manually determined position of Future Prospects oriC-regions and the cyan dots show the manually determined position of DnaB. Middle image shows the oriC region positions (lacO cassette bound by GFP-LacIq) and the far • We aim to publish the first FRET-system reporting on Galfa-Q activation. right image shows the labeling with the pAb against DnaB and secondary antibodies Galfa-Q is a major signalling node between phospholipid signalling and various conjugated to Oregon Green. The bar equals 1 mm. Swammerdam Institute for Life Science 17

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    Annual Report 2006 Research Clusters Plant Physiology Chairholder: Prof.dr M.A. Haring Plants and Health Rob Schuurink Assistant Professor Teun Munnik Associate Professor Plants are the world’s primary source for food and feed, for raw materials for industry, and they provide the oxygen we breathe. Although the health of plants is constantly challenged in both natural and agronomic ecosystems, plants Introduction can master most challenges. The Swammerdam Institute for Life Sciences is aiming to unravel the molecular mechanisms of resistance against pathogens We are studying two signal transduction cascades that help plants cope with and insects, and to study the cellular signal transduction pathways controlling biological and environmental stress conditions: phospholipid-based cellular stress responses in general. Knowledge of the genetics and biochemistry of these signalling and volatile-based external communication. processes can be translated into improvement of agricultural crops and plant protection schemes. The main character in the first pathway is the lipid second messenger, phosphatidic acid (PA), which is produced via activation of phospholipase D Two chairs contribute to the research cluster ‘Plants and Health’: ‘Plant (PLD) or indirectly, via the combined action of phospholipase C (PLC) and Physiology’ and ‘Plant-pathogen Interaction’, while the chairs “Molecular diacylglycerol kinase (DGK). By manipulating the activity of individual PLC, Cytology” and “Nuclear organization” study related topics on plant model DGK and PLD genes in tomato and Arabidopsis plants, we aim to elucidate their systems role in plant stress signalling and development. How PA exerts its effects is still unknown, mainly due to the lack of characterized target proteins. We identified several protein kinases that directly and specifically bind PA, including CTR1, a key regulator of the signalling pathway controlled by the stress hormone ethylene. In our second theme, we study the biosynthesis and regulation of volatile benzenoids. We have started to focus on benzoic acid synthesis in our model system Petunia hybrida W115 (Mitchell). We have identified, though microarray analyses, the genes that putatively encode the enzymes that shorten the C3 side chain in phenylpropanoids to the C1 side chain in benzenoids. We have also identified an ABC-transporter of which the expression strongly correlates with benzenoid production and we are using an RNAi approach to identify its function in planta. Furthermore, we use Arabidopsis to study responses of plants to the wound-induced C6-volatile E-2-hexenal. Through a genetic approach we have discovered that γ-amino-butyric acid (GABA) plays an important role in transducing the perception of E-2-hexenal. Finally, we use tomato as a model system to study the regulation of terpenoid biosynthesis and to determine which terpenoids are important in attracting and repelling insects. Our goal is to engineer the synthesis of these terpenoids in cultivated tomatoes. Swammerdam Institute for Life Science 18

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    Annual Report 2006 Research Highlights In collaboration with dr Michiel Müller (SILS, Molecular Cytology) we have described the hydrodynamics and cell volume oscillations in the pollen tube Analysis of a collection of tomato and Arabidopsis transgenics revealed special apical region of Nicotiana tabacum pollen. This has resulted in a new model that features of individual phospholipase genes in these plant species. In tomato we proposes that these are integral components of the biomechanics of pollen tube discovered that LePLDβ1 responds to elicitor treatment and that RNAi cell lines growth. that no longer express this gene has reduced xylanase-induced PLD activity and respond with a disproportionate oxidative burst. This links LePLDβ1 to plant- We have now firmly established a role for GABA as a signal for E-2-hexenal pathogen interactions. The Arabidopsis AtPLDα1 and AtPLDδ1 genes were responses in Arabidopsis. GABA synthesis is highly regulated through a Ca2+- found to be involved in the wounding response and salt tolerance. Knock-out Calmodulin regulated glutamate decarboxylase and GABA levels increase upon lines for these genes were impaired in their PA response upon wounding, with E-2-hexenal treatment. Moreover, the hexenal-response mutant her1 accumulates the severest effect in the AtPLDα1 and AtPLDδ1 double knock-out. This double more GABA and by manipulating GABA levels the mutant becomes even more mutant appeared to be highly sensitive to NaCl (150mM) in the growth medium. resistant to E-2-hexenal, indicating that GABA indeed acts as a signal molecule. With regard to terpenoid synthesis, we have shown that jasmonic acid induces only the transcription of linalool synthase in trichomes while at the same time Preliminary results indicate that an inducing the direct defence responses in the other tissues of the leaf and stem. Arabidopsis diacylglycerol kinase Thus JA-responses are highly regulated through a different set of transcription knock-out line also is more sensitive to factors in the trichomes. Finally, the role of geranyldiphosphate synthase in salt treatment, while a phospholipase producing precursors for gibberellin biosynthesis has been now confirmed in C mutant displays an aberrant root Arabidopsis. phenotype upon salt treatment. In addition, we have obtained an Other Highlights Arabidopsis diacylglycerol kinase knock-out line that appears to be more VIDI dr Christa Testerink: Modulation of protein kinase function by the lipid sensitive to pathogen infection and second messenger phosphatidic acid several DGK and PLC knock-out lines that give impaired PA accumulation VENI dr Rosanna Mirabella: C6 volatiles: a new class of signal molecules in upon cold-treatment. With regard to the plants molecular mechanism of PA we further investigated PA-binding proteins. Future Prospects Characterization of the Raf-like protein kinase CTR1 revealed a novel PA- The collection of Arabidopsis knock-outs in phospholipid biosynthesis that have binding region at the C-terminus of a distinct phenotype with regard to salt or pathogen tolerance will be investigated the protein kinase domain. Binding of in more detail. Double and triple knock-outs will be made to enhance the PA was found to inhibit its kinase activity in vitro. In collaboration with Prof.dr. phenotypic effects discovered so far. Introduction of biosensors into these lines Dorus Gadella (SILS, Molecular Cytology) we have developed GFP biosensors and development of PA-biosensors will allow us to study the subcellular changes that allow the visualization of specific species of phospholipids. in distinct phospholipids pools and link these to physiological processes like salt stress. Both the molecular basis of PA-binding of the protein kinase CTR1 as well as the in vivo consequences will be studied. We aim to elucidate the 3-D structure of the kinase domain of Arabidopsis CTR1 in the presence of PA and identify the critical residues in this PA-binding site. Localization of CTR1 in response Swammerdam Institute for Life Science 19

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    Annual Report 2006 to PA in vivo will be studied using a GFP fusion of the CTR1 PA-binding Plant-pathogen Interaction domain. Our collection of PLC, PLD and DGK mutants will be screened for Chairholder: Prof.dr B.J.C. Cornelissen ethylene-response phenotypes. Now that we have Arabidopsis lines available that are incapable of sensing or producing E-2-hexenal, we can investigate the role of E-2-hexenal as signalling molecule in priming defence responses. T-DNA Frank Takken Assistant Professor insertion mutants of E-2-hexenal or GABA-inducible genes will be obtained to Martijn Rep Assistant Professor investigate their role in transducing C-6 volatile signals. Furthermore, we will start to engineer the production of terpenoids in tomato trichomes. Finally, we will use promoter-reporter constructs to determine the cis-regulatory elements Introduction that determine ODO1 expression and translate our findings from the petunia flower model to the tomato fruit model. Plant-pathogen interactions culminate either in colonisation of the plant, causing disease symptoms, or in a resistance response of the plant preventing pathogen ingress. In the latter case one speaks of resistance, in the former of susceptibility of the plant. For in depth research on the molecular basis of susceptibility and resistance we focus on interactions between soil borne pathogens and their hosts, using the interactions of the fungus Fusarium oxysporum and the root knot nematode Meloidogyne incognita with tomato (Solanum esculentum) as models. For some aspects, the model plant Arabidopsis thaliana is included. Our specific interest is focussed on basal and induced defence mechanisms of the host and on virulence and avirulence factors of the pathogen. Disease resistance of tomato to races of F. oxysporum producing avirulence factor 2 (Avr2), is mediated by the R (Resistance) protein I-2, whereas resistance against M. incognita requires the R protein Mi-1.2. Our working hypothesis is that upon recognition of a matching avirulence factor, an R protein changes conformation. This allows the R protein to form a multimeric protein complex, or activate a pre- existing protein complex, that subsequently activates a defence signalling cascade. The ability of a pathogen to infect and colonise its host depends on ‘general’ pathogenicity genes as well as on specific, secreted ‘effector’ proteins. Secreted proteins can also be ‘avirulence factors’ when they are recognized in the host plant by a matching resistance gene, thereby triggering disease resistance. Our research aims at 1) the identification and dissection of the protein complex (-es) involved in I-2 and Mi-1.2 mediated resistance, the functional analysis of individual complex-components and regulation of the downstream signalling components; and 2) uncovering the role of pathogenicity genes and effector proteins (including avirulence factors) of F. oxysporum and identification of their targets in tomato. Swammerdam Institute for Life Science 20

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    Annual Report 2006 Future Prospects All ~100 pathogenicity mutants of F. oxysporum will be analyzed in detail (number of insertions, gene identification, phenotypes). The analysis of nutritional, gene regulatory and root colonization defects of the frp1 mutant will be finalized. The F. oxysporum chromosome containing at least 8 genes for in planta secreted will be analysed in detail in collaboration with the Broad Institute (U.S.). Analysis of transcriptional regulation of the SIX1 gene and processing of the Six1 protein will be finalized. The function of several newly discovered SIX genes, including a candidate avirulence gene (SIX4) will be studied through gene disruption and complementation. The function of the tomato xylem sap protein Xsp10 will be studied in relation to resistance and susceptibility to Fusarium infection. The function of previously identified I-2 interacting proteins (especially formin and KLC) will be studied in relation to disease resistance mediated by I-2, Rx Research Highlights and Mi-1. Our nucleotide binding studies will be extended to Rx and Mi-1 and the role of regulatory N- and C-terminal domains on ATPase activity analysed. The generation of mutants of F. oxysporum with insertional mutagenesis was Related to this, the intramolecular interaction patterns of Rx and Mi-1 in relation finalized with over 10,000 transformants obtained. All have been tested in to the nucleotide binding state will be examined. bioassays and around 100 pathogenicity mutants were identified. The product of Transgenic Arabidopsis lines expressing (mutant) SUMO proteins will be one gene identified from this screen (‘5G2’) was localized to the nucleus. The frp1 analysed for altered resistance/susceptibility phenotypes to Fusarium and mutant obtained in an earlier screen was shown to have defects in expression of Pseudomonas syringae. Using tagged SUMO proteins we aim to identify in vivo genes required for root colonization. SUMO substrates. The F. oxysporum SIX1 and SIX2 genes, encoding in planta secreted proteins, were shown to be highly specific for the tomato wilt form of this fungus and may have been subject to horizontal gene transfer. These and six additional genes for in planta secreted proteins are located on the same small chromosome. VIGS (Virus Induced Gene Silencing) experiments in tomato revealed a role for the I-2 interacting proteins KLC, Formin and TRAX in disease resistance to Cladosporium fulvum. Full length cDNA’s for these proteins were isolated and functional analysis was initiated. Expression studies of the genes for three isoforms of SUMO (small ubiquitin- like modifier) in Arabidopsis using fusions of the sumo promoters with the GUS reporter gene revealed distinct expression patterns for the three isoforms. Together with the opposite phenotypes (such as early flowering, growth phenotypes etc) caused by (over)expression of the various SUMO isoforms and derived mutants, this observation suggests different functions for the different SUMO proteins in planta. Swammerdam Institute for Life Science 21

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    Annual Report 2006 Research Clusters Animal Physiology and Cognitive Neuroscience Chairholder: Prof.dr C.M.A. Pennartz SILS – Center for NeuroScience Wim Ghijsen Assistant Professor Francesco Battaglia Assistant Professor The human brain might well be the most complex control system on earth. It Sander Daselaar Assistant Professor consists of billions of nerve cells that are connected to each other in circuits of dazzling complexity. Moreover each individual nerve cell is capable of processing information that it receives from thousands of companions, finally Introduction resulting in a very complex and precisely fine-tuned response pattern. At the Swammerdam Institute for Life Sciences the brain is studied at the level of the The group’s global research aim is to elucidate how neuronal networks molecule, the cell, the network and the organism. Although still many questions distributed across the prefrontal cortex, temporal cortex and striatum cooperate are unanswered about the structure of the nerve cell, and the way transmission in a number of cognitive processes, including learning and memory consolidation, of signals takes place, the real challenge is to bridge the gap between the various attention and sensory integration. This aim is pursued using a variety of levels of integration. In particular, real breakthroughs are expected at the level techniques and at various aggregate levels, ranging from the sub-cellular to of networks. How are they organized, and how can they be influenced? Various macroscopic and behavioural domain. Nonetheless, most of the research focuses electrophysiological techniques help the researchers of the institute to answer on the level of systems physiology. General research topics include: these questions by allowing them to observe individual nerve cells and groups of nerve cells in specific parts of the brains. Such techniques will provide insight in • The short-term consolidation of memorized information of recent processes taking place during learning, but also in diseases like epilepsy and in experiences. A very promising candidate mechanism for mediating this responses to stress. process is spontaneous “off-line” reactivation of stored information. After an initial experience which is marked by highly specific firing patterns in Three chairs contribute to the research cluster ‘SILS Center for NeuroScience’: brain structures involved in memory, a replay of these firing patterns can ‘Animal Physiology and Cognitive Neuroscience’, ‘Cellular and Systems be observed, with preservation of temporally specific features such as the Neurobiology’ and ‘Hormonal Regulation of Signal Transduction in the Brain’. order in which brain cells fire. In particular, we pursue the relevance of this phenomenon for memory consolidation, and how the replay is being orchestrated amongst different brain areas, such as the hippocampus and nucleus accumbens. Technically, this project is carried out in animals by performing ensemble recordings using ´tetrode arrays´, and in humans by fMRI (functional magnetic resonance imaging) and TMS (transcranial magnetic stimulation) techniques. • The neural basis of reinforcement learning and changes in motivation and attention. In two electrophysiological projects, we study how neuronal groups in a frontal brain structure engage in the formation of representations of reward, and how networks of cells collectively learn to generate predictions about upcoming rewards, based on sensory cues that precede reward delivery. We also investigate which neurotransmitters and receptors influence the formation of neural representations of reward predictions. Technically, we Swammerdam Institute for Life Science 22

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    Annual Report 2006 use ensemble recordings in combination with local in vivo pharmacology reward-seeking behaviour. We found that two connected brain structures, the to probe these mechanisms. In addition, neural correlates of attention and hippocampus and the nucleus accumbens, reactivate coherently (i.e., together flexible shifting of focal attention are being studied with ensemble recording in time) during off-line processing. Moreover, the data indicate that reward- techniques. related information is being replayed in the nucleus accumbens, which means that emotionally valuable information is spontaneously retrieved during sleep • An important goal in studying the motivational basis of action selection is and other stages important for memory consolidation. to understand how the output of the ventral striatum to motor structures is constructed and regulated. The interaction between striatal output neurons (spiny project neurons) is an important determinant of this regulation of output and involves the release of amino acid and neuropeptide transmitters. Using brain slices of the striatum and patch-clamp techniques we will investigate how variation in presynaptic stimulation patterns leads to differentiation in release between amino acids and neuropeptides, and how these different transmitters contribute to mutual interactions such as lateral inhibition between neurons. Sub-cellular mechanisms are investigated by measuring effects of presynaptic neuropeptide receptor activation / inactivation on fast (~milliseconds) amino acid release in purified nerve terminals. In addition, adaptation of the differential transmitter release upon drug addiction will be subject of study. This research helps to understand how synaptic plasticity contributes to maladaptive changes occurring in the brain as a consequence of drug abuse. • In examining the neural basis of reinforcement learning, we found that the • The investigation of interrelationships between genes, learning and memory rat orbitofrontal cortex encodes information about the reward magnitude capacities as measured in behaviour, and the systems physiology which an animal expects after having perceived an olfactory cue associated with forms the interface between gene expression and overt behaviour. These the reward. In addition, we validated two types of ´combidrive´, i.e. an interrelationships are being studied in the context of learning and memory instrument for combining stable ensemble recordings with local, intracranial capacity, spatial navigation and behavioural impulsivity in genetically varying, drug applications. Using this novel instrument, we studied the role of NMDA recombinant mouse strains and targeted knockout mice. receptors in prefrontal excitability and discovered patterns of oscillatory activity to which spikes is phase-locked. Finally, a first series of recording • Finally, an important general aim has become to support all of the above- studies on rats engaged in attentional switching were conducted. mentioned disciplines by advanced methods of Computational Neuroscience. Furthermore, translational research is conducted by converting principles • The role of neurotransmitter transporters in presynaptic cross-talk was discovered in animal experiments to new studies on human brain (e.g. in fMRI investigated by measuring the effect of the GABA transporter substrate methodology). Gamma-Vinyl-GABA on glutamate and GABA release from purified hippocampal nerve terminals. In addition to a fast, elevation of Ca2+-channel Research Highlights dependent GABA release, a slower GABAB-receptor mediated inhibition of both GABA and glutamate release was found, indicating GABA transporter • Joint ensemble recordings have been made from two brain structures regulated presynaptic cross-talk of GABA-ergic nerve terminals with simultaneously. These recordings are being made in a study on replay, which neighbouring GABA-ergic and glutamatergic synapses via GABAB-receptors. can be observed when rats go to sleep and rest after an intensive period of Swammerdam Institute for Life Science 23

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    Annual Report 2006 • Novel instrumentation was developed for performing multi-tetrode recordings Two publications where listed in the Faculty of 1000 Biology evaluations: in freely moving, task performing mice. This technique was successfully a) Daselaar SM, Fleck MS, Cabeza R. Triple dissociation in the medial temporal applied in the mouse hippocampus, entorhinal cortex and prefrontal cortex. lobes: recollection, familiarity, and novelty. Journal of Neurophysiology 2006; Behavioural screening of several tens of recombinant-inbred mouse lines 96: 1902-11. *F1000 factor 6*. was performed to identify hereditary components in learning and memory b) Daselaar SM, Fleck MS, Dobbins IG, Madden DJ, Cabeza R. Effects of disorders with high degrees of chromosomal linkage. healthy aging on hippocampal and rhinal memory functions: an event-related fMRI study. Cerebral Cortex 2006; 16: 1771-82. *F1000 factor 6.* • We finished data collection for our first fMRI experiment. The aim of the experiment is to establish whether reactivation processes in the human brain Future Prospects can be detected in a way that is analogous to the results found in animals. The fMRI paradigm involves a training session on a visuomotor task flanked by • To establish whether, after a learning task in an fMRI scanner, reactivation two rest periods, which will allow the assessment of reactivation. The training processes in the human brain can be detected in a way that is analogous to the session is followed by a retest in the scanner 7 hours later on the same day. results found in animals. Furthermore, we aim to disrupt off-line learning and During this period, learning “consolidates”, leading to an improvement in memory consolidation by transcranial magnetic stimulation in humans, and by performance on the retest without additional training, a phenomenon known electrical intervention of hippocampal processing in rats. Initiation of further as “off-line learning”. Data analysis is currently being carried out with the forms of translational research will be pursued. intent to link reactivation of training-related activity during rest to subsequent “off-line learning”. A follow-up fMRI experiment has also been started • We aim to integrate electrophysiological and neurochemical approaches using an auditory learning task and MRI-compatible headphones. Finally, we more strongly than was hitherto feasible, first by giving the research projects aim to disrupt off-line learning and memory consolidation by transcranial combining ensemble recordings and drug application by microdialysis a magnetic stimulation in humans, and by electrical intervention of hippocampal strong push ahead, and second by extending our investigations of intra-striatal processing in rats. Initiation of further forms of translational research will be synaptic communication by new patch-clamp experiments and synaptosome pursued. studies using a newly developed instrument to measure transmitter release with a high time resolution. • In external collaborations, we have developed a model for the formation and dynamics of entorhinal grid cells in rodents, and we characterized the neural • A 2-photon imaging setup has been built up to visualize the morphology activity in the monkey amygdala in response to emotionally significant stimuli. and neural activity of neurons in the living mouse brain. Consequences of associative learning and multimodal interactions in the population dynamics Other Highlights of sensory neurons in the rat neocortex will be studied by bulk labeling of neurons with Calcium-indicator dyes in vivo. The group developed a miniaturized, multi-channel recording system with wireless data transmission: new and versatile instrumentation for cognitive and • To complete the ensemble recording studies on attention in rats, and describe physiological research in freely moving animals. on the basis of these results the neural correlates of attentional switching and the level of neural assemblies. Furthermore, the role of NMDA receptors in A grant from the Foundation for Technology & Science (STW) was awarded, mediating the formation of reward-expectancy correlates will be elucidated, for the development and application of miniaturized equipment for ensemble as well as the hitherto unknown origin of the oscillatory activity observed in recordings in mouse models of brain disease orbitofrontal cortex. Swammerdam Institute for Life Science 24

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    Annual Report 2006 • Continued behavioural screening is expected to raise indications for Cellular and Systems Neurobiology chromosomal linkage sites of aberrant learning in mice. Ensemble recordings Chairholder: Prof.dr W.J. Wadman from mouse brain should yield indications about the neural mechanisms of spatial memory, self-localization, impulsive decision making and neural coding of information about delay and magnitude of reward. Recordings Hans van Hooft Assistant Professor from genetically modified mice will be initiated (e.g. local NMDA receptor Jan Gorter Assistant Professor knockout). Taco Werkman Assistant Professor • The scope of Computational Neuroscience activities will be expanded to support new experiments and analyses in ensemble recordings, fMRI and TMS Introduction research and simulations of multimodal neuronal networks. Excitability is the most prominent property of the nervous system and its components. How ion-channels are organized and quantitatively balanced in the neuronal membrane, how they lead to neuron specific firing patterns and how these can be modulated at different time scales (plasticity) belong to the most exciting problems in neuroscience that can now be solved in a multidisciplinary approach. Neurons communicate with each other through a variety of synapses. To provide minimal functionality neurons need to be combined in small circuits. We have organized our research around a few well defined topics in the realm of neuronal excitability. Our core approach is a functional electrophysiological one (from patch-clamping to in vivo). State-of-the-art optical techniques (Ca- imaging, Voltage Sensitive Dyes) and various multi-contact electrode recordings allow the analysis of population activity. When needed, collaborations provide anatomical, immunohistochemical, molecular, genetic and behavioural expertise. The first of our three major research lines studies the fundamental properties of the 5-HT3 receptor and tries to understand its functional role in local circuits and development. Molecular techniques produced mice in which the 5HT3 receptor expressing neurons are labelled with GFP and can be studied efficiently. This has opened a wide range of possibilities to investigate the role of this receptor in functionally connected neurons and also its highly specific role in cortical column formation. The second research line studies epilepsy e.g. seizure generation, epileptogenesis (micro-array technology) and pharmacoresistance. The latter topic we approach from two sides: a) (non-) penetration of drugs via the blood-brain-barrier and b) modification of drug targets, mainly sodium channels. These studies are of high clinical relevance and we strengthen them through a side appointment at the Academic Hospital in Ghent and intense collaboration with SEIN in Heemstede. The therapeutic potential of deep brain stimulation is investigated in patients and Swammerdam Institute for Life Science 25

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    Annual Report 2006 in animal models. Technical realization is supported by collaboration with Philips that the various serotonin, dopamine and GABAB receptors have to some extent Medical Systems. a private G-protein pool which allow an intricate convergence of their activity on the GIRK channel and on the regulation of firing activity. This could explain the The third research line concentrates on specific pharmacological modulation of difference in response of VTA and SN on typical and atypical anti-psychotics. neuronal circuits. One of the aims is to improve the specificity of drugs that are We have applied for several forms of new funding in order to pursue this highly used against schizophrenia. In collaboration with Solvay Pharmaceuticals we interesting research line. study differential modulation of the firing pattern of dopaminergic neurons in the Substantia Nigra and the Ventral Tegmental Area of the rat. In addition, we Mice that have been generated in collaboration with prof. Monyer in Heidelberg support the activities of a spin-off company named Sensocom. and in which Green Fluorescent Proteins visualize the neurons that contain the 5- HT-3 receptor are used to study the functional role of this receptor in the brain. Most of our experiments are supported by computer modelling, focusing on We have quantified the calcium permeability of the ion channel that is mediated single cell excitability in relation to the direct chemical surrounding of the neuron by 5HT3 and continued the investigation of its contribution to development of as well as on the adaptive strategies for excitability that optimize the working functional columns in the cortex. It has now been established that Cajal Retzius range of active neurons. In the latter case we try to extrapolate the consequences cells receive a dominant serotonin input via this receptor in particularly in early of single cell strategies to larger neuronal networks. The combination of life. Manipulating this signal pathway has a strong effect on the morphology of theoretical and experimental work has proven to be very fruitful in the scientific cortical pyramidal neurons. setting of SILS and the FNWI. The study on the interaction of various inputs on subiculum neurons in the Fig 1: GFP labelled interneurons hippocampal region was completed and submitted for publication. It proved in cortical slices from animals the usefulness of the V(oltage) S(ensitive) D(ye) technique for the analysis of 1 week, 2 weeks and adult. of spatial/temporal integration of neuronal signals in the in-vitro slice. This Lower panel gives the (5HT3 permits us to analyze network activity from a different perspective. The studies specific) synaptic serotonin have been continued by analyzing the spatial organization of theta rhythm in a response of a Cajal-Retzius cell, combined hippocampus-entorhinal cortex slice. Special attention was given to illustrated in the panel to the human slice preparations that became available due to epilepsy surgery in the right. Vrije Universiteit (prof.dr.M.Witter) and in which we investigated spontaneous seizure-like activity and its response to anti-epileptic drugs. Fig 2: Morphology of typical Research Highlights cortical neurons that developed under normal conditions (right The studies on homeostatic scaling of neuronal excitability as modulated by panel) or in the absence of synaptic activity were finished with the publication of a series of papers and a serotonergic innervation (left modelling study that resulted in the promotion of dr. Michiel Remme. We started neuron). The latter one has a new collaboration with prof.dr.Tallie Baram from the University of Irvine restricted morphology California in order to elucidate the underlying molecular mechanisms of Ih homeostatic regulation. The investigation of the interactions between serotonin and dopamine / GABA in the Substantia Nigra and Ventral Tegmental Area was completed. We hypothesize Swammerdam Institute for Life Science 26

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    Annual Report 2006 The micro array study on gene expression in different brain regions Fig 3: Schematic overview of (CA3, Entorhinal Cortex and Cerebellum) during the prominent phase of a computational model of a epileptogenesis in a rat model of Temporal Lobe Epilepsy (1 day to 3 month branched neuron that interacts after status) was completed and published. It has given us a huge data base of with a glia surrounding interesting biological “processes” that are affected by epileptogenesis. We will use compartment through an them as new leads in the investigation of epileptogenesis. The first process that extracellular space of restricted is now under investigation is inflammation. This research line also studies the dimensions. regulation of the blood brain barrier, where we found that multi-drug-transporter proteins are up-regulated during epileptogenesis. This could explain why in some cases pharmacoresistance develops, as it implies that drugs like phenytoin are strongly extruded from the brain under these conditions. Further studies will have to show, whether this is a universal mechanism or whether large differences exist between anti-epileptic drugs. The studies on the use of deep brain stimulation have been continued on patients as well as in experimental animals. In the latter a modulating effect of continuous background stimulation in the hippocampus with 130 Hz could be demonstrated on several parameters of evoked seizures during kindling. Moreover 130 Hz Other Highlights stimulation seems to be more effective than low frequency stimulation. A first trial with 10 pharmacoresistant patients was also successfully completed and A new research grant from ALW (dr. Hans van Hooft) was obtained that will published. It demonstrates the potential of DBS for epilepsy treatment. A new allow to intensify the research on the 5HT3 receptor. alliance with Philips Eindhoven was started to realize practical application and fundamental support for DBS. A new NEF grant was obtained that will allow studying the role of inflammation in epileptogenesis. The theoretical studies on scaling of neuronal excitability using a combination of Ih and IA were finished. Analytical as well as computational analysis Two new collaborators were hired on positions made available from NEF for demonstrated that such a system of regulation can work and has several epilepsy research. A post-doc will investigate seizure generation, while a PhD advantages over other described ways of regulation. In addition we investigated student (AIO) will continue the work on sodium channel modulation. whether such a scaling mechanism could interfere with learning rules that employ Spike Time Dependent Plasticity in order to implement learning and pattern The collaboration with Philips Research was finalized and projects (three PhD recognition. We conclude that such interaction are insignificant, which implies students {AIO’s}) are being started in Eindhoven and in Amsterdam. that excitability scaling with intrinsic potassium channels could be a reality. We also investigated the consequences of scaling in a neuronal network that contains Our group will participate in a large research program on the role of the excitatory and inhibitory neurons. The studies that investigated the role of endocannabinoid receptor. This program will continue our collaboration with the extracellular space and neuron-glia interactions in seizure generation were Solvay Pharmaceuticals, the leading industrial partner in the program. completed. Our group participates in an international attempt to investigate and compare 3 different animal models of temporal lobe epilepsy. Two labs for each model will generate epileptic animals and a centralized analysis using micro arrays to quantify gene expression in the Dentate Gyrus will be undertaken. This effort Swammerdam Institute for Life Science 27

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    Annual Report 2006 should tell us which processes all models have in common and how large inter- the recording of membrane voltages and employ it to investigate the spatial lab and inter-model variation is. properties of oscillatory behaviour in hippocampal-EC brain slices as well. The computer modelling studies on excitability scaling and seizure generation The spin-off company Sensocom was provided with a state-of-the-art set up in will be completed and new lines that support e.g. deep brain stimulation will which depth EEG signals from several relevant brain regions can be quantified need to be formulated. Financing will be needed, probably with the new Dutch under strict behavioural conditions. frameworks for NeuroInformatics, Cognition or Systems Biology. Future Prospects In 2007 several new projects will start as soon as new collaborators have been recruited. Our main mission will be the control and organization of excitability under basic and pathological conditions. We concentrate on our three main research lines: 1) Functional role of 5HT3 receptors in development and under adult conditions. The mice with GFP-labeled 5HT3 receptors will be used to further elucidate cell type specific properties of neurons that contain this receptor (focus on Cajal Retzius cells and on cortical pyramidal neuron morphology). Our VSD and calcium imaging expertise will be extremely useful. 2) We will substantiate the hypotheses that were formulated to explain pharmaco- resistance on the basis of (local) modulation of the permeability of the blood brain barrier and we will investigate the possibilities for using multi-drug transporter proteins regulators in the control of epilepsy / epileptogenesis. The leads that have come out of the micro-array experiments on epileptogenesis will be followed up and we will investigate the role of inflammation in epileptogenesis. We will also continue the investigations into the potential use of Deep Brain Stimulation as an alternative treatment against epilepsy. We have created new possibilities to link it with the clinic. The epilepsy research line will be merged with the one on intrinsic scaling of excitability. 3) The continuation of our collaboration with Solvay Pharmaceuticals will take place within the framework of TIPharma and will concentrate on the role of eCB receptors in specific behavioural conditions. We will seek new funding for the project that studies interaction between serotonin and dopamine which formed our hypothesis for different responses from SN / VTA. In addition the following points will also merit attention: We will further strengthen and integrate the use of optical techniques for Swammerdam Institute for Life Science 28

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    Annual Report 2006 Hormonal Regulation of Signal Transduction in the Brain modified mutants) and examine changes in neurogenesis, morphology, electrical Chairholder: Prof.dr M. Joëls properties and behaviour at various ages. Paul Lucassen Associate Professor Harm Krugers Assistant Professor Henk Karst Researcher Introduction The main aim of our research is to delineate how stress hormones affect the function of rodent brain cells and how this can explain behavioural adaptation. Corticosterone promotes GluR2 trafficking to the membrane. Corticosterone applied The emphasis of our research is on the cellular processes that are altered by stress to primary hippocampal cultures for 3 hours enhances expression of GluR2 subunits hormones like corticosterone. To investigate this we use electrophysiological (red label) in the membrane (right panel) in comparison to vehicle treated cells (left recording techniques, mostly in vitro. In collaboration with others this is panel). Membrane expression of GluR1 subunits remains unaltered after application combined with methods that monitor gene expression patterns. The latter is of corticosterone (green label). Experiments were performed together with Casper highly relevant, as corticosteroid hormones act via their nuclear receptors as Hoogenraad (Erasmus University Rotterdam). regulators of transcriptional activity. Recently, however, we discovered that corticosteroid hormones also change neuronal function in a rapid non-genomic manner. It is the combined rapid (non-genomic) and delayed (genomic) pathways Research Highlights which make corticosteroid hormones such powerful tools to change brain function over a prolonged period of time after stress. In addition to examining Over the past year we have studied the rapid non-genomic effects of neuronal cell function and the underlying molecular mechanism, we also corticosterone in more detail. On the one hand we examined the intracellular investigate the effect of stress on the morphology of brain cells. The theories signalling pathway. We observed that the previously described rapid effect that come forward from the experimental work in animals are tested also in the of corticosterone on glutamate release probability is most likely mediated by human brain, in collaboration with other research labs. mineralocorticoid receptors inserted into the presynaptic membrane. Via these receptors corticosterone activates the ERK pathway, which then results in fusion While most of the research focuses on the effect of a single exposure to stress, of vesicles with the presynaptic membrane. In addition to these presynaptic we are also interested in long-term consequences of stress. One series of pathways, corticosterone can also change a particular K-conductance, via a investigations therefore specifically examined how repetitive stress during postsynaptic non-genomic pathway. adulthood can change brain function, neurogenesis and the shape of neurons for a prolonged period of time. Another research line is dedicated to the long-term On the other hand we examined the importance of non-genomic effects for the consequences of stressful situations early in life. The latter can pertain to maternal interaction of corticosterone with other stress hormone systems in brain. We deprivation of neonatal rat pups; however, we are also interested in extremes observed that corticosterone (non-genomically) acts in the same direction as within the natural variation of maternal care. noradrenaline when the two hormones are present at the same time. However, when genomic actions of corticosterone are allowed to develop, the hormone A separate research line concerns the role of structural proteins and cell cycle completely suppresses the effect of noradrenaline. This has led to the hypothesis factors in neurodegenerative diseases, like Alzheimer’s disease. Here we use that stress hormones like corticosterone and noradrenaline in concert strengthen experimental animal models for neurodegenerative diseases (usually genetically synaptic contacts shortly after the stress and thus help to encode the information. Swammerdam Institute for Life Science 29

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    Annual Report 2006 The subsequent delayed effect of corticosterone makes it very hard for new With respect to the delayed corticosteroid effects, we will investigate the ways information to change synaptic contacts, which helps to preserve the original in which stress hormones affect the glutamatergic signalling in the hippocampus message. and the consequences of these effects for behaviour. We will furthermore study the mechanism underlying delayed effects of corticosteroid hormones on cellular Other Highlights morphology in brain. Members of the group organized 3 SILS-CNS Masterclasses (March 2nd, We will examine the long-term consequences of maternal care and maternal September 26th, December 11th); a symposium in the Biological Centre Haren; deprivation for the physiology, morphology and proliferation in various an international workshop on Stress, Plasticity and Memory (Nervi, Italy); a hippocampal areas. symposium during the 5th FENS Forum Meeting in Vienna; several symposia during the 5th ENP Meeting (Doorwerth); Marian Joëls served as Vice-Chairman Mutant mouse models for Alzheimer’s Disease or Frontotemporal Dementia of the FENS Forum 2010 Local Organizing Committee. will be tested for cell cycle, morphology and functional properties in the hippocampus. Paul Lucassen received a grant from the HersenStichting Nederland; Marian Joëls received an HFSP programme grant. Marian Joëls was the main applicant of a EUROCORES on “Stress and Mental Health”, which was approved by the ESF. Members of the group served as referee for many international journals (including the top-tier journals) and funding agencies; Marian Joëls was elected on the Editorial Board of Neural Plasticity. Marian Joëls served on the Board of the KNAW Science Division; the Board of the Division Earth and Life Sciences ALW/NWO; acted as chairman of the National Initiative Brain & Cognition; as Departmental Head Earth and Life Sciences UvA; and was elected as chairman of the Dutch Neurofederation. Future Prospects For the upcoming year we have the following aims: We aim to better understand the mechanism and physiological importance of the rapid non-genomic corticosteroid effects. We will examine the rapid and delayed interactions between corticosteroids and other stress hormone systems, in the hippocampus as well as other areas. This will be done both with electrophysiological techniques in rodent tissue and in the human brain using fMRI approaches. Swammerdam Institute for Life Science 30

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    Annual Report 2006 Research Clusters Mass Spectrometry of Biomacromolecules Chairholder: Prof.dr C.G. de Koster Life Science Technologies Luitzen de Jong Associate Professor Leo de Koning Assistant Professor The Swammerdams’ Life Science Technologies Cluster carries out fundamental biological research and makes a wide range of techniques available to the SILS researchers. Advanced microscopy enables us to bridge the gap between the level Introduction of single molecules and the level of aggregates of molecules in single/multiple cells, and to make the life processes visible in time (4D imaging). The institute Future progress in the life sciences will heavily depend on the integration of works with the newest modes of advanced optical microscopy, for instance chemistry, physics, mathematics, (bio)informatics and biology. Our group FRET-microscopy, enabling the measurement of distances between molecules in combines mass spectrometry with biomolecular and organic chemistry. We focus a range of one to eight nanometres in intact living cells. on four research themes that are carried out in close collaboration with other groups within and beyond the Swammerdam Institute for Life Sciences, i.e., Micro-array technology simultaneously analyses all genes of a particular (i) systematic analysis of protein-protein interactions, (ii) post-transcriptional organism, in a particular cell type or under specific growth or stress conditions regulation of gene expression, (iii) host-fungal pathogen interactions and (iv) or other internal or external stimuli. The Micro-Array Department (MAD), a identification of biomarkers to discriminate between healthy and diseased state. semi commercial facility, produces micro-arrays for the spotted-array technology We are developing innovative, mass spectrometry-based experimental approaches platform, for example for the genes of man, mouse, yeast, tomato, petunia plants, that are designed for these research areas, but which are also more widely etc. and makes GeneChips from Affymetrix available. The MAD is accessible applicable. to all academic, as well as industrial research groups, and can be found on the internet at www.microarray.nl. The large stream of data, produced by micro array Research Highlights technology, needs to be processed and analysed by means of bioinformatics, for which a bioinformatics support group has been established. Chemical crosslinking is used to identify nearest neighbours in protein complexes, while identifying crosslinked amino acids residues is a powerful Mass spectrometry is essential to characterize proteins and to analyze the method to validate models of the 3-D structure of proteins. Mapping of cross- relationship between the structure and function of a protein (proteomics). links in proteolytic digests of large protein complexes requires cleavable Mass spectrometry is a key technology at SILS in which we have a long history cross-linkers that enable isolation of the cleavage products with preservation and substantial expertise. An important part of the research involves the of information about connection. We discovered an amine-specific cross- identification, quantification and functional characterization of proteins. Bio- linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these informatics also plays an important role in this by efficiently analysing the data requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)- streams. Bioinformatics and data analysis are essential to collect, compare and phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. integrate all available data, and to present them to the researchers in a way that One pathway leads to cleavage in a fraction of the cross-linked species and in the the information is summarised and visualises the underlying biological processes. other pathway the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by BAMG without actual Three chairs and one department contribute to the research cluster ‘Life Science cross-linking yield distinct sets of TCEP-induced reaction products that can Technologies’: ‘Mass Spectrometry of Biomacromolecules’, ‘Micro Array be isolated by reverse-phase diagonal chromatography and identified by mass Department and Integrative Bioinformatics Unit’, ‘Biosystems Data Analysis’ spectrometry to reveal the identity of parent compounds. The ease by which and ‘Molecular Cytology’. cross-link-derived reaction products can be isolated and identified opens avenues Swammerdam Institute for Life Science 31

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    Annual Report 2006 for mapping cross-links in complex biological assemblies and mixtures of protein ii) In collaboration with Prof.dr K.J. Hellingwerf and dr M.J. Teixera de Mattos complexes. we will aim at unraveling the regulatory circuit underlying the transition of aerobic to (semi)-anaerobic metabolism in E. coli. Cultures of E. coli will be pulse labeled with AZHAL before, during and after the transition from aerobic to anaerobic metabolism. Within the scope of IOP Vertical Genomics we will quantitatively map glycolytic enzymes and compare data with mRNA concentration to unravel new post-transcriptional control mechanisms for regulation of glycolysis. iii) The MS group will further explore the question how mass spectrometry in combination with novel purification strategies and bioinformatics tools can provide detailed quantitative structural and functional information about cell wall proteins of Candida albicans and Saccharomyces spec. With the group of Prof.dr S. Brul we will aim at characterization of the spore proteome to gain insight into the development of Bacillus subtilus spores. We will continue the collaborations with the groups of the SILS-plant cluster. iv) In the framework of our collaborative clinical proteomics research with the AMC, biomarker identification is an important research theme. We will focus on identification of diagnostic and prognostic markers for ovarian cancer in collaboration with Dr M.R Buist (AMC), for idiopathic nephrotic syndrome with Dr. J. Aten (AMC) and within SILS-LST with the Biosystems Data Analysis group of Prof.dr A. Smilde. Together with the group of Prof.dr J. Aerts (AMC) Future Prospects further research is planned for the identification of biomarkers for Gaucher and Fabry disease. Focal points in our MS research program are as mentioned above i) systematic analysis of protein-protein interactions, ii) post-transcriptional regulation of gene expression, iii) host-fungal pathogen interactions and iv) biomarker identification. i) In our projects where characterization of protein-protein complexes plays an important role we will move towards functional characterization of larger assemblies using our new cross-linkers. For this purpose novel and innovative cross-linkers will be further developed and improved in close collaboration with organic chemists (J. van Maarseveen, HIMS) to enhance quantitative, selective and sensitive detection of protein assemblies in complex biomatrices. In collaboration with others several complexes will be structurally characterized, id est the divisome and subcomplexes thereof from E.coli (Dr T. den Blaauwen), the HGF/SF – MET complex (with Dr E Gherardi, MRCC, Cambridge, UK) and the yeast exosome (with Dr D. Tollervey, WTCCB, Edinburgh, UK). Swammerdam Institute for Life Science 32

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    Annual Report 2006 Biosystems Data Analysis Other Highlights Chairholder: Prof.dr A.K. Smilde Prof.dr A. Smilde was the Eastern Analytical Symposium 2006 Award Recipient for Achievements in Chemometrics, Nov 2006, NJ, USA. Huub Hoefsloot Associate Professor Johan Westerhuis Assistant Professor Future Prospects In the coming year we will be: Introduction 1) Developing a method for analyzing dynamic metabolomic data: modelling differences in hormone dynamics of control and narcoleptic patients. The general aim of our research is developing and validating methods for 2) Developing methods for incorporating a priori biological information in summarizing and visualizing complex biological data. metabolomics data analysis tools: generating more realistic models. 3) Developing methods for identifying time-course patterns in clinical proteomics Research Highlights data: tracking the efficacy of treatments. A key question in omics data analysis is whether the results of a data analysis can be trusted (this is called validation in our jargon). A validation method has been developed for a commonly used metabolomics data analysis method (ASCA). Moreover, an existing validation method in metabolomics – cross-validation – has shortcomings, which have been investigated and reported. In clinical proteomics, a key question is to find reliable biomarkers for a given disease. A biomarker selection and validation method has been developed which showed good results. In many cases, clinical biomarker discovery is based on disease classification methods. In a national collaboration (with seven other groups) a strategy for finding and validating the best classification method for a given problem has been established. Swammerdam Institute for Life Science 33

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    Annual Report 2006 Micro Array Department and Integrative Bioinformatics Unit transcriptomics. IBU’s main research efforts are: i) domain interaction with Group leader: Dr T.M. Breit the aim to extend the possibilities for computer-assisted experimental biology research by building an actual e-BioScience laboratory (e-BioLab) with a virtual lab e-bioscience problem solving environment; ii) domain modelling with the Floyd Wittink Project management “wet-lab” aim to enable biological problem-solving and phenomenon-discovery via data Mattijs Jonker Project management “dry-lab” integration and computational experiments. To this end, IBU participates in three Jenny Batson Project Administration nationwide projects: “BioRange”, a nationwide bioinformatics research project; Marco Roos Senior Researcher IBU “BioAssist”, a national bioinformatics support programme and “Virtual Lab for Han Rauwerda Senior Researcher IBU e-Science (VL-e)”, the Dutch e-science project in the field of ICT infrastructure and methods. Introduction Research Highlights MicroArray Department (MAD): Microarray technology has become a well- The most important achievements of the MAD during 2006: established tool in the analysis of genome-wide gene expression studies. The • Installation of support-bioinformatics Dry-lab with staff, microarray data- ultimate goal of a microarray experiment is simultaneous examination of the analysis methods and infrastructure. expression of all genes of a specific organism, in a cell type in a specific growth • As official Affymetrix Service provider, the MAD has introduced and validated or stress condition, to unravel complex cellular mechanisms or identify and SNP analyses. use biomarkers. Transcriptomics biomarkers are genes whose expression • Started procedure to become a certified Agilent Service Provider. profile can be used for diagnostic purposes or to monitor and predict cellular • Started development of microRNA array, universal array and combination processes. Another purpose is to examine the DNA of tissues or individuals array. for mutations that can be related to complex diseases like cancer or diabetes. • Ongoing acquisition of new industrial and academic clients, many new Because microarray experiments produce a vast amount of data, extensive projects. bioinformatics infrastructure, methods and expertise are needed to cope with • Organized a nation-wide jubilee microarray competition for life sciences these data effectively. Microarray bioinformatics comprise data-handling researchers. (storage and exchange), data-preprocessing (normalization and validation), and • Execution of projects and start of research initiatives with SILS groups (van data-analysis (clustering, biomarker selection, etc.). The MAD consists of a Driel, Brul, Crielaard, Wadman). microarray technology section (Wet-lab) with 3 specialists, a microarray data- • Execution of projects and start of research initiatives with AMC groups analysis section (Dry-lab) with 5 bioinformaticians, and management with 2 staff (Versteeg, van Kampen) members. The MAD operates as a technology and bioinformatics core facility for • Execution of strategic collaboration with the RIVM on p53 research. UvA scientists, as well as service provider for external academic and industrial • Start of strategic collaboration with UMC on medical microbiology. customers. The MAD is an official Affymetrix Service Provider and is in the • Construction of an e-BioScience Laboratory (eBioLab) with a VL problem- process of becoming a certified Agilent Service Provider. solving environment. • First software applications running: R on Grid and SigWin-finder workflow Integrative Bioinformatics Unit (IBU): To be able to perform true analyses tool of transcriptomics data, these data must be combined with other biological • Partner in initiation of virtual e-BioScience institute. -omics data. The bioinformatics research group consists of ~10 staff members • First integrative experiments with semantic web-technology. with expertise in experimental biology, bioinformatics, informatics, statistics, • First research articles accepted. plus mathematics, and combines multidisciplinary life sciences research with bioinformatics research in a collaborative setting, with a strong focus on Swammerdam Institute for Life Science 34

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    Annual Report 2006 Other Highlights Dr T.M. Breit is a permanent member of the National Advisory Committee as well as the Management Team of the Netherlands BioInformatics Centre (NBIC). Dr T.M. Breit became a member of the NGI Horizon program committee. Dr S. Marshall is Advisory Committee Representative for the UvA in the world wide web consortium (W3C); Semantic Web Health Care and Life Sciences Interest Group. Future Prospects The challenges for 2007 are to: MAD Technology; - implement a bead based microarray technology platform. - develop, test, evaluate and implement relevant microarray technology implementations such as: microRNA, universal, combination and protein arrays. MAD Bioinformatics; - test, evaluate and implement relevant bioinformatics methods, tools and infrastructure such as advanced biomarker selection, data preprocessing and analysis. - extend our current life sciences research programs on gene-expression regulation, food-safety and toxicogenomics by focused research. MAD Research; - develop a problem solving environment for the complete chain of microarray data analysis. - perform true integrative bioinformatics computational experimentation. - further develop the actual e-BioLab. - develop an information resource model within the Huntington use case. Swammerdam Institute for Life Science 35

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    Annual Report 2006 SILS and society 3. Societal Activities Introduction The Swammerdam Institute for Life Sciences is active in the study of life processes that create and determine a living organism. Life Sciences are developing rapidly, and the developments in life sciences continue to have a huge societal impact, for instance in the areas of nutrition, medicine, and industrial applications. The Swammerdam Institute carries out fundamental research, with attention for applied oriented research. This is illustrated for instance by our collaborations with several companies and TNO. Communication with the public Next to training scientists, and making them aware of the need for societal communication, scientists from the institute directly engage in communication with the public. One form this takes is by participating in the beta-festivals the faculty collaborates in, and in the open day at the Science Park. At the open day the people of Amsterdam, and especially the Watergraafsmeer are invited to see what research is carried out at the sciencepark. NEMO, the children’s science centre and the Universiteit van Amsterdam collaborate in organising the “Wake Up!” children’s lecture series. Dr Gertien Smits, microbiologist of the Swammerdam Institute has given a lecture with the title “Can you eat bacteria?” (Kun je bacteriën eten?) in this lecture series on March 12, 2006. In this lecture she explained the children that bacteria are present everywhere, and that, together with our food we eat many bacteria. However, inside our body we already have millions of bacteria that help us to digest our food. Most of the time these bacteria make sure that if we accidentally eat bad bacteria, they don’t take over and make us ill. Collaboration with industry Contacts with industry are very important for the Swammerdam Institute. Therefore we stimulate our staff when they are asked as advisors by industry. A few examples: Dr Frans Klis advises DSM (Delft), Prof.dr Klaas Hellingwerf advises Purac Biochem on the bioinformatics of a lactic acid bacterium. Dr. Swammerdam Institute for Life Science 36

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    Annual Report 2006 Joost Teixeira de Mattos is external adviser of Heineken-Zoeterwoude and DSM-Delft. Prof.dr Michel Haring advises Rijk Zwaan Breeding, prof.dr Arie Otte collaborates with and advises Crucell and prof.dr Stanley Brul has several collaborations with Unilever. Prof.dr Wytse Wadman, in the area of neurobiology collaborates with Solvay Pharmaceuticals and Philips and has a strong link with the clinical through a side appointment in the Academic Hospital Ghent. Prof.dr Ben Cornelissen advises Arcadis in the area of Dutch Elm disease (“Dutch Trig”). Our commitment to collaborating with industry is also shown in our projects with shared grant applications, for instance prof.dr Michel Haring and dr Rob Schuurink collaborate with Keygene, and have expanded this collaboration by a successful STW application. The longstanding collaboration of prof.dr Wytse Wadman with Solvay Pharmaceuticals is built on by shared participation in a TTI Pharma project. Prof.dr Age Smilde and dr Huub Hoefsloot collaborate with Unilever and other (academical) partners in an EU Transfer of Knowledge project. Innovation Innovative results are protected in patent applications, to ensure full societal use of important findings. The institute participates in the Technopartner funded collaboration I Am Starter, in which the UvA and the AMC collaborate with the Vrije Universiteit and its academic Medical Centre (VUMC). Swammerdam Institute for Life Science 37

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    Annual Report 2006 Finance, Personnel and Infrastructure 4. Management Finance Due to the introduction of new financial systems at the UvA, the financial results for 2006 will be shown either in a separate addendum to this annual report, or in the report for 2007. Revenues and costs for previous years* 2005 2004 2003 2002 2001 university funding 7577 8987 7364 6131 5838 external funding 4515 6167 4474 3883 3852 total revenues 12092 15154 11838 10014 9690 personnel costs 9122 9626 8919 7465 7096 equipment 803 1775 998 782 600 bench fees 2729 3214 2312 1668 1636 total costs 11851 14614 12229 9915 9332 result 241 540 -391 99 358 * All amounts are given in K Euro. In this table –starting from 2004-external funding is considered to be 2nd and 3rd funding source only. Funding The funding results for 2006 will be shown either in a separate addendum to this annual report, or in the report for 2007. The funding system of Netherlands universities distinguishes three different kinds of funding sources. These funding sources are numbered one to three. Resources originating from the Netherlands government and directly given to the university are referred to as the first funding source. External funding is divided into funding originating from the Netherlands Organization for Scientific Research (second funding source) and money originating from all other resources such as EU and contract research (third funding source). Swammerdam Institute for Life Science 38

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    Annual Report 2006 Income and costs in the 1st funding source, in ka, for the years 2003-2005. Reserves for the Swammerdam Institute, in ka, for the years 2005-2006 2005 2004 2003 01-01-06 01-01-05 Revenues 7577 8987 7793 Reserves (1st funding stream) 558 233 Costs 7357 8902 8291 Reserves (3rd funding stream) 1005 1089 Result 220 85 -498 Total financial reserves 1563 1322 Income and costs in the 2nd funding stream, in ka, for the years 2003-2005. Private and Public Partnerships 2005 2004 2003 Revenues 2160 2303 2279 The Swammerdam Institute actively establishes collaborations with industry. Costs 2160 2303 2279 Result 0 0 0 Universities play a role of increasing importance in creating innovations, either directly by establishing spin-off companies, or indirectly by making its results publicly available. The life sciences results from the Swammerdam Institute can Income and costs in the 3rd funding stream, in ka, for the years 2003-2005. for instance be used in the areas of medicine development, food development and food safety, and environmental processes. In order to protect its intellectual 2005 2004 2003 properties the Swammerdam Institute follows an active patenting policy. In most Revenues 2355 3864 1766 causes our IP forms the basis for collaborations with industrial partners. Costs 2334 3409 1659 Result 21 455 107 To achieve our aims, the Institute seeks further partnerships with industry. These collaborations can have different forms like consultancies for industrial R&D organizations, transfer of biological materials, patent licensing, sponsored Financial reserves of the Swammerdam Institute for Life research and services using advanced techniques. In some cases we consider Sciences and set up spin-off companies. In the collaborations with industry the institute aims to establish long lasting relations with partners all around the world As stated before, the institute’s resources can be divided into university funding that are based on confidence and mutual respect, and in which benefits, both and external funding. In a similar way the financial reserves of the institute can technological / scientific and financial are mutually shared. Upon request a be discerned by their origin. Profits or losses that are made on externally funded brochure of the institute and a description of our research portfolio can be send. projects increase or decrease the reserves of the 3rd funding stream. Because For further information please contact: sande@science.uva.nl. most groups have externally funded projects they also possess a certain amount of these reserves. They can spend money from these reserves if additional resources are lacking but required for continuation of their research. Results from Personnel university funded activities of the institute increase (or decrease) the 1st funding stream reserves. Because the resources of the 1st funding stream are spent by the The university aims at a more equal division of males and females in the staff at management these reserves can be considered to belong to the management. It all levels. At the level of PhD students and post doctoral fellows we have roughly must be stated that the management is responsible for the entire financial reserves an equal division. At the level of assistant professor, associate professor and at all times. professor however, the majority of staff is male. In application procedures for new staff the Swammerdam Institute for Life Sciences actively looks for female staff at these levels. Age-wise our staff is spread over the full range from starting PhD, to people who Swammerdam Institute for Life Science 39

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    Annual Report 2006 are (close to) retiring. In recent years a lot of young people have been appointed, bringing new ideas and enthusiasm. We also feel it is very valuable to keep the experience and network of retiring staff accessible. Therefore we keep the connections, and use our retiring staff in advisory roles. Infrastructure The Swammerdam Institute for Life Sciences is divided over two locations and looking forward to completion of the new planned building of the faculty in 2008/2009. The new green house complex was ready for use in the spring of 2006. Swammerdam Institute for Life Science 40

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    Annual Report 2006 Evaluation of the Swammerdam Institute for Life Sciences 5. Scientific Advisory Board Composition of the SILS Scientific Advisory Board Chairman: Dr N.C.M. Laane DSM Food Specialties Prof.dr P. de Wit Laboratory of Phytopathology Wageningen University Prof.dr J.P. Armitage Department of Biochemistry University of Oxford Prof.dr J. Garthwaite Wolfson Institute for Biomedical Research University College London Prof.dr C. Cremer Interdisciplinary Center for Scientific Computing Ruprecht-Karls-University Evaluation in 2006 On Thursday 20 and Friday 21 April the third meeting of the Scientific Advisory Board (SAB) of the Swammerdam Institute for Life Sciences took place. The SAB members were asked to advice the dean of the Faculty of Science and the director of SILS on the quality of the SILS’ research and research groups, in preparation for an external research evaluation. In a concluding session at the end of the second day the SAB and the director of SILS briefly discussed the main subjects that had caught the attention of the Board. The SAB feels two day visits every two years are the best way to evaluate SILS. The SAB felt very pleased with the research at the Swammerdam Institute. The morale of the groups is considered to be very good, and the staff is felt to be enthusiastic and professional in their research. This includes the PhDs and the postdocs the SAB has met. Swammerdam Institute for Life Science 41

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    Annual Report 2006 The general level of the research is very good (“pretty outstanding international level research”), and when science moves forward, in an active manner so do the researchers at SILS. The SAB recommends SILS to make a strategic plan. The SAB, as in the previous two evaluations, stresses their appreciation for internal interactions, and encourages the SILS management to stimulate these, and to stimulate joint publications. The SAB is of the opinion that some excellent interactions exist around the technology groups. The SAB feels the attention given to Systems Biology is very positive, and thinks it can help to further stimulate internal interactions. The SAB feels Systems Biology at SILS should be open for all groups to participate in. The SAB feels it currently cannot give much advice on Organisation and Finances, because the program concentrated on scientific matters. The committee members feel they would like to spend some time during the next visit on organizational matters, to be able to give advice again on these aspects. The SAB noted a very good level of personal grants with the staff of SILS, and feels this is well above the average in the Netherlands. The SAB commends SILS on this, and considers it a mark of good quality. The SAB feels National and International collaborations are essential for a research institute, and recommends SILS to keep investing efforts in maintaining and expanding its external collaborations. The SAB feels the number of collaborations SILS has is very good. The SAB is of the opinion it is very important to different disciplines, and has recommended SILS (also during its previous evaluations) to keep links with (bio)-chemistry, (bio)-physics and mathematics. The SAB realized the difficulty this can give because of the organizational and financial structure of the university, and the restraints this can give. The SAB welcomes the discussions that are being held in the framework of Systems Biology, and hopes this will strengthen SILS in these areas. Close interactions are needed between biology and mathematicians, and both kinds of scientists must be encouraged to be open minded. Swammerdam Institute for Life Science 42

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    Annual Report 2006 Peer Review: outcome at the Institute Level 6. Assesment of Research Quality The Peer Committee Prof.dr P.R. Cook (University of Oxford); Prof.dr W.P.M. Hoekstra (University Utrecht, Royal Academy of Arts and Sciences, Chair); Prof.dr J.G.R. Jefferys (University of Birmingham); Prof.dr D.B. Kell (University of Manchester); Prof.dr D. Scheel (Leibnitz Institute of Plant Biochemistry). Drs Klaas Deen acted as secretary. The Swammerdam Institute was assessed at the level of the research institute and its programmes. The assessment is based on a thorough self evaluation report by the institute and on interviews which the peer committee had with the management of the faculty, the director of the institute and the program leaders. Next to these the committee received a bibliometric study of the output from the institute. In their assessment the committee evaluated the past performance and whenever possible gave comments towards the future of the institute. The committee followed the instructions of the Standard Evaluation Protocol (SEP) in a strict way. With regard to the research programmes, the committee had given ratings for the ‘Quality’, ‘Productivity’, ‘Relevance’ and ‘Prospects’. The part of the report on the evaluation of the institute is represented below. Evaluation of the institute Quality: 4 Productivity: 3/4 Relevance: 4/5 Prospects: 4 Swammerdam Institute for Life Science 43

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    Annual Report 2006 Quality in order to act as a reputable research institute within a university that is proud to be acknowledged as research-university, the balance between research and The Swammerdam Institute for Life Sciences (SILS) started in 2000 as a result teaching is delicate. Fact is that a nominal teaching time of 0.5 is vastly in excess of implementing within the Science faculty a new model for organising research. of that of its international competitors. SILS is organised in four clusters, each encompassing a number of research groups. Although the clusters are certainly helpful for the organisation of the research, they are not the cornerstones of the institute. The very informative self evaluation and the open discussions with the group leaders during the site visit revealed that the various research groups and expertise centres are the real cornerstones of SILS. Although the committee met some group leaders with a slight reservation towards SILS as an appropriate umbrella for their research and their ambitions, it should be stressed that SILS is seen by the research leaders in general as a useful organisation within the faculty to cope with the opportunities and the threats for the life sciences. Within SILS, research groups and expertise centres of high quality are brought together. There are many groups with a very sound conceptual view on fundamental as well as on applied science. The concept of system biology is illustrative in this respect. The quality of the research is also shown by the fact that SILS is very successful in finding research funds, and especially the many prestigious personal grants are worth mentioning. Moreover, SILS is operating in a surrounding (AMOLF, CWI, AMC, NIN) that offers many opportunities for fruitful co-operations. There is a bright outlook for life sciences in Amsterdam and SILS could be very instrumental to realise this. However, the committee has noticed that most of the research groupings are comparatively small, and this can make it hard to be internationally competitive. Collaborations are therefore seen Relevance as an important part of the SILS portfolio for the future. The relevance of SILS as an institute where forefront research in life sciences is performed and where many efforts are done to stimulate public engagement with Productivity science is very good. The quality of the output is high, which was also confirmed by the extensive bibliometric analysis. Prospects Many SILS groups reach with their papers frequently top journals. However, the productivity is open for improvement. Although in general the productivity is The prospects for SILS are very good, although not unconditional. good to very good, it is quantitatively not up to the international standard. An explanation for this could be, as indicated by many group leaders, the heavy (and The present director of SILS was appointed in 2005. He has no personal research unevenly spread) teaching load they experience. Although the committee tends group, but brings in managerial expertise in academic organisations. It is too early to accept this explanation, it is very remarkable that hardly any one could give to evaluate his performance, but extensive talks during the site visit taught the an insight as to the factual teaching load. The committee wants to stipulate that committee that the director is well aware of the strengths and the weaknesses of Swammerdam Institute for Life Science 44

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    Annual Report 2006 the institute as well as of the opportunities and threats facing it. He clearly shows As a consequence of modern biology, data handling and modelling are very dedication and ambition to lead SILS, mainly by supporting the group leaders important. Within SILS there is a very active group dealing with these aspects, - respecting their authentic academic responsibilities - and by creating a more but there is a need for more. The committee believes it is not realistic to expect transparent and coherent organisation. that the BDA, with a part time group leader, is able to fulfil all the needs. Although there seem to be a lot of ideas about e-Bioscience, the committee could SILS is advised regularly by a Scientific Advisory Board (SAB). The SAB reports not find a clear and coherent vision in this respect. The committee feels that the reveal that the SAB advises are very instrumental for SILS. In retrospect it would problem of proper data handling and adequate modelling within SILS should be a have been nice if the committee during the site visit had an opportunity to main point for the future strategy. interview the chairman of the SAB. A main point for the future strategy is also the focus of the institute. Some groups The committee has spoken with a delegation from PhD students and post docs. feel that they heavily depend on external funding. To find more sources, they There the committee noticed that SILS has not yet succeeded to become visible as tend to expand their research in many different lines. This may cause lack of a coherent institute for these groups. Although the location of the institute at two focus. The committee advises SILS to develop a strategy where the groups focus different places certainly is a problem, the main problem seems that not enough their research mainly on the areas where SILS members are unique and really is done to bridge the gaps between the various clusters. Especially the Silly talks competitive. were blamed for not being very communicative. The committee strongly feels that SILS should look for more effective tools to inform young scientists about Another concern with regard to the funding deals with lack of flexibility. As it is what is going on in SILS. This could be very helpful for creating corporate now there is hardly seed money in SILS for an adequate and proactive strategic identity and could, even more importantly, serve as a vital aspect in the training policy. The committee believes it should be worthwhile to strive for more seed of future researchers. Regular seminar lectures, master classes, SILS symposia etc. money at the level of the institute. could, and in fact should, be organised SILS wide. There is no need to wait till the new housing is effective. Last but not least, SILS is very depending on highly advanced equipment. The committee met all over SILS great concern that it might be very difficult to keep up with the very expensive equipment development. This is indeed also in the view of the committee a point of great concern. Biology is Big Science now and universities and funding agencies need to recognize this. Swammerdam Institute for Life Science 45

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    Annual Report 2006 Appendix 1a Appendix 1 Molecular Microbial Physiology Chairholder: Prof.dr K.J. Hellingwerf Research Results Research Results in Numbers Peer reviewed publications 16 Non-peer reviewed publications 0 PhD Theses 0 Patent applications 0 Staff (Research input in fte during 2006) Klaas Hellingwerf Chairholder Jeroen Hugenholtz Honorary Professor (bijzonder hoogleraar) Joost Teixeira de Mattos Associate Professor Position FS1* FS2** FS3º Total Chairholder 0.5 0 0 0.5 Associate/Assistant professor 0.6 0 0 0.6 Research Fellow 0.9 0.9 0.35 2.2 PhD Student 0 1.5 1.5 3 Technician 0.6 0 0.5 1.1 Total 2.6 2.4 2.4 7.4 * FS1 = University Funding, ** FS2 = External funding, governmental grants º FS3 = External funding, e.g. EU grants, commercial funding Result research evaluation 2006 according to VSNU protocol Quality: 4/5 Productivity: 4 Relevance: 5 Prospects: 4 Swammerdam Institute for Life Science 46

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    Annual Report 2006 Publications Hellingwerf, K.J. (2006). On the mechanism of activation of the BLUF domain of AppA. Biochemistry, 45, 51-60. Key Publications Linden, H.P. van der, Burgdorf, T., De Lacey, A.L., Buhrke, T., Scholte, M., Fernandez, V.M., Friedrich, B., & Albracht, S.P.J. (2006). An improved Yeremenko, S., Stokkum, I.H.M. van, Moffat, K., & Hellingwerf, K.J. (2006). purification procedure for the soluble [NiFe]-hydrogenase of R.eutropha: new Influence of the crystalline state on photoinduced dynamics of photoactive insights into its (in)stability and spectroscopic properties. J.Biol.Inorg.Chem., yellow protein studied by ultraviolet-visible transient absorption spectroscopy. 11, 247-260. Biophys.J., 90(11), 4224-4235. Roseboom, W., De Lacey, A.L., Fernandez, V.M., Hatchikian, E.C., & Albracht, S.P.J. (2006). The active site of the [FeFe]-hydrogenase from D. desulfuricans. Avila-Perez, M., Hellingwerf, K.J., & Kort, R. (2006). Blue light activates the II. Redox properties, light sensitivity and CO-ligand exchange as observed by sigmaB-dependent stress response of Bacillus subtilis via YtvA. J.Bacteriol., infrared spectroccopy. J.Biol.Inorg.Chem., 11, 102-118. 188(17), 6411-6414. Stokkum, I.H.M. van, Gobets, B., Gensch, T., Mourik, M., Hellingwerf, K.J., Grondelle, R. van, & Kennis, J.T.M. (2006). (Sub)-picosecond spectral Academic publications (refereed) evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system. Photochem.Photobiol., 82(2), 380-388. Albracht, S.P.J., Roseboom, W., & Hatchikian, E.C. (2006). The active site of the Veening, J.W., Kuipers, O.P., Brul, S., Hellingwerf, K.J., & Kort, R. (2006). [FeFe]-hydrogenase from D.desulfuricans. I. Light sensitivity and magnetic Effects of phosphorelay perturbations on architecture, sporulation, and spore hyperfine interactions as observed by electron paramagnetic resonance. J.Biol. resistance in biofilms of Bacillus subtilis. J.Bacteriol., 188, 3099-3109. Inorg.Chem., 11, 88-101. Vengris, M., Larsen, D.S., Horst, M.A. van der, Larsen, O.F., Hellingwerf, K.J., Avila-Perez, M., Hellingwerf, K.J., & Kort, R. (2006). Blue light activates the & Grondelle, R. van (2006). Ultrafast dynamics of isolated model photoactive sigmaB-dependent stress response of Bacillus subtilis via YtvA. J.Bacteriol., yellow protein chromophores: “Chemical perturbation theory” in the 188(17), 6411-6414. laboratory. J.Phys.Chem.B., 109(9), 4197-4208. Bekker, M., Teixeira De Mattos, M.J., & Hellingwerf, K.J. (2006). On the role of Visser, W.F., Roermund, C.W.T. van, Hellingwerf, K.J., Waterham, H.R., & Two-component regulation systems in bacterial physiology. Science Progress, Wanders, R.J.A. (2006). First identification of a 2-ketoglutarate/isocitrate 89(3/4), 213-242. transport system in mammalian peroxisomes and its characterization. Biochem. Deutscher, J., Francke, C., & Postma, P.W. (2006). How phosphotransferase Biophys.Res.Commun., 348(4), 1224-1231. system-related protein phosphorylation regulates carbohydrate metabolism in Wilderen, L.J. van, Horst, M.A. van der, Stokkum, I.H.M. van, Hellingwerf, bacteria. Microbiol.Mol.Biol.Reviews, 70(4), 939-1031. K.J., Grondelle, R. van, & Groot, M.L. (2006). Ultrafast infrared spectroscopy Grinstead, J.S., Avila-Perez, M., Hellingwerf, K.J., Boelens, R., & Kaptein, R. reveals a key step for successful entry into the photocycle for photoactive (2006). Light-induced flipping of a conserved glutamine sidechain and its yellow protein. Proc.Natl.Acad.Sci.USA, 103(41), 15050-15055. orientation in the AppA BLUF domain. J.Am.Chem.Soc., 128(47), 15066- Yeremenko, S., Stokkum, I.H.M. van, Moffat, K., & Hellingwerf, K.J. (2006). 15067. Influence of the crystalline state on photoinduced dynamics of photoactive Grinstead, J.S., Hsu, S-TD., Laan, W.W.J., Bonvin, A.M.J.J., Hellingwerf, yellow protein studied by ultraviolet-visible transient absorption spectroscopy. K.J., Boelens, H.F.M., & Kaptein, R. (2006). The solution structure of the Biophys.J., 90(11), 4224-4235. AppA BLUF domain: Insight into the mechanism of light-induced signaling. ChemBioChem, 7, 187-193. Invited Lectures Hazra, P., Inoue, K., Laan, W.W.J., Hellingwerf, K.J., & Terazima, M. (2006). Tetramer formation kinetics in the signalling state of AppA monitored by Crielaard, W. (2006, oktober 10). Multispecies biofilm communities in the oral time-resolved diffusion. Biophys.J., 91(2), 654-661. cavity. Wageningen, Wageningen Center for Food Sciences. Laan, W.W.J., Gauden, M, Yeremenko, S., Grondelle, R. van, Kennis, J.T.M., & Crielaard, W. (2006, april 10). Response of Streptococcus mutans towards Swammerdam Institute for Life Science 47

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    Annual Report 2006 environmental stress and interaction of Streptococcus mutans with Veillonella Saccharomyces cerevisiae. Papendal, Voorjaarsvergadering Ned.Ver. parvula grown in dual species biofilm. Papendal, Wet.Voorjaarsvergadering Microbiologie NVVM/NVvM. Hellingwerf, K.J. (2006, maart 15). Systems microbiology. Amsterdam, Sils research day. Hellingwerf, K.J. (2006, april 11). Effects of phosphorelay perturbations (and light?) on architecture, sporulation and spore resistance in biofilms of Bacillus subtilis. Papendal, Annual NVvM meeting. Hellingwerf, K.J., & Avila-Perez, M. (2006, mei 01). On the biological function of YtvA from B.subtilis. Il Ciocco, Italy, Gordon Res.Conf.on Photosensory Receptors & Signal Transduction Hellingwerf, K.J. (2006, mei 11). Transient functional unfolding of photoactive yellow protein (PYP). Bedlewo, Poland, Workshop Polish Acad.of Sciences on the Structure and Function of Biomolecules II. Hellingwerf, K.J. (2006, juni 04). Signal generation in photoactive yellow protein (PYP). Hyogo, Japan, 12th Int.Conference on Retinal Proteins. Hellingwerf, K.J. (2006, oktober 05). Systems microbiology. Amsterdam, SILS Research Disc. on Systems Biology. Hellingwerf, K.J. (2006, oktober 14). From structural- and systems biology to ‘synthetic’ biology. San Feliu, Spain, ESF-EMBO Symp. “Bacterial networks”- Joining the strengths of structural-and systems biology to reach ‘synthetic’ biology. Hellingwerf, K.J. (2006, oktober 27). On signal transduction networks, biofilm formation and sporulation in Bacillus subtilis. Amsterdam, Autumn meeting MMP Section of NVvM. Hellingwerf, K.J. (2006, november 10). Photo-signaling. Wageningen, Capita selecta “Structure & Dynamics of biomolecules in vivo and in vitro, Wageningen University. Hellingwerf, K.J. (2006, november 21). Signal transduction, biofilm formation and sporulation in Bacillus subtilis. Vlaardingen, Progress report Unilever Research. Hellingwerf, K.J. (2006, december 14). Signal transduction networks, biofilm formation and sporulation in Bacillus subtilis. Rosario, Argentina, Lecture Universidad de Rosario, Inst. de Biol.Mol. y Cellular de Rosario. Hellingwerf, K.J. (2005, december 19). The mechanism of photo-activation of Photoactive Yellow Protein. Honolulu, USA, Pacifichem. Teixeira De Mattos, M.J. (2006, maart 19). On the redox state and size of the quinone pool in Escherichia coli. Tomar, Portugal, Meeting on Microbial Respiratory Chains. Teixeira de Mattos, M.J. (2006, april 11). Modelling the heat response of Swammerdam Institute for Life Science 48

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    Annual Report 2006 Appendix 1b Publications Key Publications Molecular Biology and Microbial Safety Chairholder: Prof.dr S. Brul Brul, S., Schuren, F., Montijn, R., Keijser, B.J.F., Spek, H. van der, & Oomes, S.J.C.M. (2006). The impact of functional genomics on microbiological food quality and safety. Int. J. Food Microbiol. 112, 195-199. Research Results in Numbers Peer reviewed publications 11 Smits, G.J. Schenkman, L.R., Ende, H. van den, Brul, S., Pringle, J.R. & Non-peer reviewed publications 1 Klis, F.M. 2006. Role of cell-cycle regulated expression in the localized PhD Theses 0 incorporation of cell-wall proteins in yeast., Mol. Cell. Biol. 17, 3267-3280. Patent applications 2 Klis, F.M., Boorsma, A., & Groot, P.W. de (2006). Cell wall construction in Saccharomyces cerevisiae Yeast 23, 185-202. Staff (Research input in fte during 2006) Stanley Brul Chairholder Patent applications Hans van der Spek Assistant Professor Gertien Smits Assistant Professor Beek, A.S. ter, Brul, S., & Vaart, M van (19-12-2006). Screening method for the Frans Klis Associate Professor (senior researcher) identification of a preservative. Keijser, B.J.F. (2006). Control of preservation by biomarkers. no EP 05077246.6. Position FS1* FS2** FS3º Total Chairholder 0 0 0.3 0.3 Academic publications (refereed) Associate/Assistant professor 0.5 0.5 0 1.0 Research Fellow 0 1.5 0.2 1.7 PhD Student 0.4 0 2.3 2.7 Albrecht, A., Felk, A, Pichova, I, Naglik, J.R., Schaller, M., Groot, P.W.J. de, Technician 3.3 0 0.3 3.6 Maccallum, D., Odds, F.C., Schafer, W., Klis, F.M., Monod, M, & Hube, B. Total 4.2 2.0 3.1 9.3 (2006). Glycosylphosphatidylinositol-anchored proteases of Candida albicans target proteins necessary for both cellular processes and host-pathogen * FS1 = University Funding, ** FS2 = External funding, governmental grants interactions. J.Biol.Chem., 281(2), 688-694. º FS3 = External funding, e.g. EU grants, commercial funding Brul, S., Schuren, F., Montijn, R., Keijser, B.J.F., Spek, H. van der, & Oomes, S.J.C.M. (2006). The impact of functional genomics on microbiological food. Int.J.Food Microbiol., 112, 195-199. Result research evaluation 2006 according to VSNU protocol Meijer, H.J., Vondervoort, P.J. van, Yin, Q., Koster, C.G. de, Klis, F.M., Govers, Quality: 3/4 F., & Groot, P.W.J. de (2006). Identification of cell wall-associated proteins Productivity: 3 from Phytophthora ramorum. Mol.Plant Microbe Interact., 19(12), 1348-1358. Relevance: 4 Pardini, G., Groot, P.W.J. de, Coste, A.T., Karababa, M., Klis, F.M., Koster, Prospects: 2/3 C.G. de, & Sanglard, D. (2006). The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans. J.Biol. Chem., 281(52), 40399-40411. Ram, A.F.J., & Klis, F.M. (2006). Identification of fungal cell wall mutants Swammerdam Institute for Life Science 49

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