Annual Report 2008 NCMLS

Annual Report 2008 NCMLS

Postal address 259 NCMLS P.O. Box 9101 6500 HB Nijmegen The Netherlands Visiting address Geert Grooteplein 28 6525 GA Nijmegen T: +31 (0)24 361 07 07 F: +31 (0)24 361 09 09 E: info@ncmls.ru.nl I: www.ncmls.eu Editing: Judith Burgers, Dr. Adrian Cohen Design: Final Design Front cover: An artistic image created by leakage of fluorescent FITC dextran from a dyed tumor, Prof. P. Friedl, Department of Cell Biology, NCMLS Photography: Dr. Adrian Cohen, Theo Hafmans Printed by: Thieme MediaCenter, Nijmegen 2 NCMLS

The 2008 annual report of the Nijmegen Centre for Molecular Life Sciences (NCMLS) provides an overview of education and research activities at the institute, and highlights a number of recent exciting als. Foreword The 2008 annual report of the Nijmegen Centre for Molecular Life Sciences (NCMLS) provides an overview of education and research activities at the institute, and highlights a number of recent exciting research discoveries published in top international scientific journals. This year has been particularly important for NCMLS for two reasons. In order to realise our ambitions in education and research, periodic evaluations of strategy and progress are a necessity. During 2008, NCMLS has taken the time to conduct an internal evaluation of its research and education activities and review changes implemented as a result of recommendations since the 2005 external evaluation. Looking at a wide range of aspects, such as the structure and coherence of research themes and international visibility of the institute, we were pleased with the outcome. The institute is built on a solid foundation of core departments and investigators providing ample opportunities for further expansion of the institute. All in all a positive step towards the external review in 2011, and renewal of the recognition of NCMLS as a Graduate School. In the words of Benjamin Disraeli, change is inevitable. Change for the better, of course. Termed Beter worden in het Radboud (literally translated as Getting better at the Radboud ), the Radboud University Nijmegen Medical Centre has critically reviewed its three core functions of patient care, research and education. The formation of new research institutes each with a clear mission and goals, in addition to NCMLS, provides an improved climate for inter-institutional collaborations between fundamental and clinical researchers, crucial for translational research. The new research infrastructure, together with a new financial model rewarding excellent researchers, is seen as a positive step forward in putting NCMLS on the international map. Other particular highlights for 2008 include: the appointment of Dr. Ronald van Rij as the second awardee of the NCMLS tenure-track research fellowship, demonstrating NCMLS as an attractive environment for talented young investigators; separate science retreats for PhD students, post-doc researchers and NCMLS principal investigators; and the NCMLS New Frontiers in Ubiquitination international one-day symposium that attracted over 350 scientists and clinicians from all over Europe to discuss the roles of ubiquitination in health and disease. Our 2008 annual report focuses on some of the younger researchers within the various research themes of our centre and their contribution to the understanding of the molecular and cellular basis of disease. Wishing you an enjoyable read, Carl Figdor Scientific Director NCMLS Annual Report 2008 Carl Figdor Foreword 3

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Table of contents Foreword 3 Nijmegen Centre for Molecular Life Sciences 7-24 Nijmegen Centre for Molecular Life Sciences 7-8 Mission 7 Introduction 7 Research and management organisation 8 Research 9-10 Research themes 9 Output 11 Social Impact & Selected Awards 12 New Frontiers in ubiquitination symposium 13 Scientific Retreats 14 PhD retreat 14 Post-doc retreat 14 Principal Investigator retreat 14 Molecule-2-Man 15 Technology Platforms 16 The NCMLS as Graduate School 17-19 Introduction 17 MSc Molecular Mechanisms of Disease 18 International PhD programme 19 NCMLS Members 20 NCMLS Committees 21-23 Selected Research Highlights NCMLS 2008 25-63 Theme 1: Infection, immunity and regenerative medicine 25-39 Theme 1a Infection and autoimmunity 26 Theme 1b Immune regulation 30 Theme 1c Tissue engineering and pathology 37 Page Theme 2: Cell metabolism, transport and motion 41-51 Theme 2a Energy and redox metabolism 42 Theme 2b Membrane transport and cell dynamics 46 Theme 3: Cell growth and differentiation 53-63 Theme 3a Genetic and epigenetic pathways of disease 54 Theme 3b Chemical and physical biology 62 Scientific publications 2008 65 Annual Report 2008 Table of contents 5

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The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals Nijmegen in the Centre interests forof Molecular curiositylife driven Sciences research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Mission Understanding the cellular basis of disease The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals in the interests of curiosity driven research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Introduction The NCMLS was first established in 1994 under the name Institute of Cellular Signalling (ICS), and subsequently in 1995 and again in 2000 recognised by the Royal Netherlands Academy of Arts and Sciences (KNAW) as a research school. In 2000 the name Nijmegen Centre for Molecular Life Science (NCMLS) was coined, the NCMLS and ICS organisations were merged and placed under a collegial board of directors to reflect a further integration of Nijmegen research themes and the multidisciplinary and translational research approach of the organisation. New approval for the new name and organisation for the NCMLS was obtained during the 2004 recognition-round by the KNAW. The research school continues to represent a growing research staff, both from the Radboud University Nijmegen Medical Centre (RUNMC) and the Faculty of Science, Mathematics and Computing Sciences (FNWI) at the Radboud University Nijmegen (RU). The NCMLS is a leading multidisciplinary research school within the domain of molecular mechanisms of disease and particularly in the fields of molecular medicine, cell biology and translational research. NCMLS aims to develop as a unique centre of excellence with innovative approaches to research and education. In order for the NCMLS to rank as one of the top European research institutes/graduate school, a competitive research environment should be created where quality and excellence are foremost considerations. By offering researchers a stimulating and challenging environment, NCMLS aims to foster talent and further increase international visibility. its research themes and in the Master s and PhD programmes offered. As such, NCMLS provides a stimulating and challenging environment for young scientists to learn the vital skills necessary for an independent and productive career in science. The NCMLS Graduate School operates a two-year Research Masters M.Sc programme, Molecular Mechanisms of Disease, accredited by NVAO, the Dutch-Flemish accreditation organisation, which is a member of the European Consortium for Accreditation (ECA). The NCMLS also runs a nationally approved PhD research training programme which attracts an increasing number of foreign students to the institute. NCMLS researchers collaborate at local, national and international levels. The research school is allied with the Institute for Molecules and Materials (IMM) and the Donders Institute for Brain, Cognition and Behaviour (DI-BCB), providing a solid platform for integrating the chemical synthesis, nanoscience and neuroscience with molecular life sciences. Furthermore, incorporating the Centre for Molecular and Biomolecular Informatics (CMBI) within the NCMLS has strengthened the multidisciplinary approach to solving biomedical research problems. The NCMLS houses The Netherlands Bioinformatics Centre (NBIC) which aims to stimulate the development of bioinformatics in the Netherlands, further strengthening NCMLS links in bioinformatics. The NCMLS also has associations with the Dutch Programme for Tissue Engineering (DPTE) and the Netherlands Proteomics Centre. The NCMLS contributes to the Top-Institute Pharma and has several academic and industrial partners in this context. In addition, the NCMLS is actively involved in national programmes, such as Centre for Societal Genomics (CSG), Center for Translational and Molecular Medicine (CTMM), Netherlands Genomics Initiative (NGI), and international multipartner programmes such as EU FP6/7. The major goal of the NCMLS is to generate basic knowledge in molecular medical research and to translate this knowledge into clinical applications, into the development of diagnostics and into the treatment of patients through translational research programmes. The multi-disciplinary approach to research in molecular life science is reflected both in Annual Report 2008 Nijmegen Centre for Molecular Life Sciences 7

The NCMLS seeks to achieve greater insights into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The NCMLS will pursue its goals Nijmegen in the Centre interests for of Molecular curiositylife driven Sciences research and education. The NCMLS aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. Research and management organisation Research and education are focused along three main research themes in keeping with our mission towards Understanding the cellular basis of disease : (a) Infection, immunity & tissue regeneration; (b) Metabolism, transport & motion; and (c) Cell growth & differentiation. These themes are further divided into seven sub-themes. NCMLS Principal Investigators are organised within these (sub)themes providing critical mass of expertise per subtheme and a visible structure for the institute on which to base science research and education policy. The NCMLS Research Council meets monthly and is involved with all decisions and policy-making with respect to NCMLS research and education activities. The Research Council comprises representatives of the three research themes, representatives of the seven sub-themes and representatives from the various NCMLS Committees. A number of NCMLS Committees are established, each focusing on particular aspects of NCMLS research and education. All Committees report to the Research Council, directly or via the Scientific Manager. The Scientific Director of the NCMLS institute, Prof.dr. C. Figdor, consults with the Dean of the Medical Faculty (Prof.dr. Frans Corstens) and the Dean of the Science Faculty (Prof.dr. Jan Kuijpers) during regular meetings in which research and education policy is discussed in relation to Radboud University and its Medical Centre research strategies. NCMLS PI retreat (see page 14) 8 Nijmegen Centre for Molecular Life Sciences NCMLS

Within the NCMLS institute, all research and education is linked to the study of molecular life sciences Research in relation to disease. NCMLS focuses on three thematic research areas, which are further divided into sub-themes. Each Research themes Within the NCMLS institute, all research and education is linked to the study of molecular life sciences in relation to disease. NCMLS focuses on three thematic research areas, which are further divided into sub-themes. Each subtheme and theme is headed by a prominent NCMLS Principal Investigator. Theme 1 Theme 2 Theme 3 Infection, immunity and Cell metabolism, transport and motion Cell growth & differentiation regenerative medicine (Adema) (Wieringa) (van Bokhoven) 1a: Infection and autoimmunity (Schalkwijk) 2a: Energy and redox metabolism (Wieringa) 3a: Genetic and epigenetic pathways of disease 1b: Immune regulation (Adema) 2b: Membrane transport and cell dynamics (van Bokhoven) 1c: Tissue engineering and pathology (van Kuppevelt) (Bindels) 3b: Chemical and physical biology (van Hest) Theme 1: Infection, immunity and regenerative medicine Infection and autoimmunity (Prof.dr. J. Schalkwijk) Immune regulation (Prof.dr. G. Adema) Tissue engineering and pathology (Dr. A. van Kuppevelt) The immune system has the task of eliminating pathogens and eradicating arising tumours, while preventing auto-reactive responses harmful to the host. In keeping the balance of this dual task, a complex interplay between immune cells and tissue cells exist and many stimulatory and inhibitory circuits operate simultaneously. The functional outcome is further shaped by genetic and environmental factors, such as pathogens and tumours. Deregulation of this intricate balance is directly associated with human diseases, ranging from inflammatory and autoimmune disorders to cancer, infection and transplantation disorders. In every case, prolonged deregulation can initiate a cascade of events ultimately leading to tissue damage and destruction. Tissue engineering is a relatively new field of research aiming at repairing or replacing damaged tissues applying implantation of smart synthetic biomatrices or stem cells. Immune control is intrinsically involved in these processes to allow acceptance- and prevent immune attack of engineered tissues. The central questions in theme 1 are dealt with in a multi-disciplinary approach (from molecule-2-man) and aim to define the molecular basis of: (i) events that trigger or fuel immune-related disorders and infectious diseases (sub-theme 1a); (ii) immune regulatory circuits (sub-theme 1b); and (iii) tissue pathology and regeneration and stem cell behaviour and differentiation (sub-theme 1c). Insight into immune regulatory and differentiation pathways, and how they are deregulated in diseases, allows the design of novel treatments leading to a translational research aim of (iv) designing novel clinical applications in inflammatory and autoimmune diseases (e.g. SLE, rheumatoid arthritis, diabetes, psoriasis), infectious diseases (e.g. fungi, viruses), transplantation (e.g. stem cell, kidney), immunotherapy of cancer and tissue regeneration (sub - themes 1a/b/c). Annual Report 2008 Research 9

Within the NCMLS institute, all research and education is linked to the study of molecular life sciences Research in relation to disease. NCMLS focuses on three thematic research areas, which are further divided into sub-themes. Each Theme 2: Cell metabolism, transport and motion Energy and redox metabolism (Prof.dr. B. Wieringa) Membrane transport and cell dynamics (Prof.dr. R. Bindels) Theme 3: Cell growth and differentiation Genetic and epigenetic pathways of disease (Prof.dr. H.van Bokhoven) Chemical and physical biology (Prof.dr. J. van Hest) In depth knowledge of the behaviour and fate of macromolecules, metabolites and ions, and the different pathways for intermolecular relationships, is required because the genome only (read: transcriptome and proteome) has been a relatively poor source of explanation for the differences between cells or between healthy and sick people. Study of disease at the small molecule level but in the context of the macromolecular world of the cellular organelles, the intact cell, or organs and tissues in the entire organism is therefore a central theme in the theme 2 research programme of the NCMLS. Intrinsic genetic problems or extrinsic factors causing cellular energy deprivation, ion and metabolite and water transport failure, toxic accumulation of intermediates, or ischemia and anoxia caused by cerebro-vascular obstruction are underlying a broad range of diseases, including for example cancer, neuropathy and myopathy, degenerative disorders like Alzheimer s and Parkinson, ischemic/anoxic organ failure, exercise intolerance and fatigue or renal tubulopathy and retinopathy. Also for very frequent problems like obesity and diabetes type II and even aspects of ageing it is now well established that there is a direct connection to metabolism and molecular transport and motion. Within its research theme 2 the NCMLS bundles studies in two particular relevant and strongly overlapping areas (a) Energy and redox metabolism and (b) Membrane transport and cellular dynamics. Between these topics links exist at many levels. Metabolites like ATP and NAD(P)(H) produced by in key pathways like glycolysis and mitochondrial respiratory complexes are consumed as fuel or needed as co-factors for ion-transport ATPases or drug-transporters and the acto-myosin motor machinery involved in organelle dynamics and cell movements. Renal disease, cardiomyopathy and brain and muscle disorders have now been demonstrated to be caused by defects in the production or assembly of ATPases, water channels, or the mitochondrial machinery. Also sometimes defects in metabolic signalling are involved. The NCMLS already provides unique facilities and a strong combination of knowledge in medical, physical and chemical sciences to work in this molecule and disease area, with direct diagnostic and therapeutic relevance. Our future efforts will be directed to creating an even stronger focus on the particular fields of strength, with emphasis on renal disorders, mitochondrial medicine, muscle dystrophies, neurodegeneration and cancer, particularly in overlap with studies within Themes 1 and 3. The fate of all cells lies in the fine balance between growth and differentiation. If this balance is disturbed, uncontrolled growth and deregulated cellular development can lead to disease. Studying the processes that underlie growth and differentiation is pivotal to a basic understanding of the causes of many diseases and malfunctions. Multi-level analysis is used to study the functional blueprint of all cellular decisions, a functional genomics approach is pursued that ranges from deciphering the genome in terms of actively transcribed genes under defined cellular circumstances (such as normal differentiation versus unregulated proliferation) to specific disease-linked genomic studies. Since the single cell cannot be viewed in isolation from its cellular surrounding, decisions within the cell need to be linked to external cues and constraints, and the translation of this approach within cells is at the core of research on signalling networks. In order to understand the molecules that convey the information packaged in the functional genomic blueprint as well as the signals from the cellular outside world, it is also necessary to gain a better understanding of the protein structure and design of these molecules that finally convey the growth and differentiation decisions. Valuable insights can be gained from investigating a specific differentiation programme and neural development is studied as a special case. Our research activities aim to: Unravel the molecular basis of cell behavior, which emanates from the genetic and epigenetic code contained in the nucleus in the context of health and disease (e.g. cancer, developmental disorders, mental handicap, cognitive impairments, neurodegenerative disorders and age-related bone diseases). Elucidate protein structure and protein-protein interactions within cellular signaling pathways that control cell proliferation and differentiation. Exploit the potential of molecular chemistry to modify, design and mimic proteins and their building blocks with the purpose to modulate and analyze their activities and properties in the cellular environment. 10 Research NCMLS

The output of an institute, measured in publications, prestigious awards, patent applications etc, is an Output important measure of the growth and performance of an institute. Furthermore, such information can be used to determine Output The output of an institute, measured in publications, prestigious awards, patent applications etc, is an important measure of the growth and performance of an institute. Furthermore, such information can be used to determine internal scientific policy, to facilitate comparisons with other similar research institutes and ultimately provide a source of quantitative information for institute evaluations. In recent years, NCMLS has been growing steadily with increasing numbers of yearly publications, from just under 500 in 2004 to more than 600 in 2008 (Source: Metis). Of course, the international relevance of these publications is also important and indeed may be quantitatively measured. All scientific journals have a so-called impact factor (Thomson Institute for Scientific Information), which is a measure of their international importance judged by a citation analysis of publications in the journal of interest. Ten journals were selected that have high impact factors (>10) and an analysis of the number of NCMLS publications per journal was conducted. Since 2004 there has been more than a three-fold increase in publications in these top journals demonstrating a continuing improvement in the external profile of NCMLS. The top three high-impact factor journals in 2008 for NCMLS publications were: Nature & Nature-derived journals, American Journal of Human Genetics and Blood. Publications within NCMLS are well distributed across the 7 different subthemes, ranging from 10% - 19% of the total NCMLS publications. Notably, Theme 1 contributes nearly half of all publications, whilst Theme 2 and Theme 3 contribute nearly one quarter and nearly one third of all publications, respectively. Annual Report 2008 Output 11

Various members of the NCMLS are funded by national and international patient-oriented nonprofit organisations, such as the Kidney Foundation, Dutch Cancer Society, the Diabetic Foundation, and the Rheumatoid Arthritis Societal Impact & Selected Awards Societal impact Various members of the NCMLS are funded by national and international patient-oriented non-profit organisations, such as the Kidney Foundation, Dutch Cancer Society, the Diabetic Foundation, and the Rheumatoid Arthritis Foundation. In addition, NCMLS members have advisory functions or are board members within these organisations or at the Royal Netherlands Academy of Arts and Sciences (KNAW). Moreover, clinical groups which are in daily interaction with patients or their relatives, inform patient organisations and are involved in public and strategic policy. Selected awards Dr. Frank van Kuppeveld (Dept. Medical Microbiology) and Prof.dr. Gosse Adema (Dept. Tumour Immunology) have been awarded 675.000 USD by the Juvenile Diabetes Research Foundation (JDRF) for the investigation of the interplay between enterovirus, pancreatic beta cells, and dendritic cells in the development of type 1 diabetes. On the 17th January, TI-Pharma announced a new project in the fight against malaria. The TI Pharma project has a budget of 16 million Euros over a period of 4 years and will be carried out by a large consortium that includes Prof. Robert Sauerwein, Dept. Medical Microbiology, NCMLS. This project aims to develop a highly protective malaria vaccine that would save the lives of millions of people in developing countries, especially infants and children. Dr. Ronald van Rij is the second person to receive the NCMLS tenuretrack research fellowship. Dr. Ronald van Rij is group leader in the section Virology at the Dept. Medical Microbiology and is interested in the mechanism of RNAi-mediated antiviral defense, its role in viral pathogenesis, and its relation to other defense mechanisms. Prof.dr. Roland Brock (Dept. Biochemistry) has been appointed as visiting professor at the Shanghai Medical College, Fudan University, China. Dr. Joost Hoenderop (Dept. Physiology) was awarded a ZonMw TOP grant of 675.000 to study the molecular regulation of the magnesium transporters in the kidney. Dr. J. de Vries (Dept. Tumour Immunology) was awarded a 500.000 subsidy for 2yrs within the ZonMW Translational Research pilot program. The project title is: Boosting the immune response to prevent cancer. Prof.dr. Carl Figdor (Dept. Tumour Immunology) & Prof.dr. John Jansen (Dept. Periodontology and Biomaterials) were elected as members of The Royal Netherlands Academy for Arts and Sciences (KNAW). Prof.dr. Peter Friedl (Dept. Cell Biology) received the prestigious German Cancer Award at the occasion of the 28th German Cancer Congress in Berlin. Dr. Ester Piek (Dept. Applied Biology) received a MEERVOUD grant from NWO. The MEERVOUD programme aims to help women postdocs become university lecturers. Prof.dr. Joost Schalkwijk (Dept. Dermatology) has recently been awarded the Wyeth Award ( 100.000) within the programme 'Advances in psoriasis research'. Dr. Frank Wagener (Dept. Pharmacology and Toxicology) was awarded a 500.000 grant from the Dutch Burn Foundation for a 4-year project to perform research entitled: Induction of cell protective proteins as novel strategy against oxidative stress- and inflammation-induced hypertrophic scar formation. The Veni, Vidi and Vici grants are awarded by the NWO as part of the Vernieuwingsimpuls (Renewal impulse) and is supported by the Ministry of Science, Education and Culture (OCW), and the Royal Academy of Sciences and the Arts (KNAW). A number of these grants were awarded to members of the NCMLS and will be the basis of important future research. The following 4 VIDI grants which were awarded to members of NCMLS in 2008. Dr. Annette Schenck (Dept. Human Genetics) received a VIDI award for the study of mental retardation, one of the largest unsolved medical problems of our time. Dr. Anneke den Hollander (Dept. Human Genetics) received a VIDI award for the study of macular (central part of retina) degeneration, the main cause of poor vision in the elderly. Dr. Ronald van Rij (Dept. Medical Microbiology) received a VIDI award for research immune defense mechanisms in the fruitfly (Drosophila). Dr. Jan Koenderink (Dept. Pharmacology & Toxicology) received a VIDI award to conduct research into the applications of digitalis-like compounds as future drugs. 12 Societal Impact & Selected Awards NCMLS

Continuing the success of the first annual New Frontiers symposium in October 2007, on the 1 st New Frontiers in ubiquitination December 2008 NCMLS held its symposium second symposium, entitled, New Frontiers in Ubiquitination. Over 350 people registered for this New Frontiers in ubiquitination symposium Continuing the success of the first annual New Frontiers symposium in October 2007, on the 1 st December 2008 NCMLS held its second symposium, entitled, New Frontiers in Ubiquitination. Over 350 people registered for this unique one-day symposium. The symposium brought together top researchers from the NCMLS, Europe and America to present and discuss their latest fundamental and clinical scientific research covering altered ubiquitin states in health and disease. The Scientific Director of NCMLS, Prof.dr. Carl Figdor, opened the symposium with a Welcome word followed by key-note speaker Prof.dr. Aaron Ciechanover who presented an overview of the discovery of the ubiquitin proteosome system and the role of abnormal intracellular protein degradation pathways in the onset of disease. Other invited speakers included Bill Tansay (Transcriptional Control and the Ubiquitin- Proteasome system), Jan Hoeijmakers (The involvement of ubiquitin and histone modification in nucleotide excision repair), René Bernards (Functions of de-ubiquitinating enzymes revealed through functional genetic screens), Vishva Dixit (Ubiquitin Ligases and DUBs in Cancer), Chris Lima (Structure and function in the SUMO pathway) and Hidde Ploegh (the symbiotic relationship of herpesviruses and ubiquitin). The day was brought to a close with the awarding of the Hans Bloemendal Medal for outstanding scientific achievement to Prof.dr. Aaron Ciechanover in recognition of his Nobel prize winning discovery and groundbreaking investigations of the ubiquitin degradation system. Professor Hans Bloemendal is considered as one of the first most prominent Dutch biochemists in Molecular Life Sciences. A medal with his name serves to honour him and other scientists who have shown to be eminent and unique. Plans are well underway for the NCMLS third New Frontiers symposium on Pattern Recognition Receptors: seeing the enemy, to be held on the 5 th & 6 th November 2009. Keynote speakers include Jules Hoffmann, Institut de biologie moléculaire et cellulaire, Strasbourg, Shizuo Akira, Laboratory of Host Defense, Osaka University, Japan and Michael Karin, School of Medicine, University of California, San Diego. Annual Report 2008 New Frontiers in ubiquitination symposium 13

On April 24 th & 25 th 2008, the annual PhD student conference was held at Hotel & Congress Scientific Retreats Centre Papendal in Arnhem. This year s organising committee consisted of Jeroen Geurts and Jenny Scientific retreats PhD retreat On April 24 th & 25 th 2008, the annual PhD student conference was held at Hotel & Congress Centre Papendal in Arnhem. This year s organising committee consisted of Jeroen Geurts and Jenny van der Wijst from the Departments of Rheumatology and Physiology, respectively. The current status and enthusiasm about the NCMLS PhD program was reflected by the high attendance of 103 participants. The kick-off on Thursday was a poster session, displaying the projects of 78 PhDs - mostly in their first two years of research. The afternoon was filled with nine excellent oral presentations of fourth-year PhDs from various departments such as Physiology, Haematology, Dermatology and Human Genetics. Mind and body were refreshed during an exciting match of Pitch & Putt, a variant of golf on miniature greens. After dinner, René Bernards Head of Molecular Carcinogenesis of the Dutch Cancer Institute, gave an inspiring and impressive lecture on the improvement of cancer diagnostics and treatment using genome-wide approaches. As a result of last year s success, the Friday morning program consisted of a poster walk session. The students were mingled such that small groups of six PhDs from different departments and variable stages of their project were formed. Each PhD student got the opportunity to showcase and discuss their scientific advances. In the afternoon, six selected third-year PhD students gave a lecture on their projects from several disciplines such as Cell Biology, Molecular Biology and Biochemistry. property rights. Of course there was science as well to reflect on and tips on how to publish articles. The retreat was a great success with over 55 post-docs participating. Plans are well underway for the 2009 Post-doc retreat, Joining Forces, planned on the 21 st and 22 nd April 2009. Principal Investigator retreat The first NCMLS Principal Investigator (PI) retreat took place on the 16-18th April 2oo8 on the island of Texel. Participation of two-thirds of all NCMLS PIs enabled fruitful discussions of science and strategy. Focusing on the Molecule-2-Man project, a central theme was Makea-Wish whereby PIs presented their views on science direction and required apparatus and infrastructure. The next retreat will be planned in three years time to coincide with the external evaluation of NCMLS institute. Post-doc retreat On 21 st and 22 nd January 2008 in Vught, NCMLS held its 7 th annual retreat specifically catering for post-doctoral researchers and final-year PhD students. The aims of the retreat were to improve the NCMLS postdoc network and to show researchers how they can get the most out of their research. An attractive program of speakers and training workshops, such as Theatre techniques in Science, Turbomanagement: the 80/20 concept, and Transferable skills, was put together by the NCMLS Post-doc Committee. How to give a great presentation, How to make quick decisions (the right ones!!) or What is the position of the post-doc? were just some of the themes discussed. The evening program entailed an informal discussion on topics concerning the scientific climate in the Netherlands, and the position of postdoc s herein. Discussions were led by Prof. Peter Peters, Netherlands Cancer Institute, and a panel of experts involved in science, science management and/or intellectual 14 Scientific Retreats NCMLS

Molecule to Man (M2M) is an innovative multidisciplinary imaging platform at the Radboud University Molecule-2-Man (RU) and the Radboud (www.molecule2man.eu) University Nijmegen Medical Centre (RUNMC). M2M is built upon the strengths of leading Molecule-2-Man (www.molecule2man.eu) A leading European imaging campus in Nijmegen Molecule to Man (M2M) is an innovative multidisciplinary imaging platform at the Radboud University (RU) and the Radboud University Nijmegen Medical Centre (RUNMC). M2M is built upon the strengths of leading Nijmegen institutes in molecules and materials, molecular lifesciences, cognitive neuroscience and medical sciences. Scientists have developed an impressive range of imaging techniques capable of examining objects ranging from the dimension of a molecule to that of the entire human body. Firstly, developments in molecular imaging allow us to analyse individual molecules and their physicalchemical properties. Secondly, 21st century microscopes have gone far beyond the realms of looking at a cell for mere illustrative purposes but now allow biologists to delve deep into the cell to look at molecular function. Importantly, cell biologists at NCMLS use this new instrumentation in combination with chemical and genetic techniques to visualise molecular behaviour in live-cells in vitro and in vivo. Thirdly, advancements in techniques for body and brain imaging offer scientists and clinicians new tools for analysing functions in living systems. The RU and RUNMC have identified imaging technologies as a key strength and a focal point of attention for current and future research and development strategies. The Nijmegen campus offers a unique research environment where advanced imaging infrastructure and knowledge are all concentrated within walking distance. The M2M platform provides facilities for the imaging of molecules, cells and living organisms and thereby can offer solutions for medical and societal problems. For example, through access to novel imaging biomarkers and a multitude of imaging techniques, M2M can help develop diagnostic methodologies for diseases like Alzheimer and cancer. Additionally, the platform offers opportunities to advance personalised health care, such as the development of tailor-made synthetic drug carriers and to facilitate clinical studies of new molecular and cellular therapies using the patient s own blood, tissue or bone marrow, as the source of therapy. Furthermore, the M2M platform has the facilities and expertise to investigate the determinants of human behaviour, from the genetic and molecular building blocks to the endophenotype of neuronal systems. Relevant application domains include, among other things, the development of personalised food and the improvement of speech and language technology. research institutes, Small and Medium Enterprises and other companies. Continuing investments in M2M as an innovative research and state-of-the art technology platform are crucial to keep Nijmegen, the Netherlands and Europe at the forefront of research and innovation. Partners within M2M: The IMM conducts research in the design, study and manipulation of the properties of functional molecules and materials. It is a highly interdisciplinary research institute comprised of twenty research groups, which are active in areas ranging from condensed matter nanoscience to (bio)chemical synthesis, working with unique facilities on molecular imaging. A large number of researchers within IMM have succeeded in starting their own knowledge-based spinoff companies, some of them employing hundreds of people. The NCMLS seeks to achieve a greater insight into the complexity of living cells with the purpose of obtaining a multifaceted knowledge of both normal and pathological processes. The institute pursues its goals in the interests of curiosity driven research and aims to advance innovation in translational research based on the integration of diverse scientific expertise in molecular and medical sciences. To reach these goals it offers state-of-the-art cellular and preclinical imaging facilities. Central in this environment is the Microscope Imaging Centre (MIC), which provides researchers from academia and industry advanced technology, competence and collaboration, integrated in a joint knowledge infrastructure within the Department of Cell Biology (Profs. P. Friedl and B. Wieringa, see www.ncmls.eu). The Nijmegen Centre for Clinical & Translational Medical Research together with its partners in Oncology and Infectious diseases conduct in vivo research in healthy subjects and in clinical trials, both crucial steps in translating emerging concepts in therapeutic strategies. The Departments of Radiology and Nuclear Medicine at the RUNMC play a core role offering both fundamental and clinical research on new radiopharmaceuticals for both diagnostic imaging and therapeutic purposes. The Donders Institute focuses on basic and clinical research on cognitive neuroscience in the broadest sense covering molecular, cellular, organ and behavioural levels involved in action, perception, memory and language. It consists of hundreds of researchers and has an excellent research infrastructure for neuroimaging. M2M provides the perfect platform for knowledge exchange, access to state-of-the art instrumentation and facilitates collaborations with Annual Report 2008 Molecule-2-Man (www.molecule2man.eu) 15

Animal models are of great importance to molecular life scientists for biomedical research. The NCMLS Technology has excellent Platforms links to the Central Animal Facility (CDL) for expert advice and access to facilities for animal testing. Further- Technology platforms Research facilities available to members of NCMLS may be grouped in the following categories: Animal models Animal models are of great importance to molecular life scientists for biomedical research. The NCMLS has excellent links to the Central Animal Facility (CDL) for expert advice and access to facilities for animal testing. Furthermore, The 3V Research Centre, a new office of the CDL, provides service to scientists regarding alternatives to animal use (reduction, refinement and replacement). NCMLS has several diseaserelated models available, for example arthritis, cancer, kidney disease, tissue engineering, heart transplantation, neural disorders, metabolic disorders, osteoporosis, haematopoiesis, fungal and bacterial septicaemia and malaria (P. falciparum). Molecular imaging Imaging at the (sub)cellular level is an essential tool for molecular life scientists. The Microscopic Imaging Centre (MIC) at the NCMLS is a state-of-the-art facility for imaging of biological specimens utilizing light microscopy (bright-field, confocal and fluorescence), conventional scanning and transmission electron microscopy, and sophisticated digital imaging. The facility is available for researchers within and outside the NCMLS. NCMLS also offers access to other techniques such as, Atomic Force Microscopy Flow cytometry, FRET and FRAP. Access to magnetic resonance facilities for in vivo NMR and MRI of animals and humans (7 Tesla), as well as multi-photon microscopy, are also available. Collectively, these techniques form part of the molecule2man imaging platform. Translational research and cellular therapy A GMP facility with clean rooms is used for translational research e.g. immunotherapeutic cell therapy, stem cell transplantation and gene therapy. In November 1997 the Departments of Tumor Immunology, Medical Oncology and Haematology collectively initiated the application of dendritic cell-based anti-cancer vaccines in melanoma patients. To date, more than 250 patients have been treated with this experimental form of therapy. Genomics DNA sequencing as well as micro-array technology for gene expression profiling are rapidly becoming standard everyday laboratory tools. The Microarray Facility Nijmegen is one of the core facilities of the RUNMC. The Department of Human Genetics also harbours a sequencing facility and a genotyping facility. The facility is focusing on multiple applications such as expression profiling, genomic copy number profiling (array CGH) and high density SNP profiling. Next-generation Genome Sequencers (Roche 454 FLX Titanium and Solexa) are operational in the Departments of Human Genetics and of Molecular Biology. Proteomics The growing availability of genomic sequence information, together with improvements of protein characterisation by mass spectrometry, facilitates protein research enormously. To exploit these opportunities the Nijmegen Proteomics Facility (NPF) was established in 2004. The stateof-the-art proteomics facility offers fundamental technological tools in proteomics research and makes them available for academic and industrial researchers, both within and outside the Radboud University Nijmegen and Radboud University Nijmegen Medical Centre. Equipment available includes 2D-electrophoresis, SELDI-TOF and Mass spectrometry (MALDI-TOF, MALDI-LTQ and nano-lc LTQ-FT MS). Bioinformatics The CMBI is the Dutch national centre for computational molecular sciences and is housed on the groundfloor of the NCMLS research tower. The institute pursues a rigorous research programme with topics ranging from computational small-molecule chemistry to protein function prediction and the complete metabolism of a cell. The Centre s facilities, databases and software packages are available to external scientists and there is a helpdesk for scientists who use the service facility. Currently, the CMBI is primarily involved in bioinformatics research and in maintaining a data and software infrastructure to help scientists improve bioinformatics and/or computational small-molecule research. 16 Technology Platforms NCMLS

The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where The NCMLS students as Graduate follow a School tailormade research programmes shoulder to shoulder with professionals. NCMLS graduate school Introduction The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where students follow a tailor-made research programmes shoulder to shoulder with professionals. NCMLS graduate school offers career possibilities at all levels, from Masters students to PhD and beyond in the form of post-doctoral research positions and tenure-track positions. The regulation of cellular processes is crucial for human development, and maintenance of health throughout life. It is clear that cellular malfunction affects common multi-factorial diseases such as diabetes, immune and inflammatory disorders, renal disease, cardiovascular, metabolic and neurodegenerative diseases as well as obesity and certain forms of cancer. In the fight against such diseases, the NCMLS Graduate School which is part of Radboud University Nijmegen and Radboud University Nijmegen Medical Centre plays a key role. A major goal of the NCMLS is to translate basic knowledge generated from biomedical research into clinical application, in order to improve diagnostics and develop new treatments. All MSc and PhD students are registered junior members of the research school and have the corresponding responsibilities and privileges. Tailor-made tuition Students are guided throughout the practical training period by a supervisor and throughout the entire programme by a mentor, who stimulates them to explore your abilities and develop general research competencies, including reflection. Together with these coaches, students draw up a personal training and supervision plan. Such a broad and interdisciplinary approach to research is particularly important in the international scientific arena. Excellent career prospects There is considerable demand for specialists in fundamental molecular biology and cell biology as well as in its application to the treatment of diseases such as cancer, autoimmune and inflammatory disorders, and metabolic and neurodegenerative disorders. Our Research Master s MSc training enables students to move rapidly into an international PhD programme, giving them a more mature perspective and a broader range of experimental approaches than is possible within standard MSc programmes. They are also prepared for further training as a PhD-level researcher. Graduates are exempted from certain elements of the NCMLS PhD programme. For example, the graduate course is taught during the MSc phase and the main practical project can be incorporated into a PhD project. Education is focussed is on the domain of molecular life sciences related to disease and in particular in three main fields related to molecular medicine, cell biology and translational research. Programmes are aligned along the three research themes. Annual Report 2008 The NCMLS as Graduate School 17

The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where The students NCMLS as Graduate follow a School tailormade research programmes shoulder to shoulder with professionals. NCMLS graduate school MSc Molecular Mechanisms of Disease The NCMLS offers a high-quality Masters programme in Molecular Mechanisms of Disease (MMD), which is taught by top researchers and clinicians. A unique and challenging programme This ground-breaking programme translates disease-related basic research in cellular and molecular biology into clinical experimental research in patients. Designed to meet the needs of talented students with the drive, motivation and ambition to push forward their scientific careers, it represents a unique opportunity to develop a research project and build up an international research network. This extremely competitive programme provides a sound balance of theory and practice. We enrol just 24 students per year, each of whom is allocated a personal mentor. The selection of the top ten percent of students from all over the world results in an ambitious, highly-motivated group of students in an international atmosphere. The programme is intensive, but stimulating, with plenty of room for discussion and interaction. This selective approach guarantees excellence, especially during the research training periods. The programme offers a focused combination of didactic and practical components suited to optimally synthesize current discoveries, conceptual breakthroughs and technological advancement mandatory for the effective education of the new vanguard of multidisciplinary investigators. Currently, 21 students from 11 countries are enrolled in the programme. The MSc programme The 2-year Master s programme, which starts each September, is modular in structure and focuses on the three NCMLS research themes: Infection, immunity and tissue repair Metabolism, transport and motion Cell growth and differentiation Theoretical courses For each theme, one 4-week long module focuses on the core fundamental and translational research aspects of the field. These courses provide a solid basis of theoretical knowledge and expose students to translational research, which links basic science and the treatment of disease. One of the highlights of the programme is a series of Master classes, which are given by leading authorities from renowned international institutes. These offer valuable insights both within and beyond your chosen specialist topics, explaining the current state of the art in important scientific areas. The programme trains students not only in theoretical knowledge, but is also focused on important scientific skills such as academic writing, giving scientific presentations and writing grant applications. These skills offer our students head start in their scientific career. Research training periods The practical research skills are further developed in the two research training periods. The first research training is performed in the NCMLS. In the second year, students perform an international internship at renowned institutes which is concluded with a thesis. Table 1: Courses and study load of the Master s programme Molecular Mechanisms of Disease (1 EC = 28 hours of study, total programme is 120 EC) Year 1 Year 2 Introductory course (1.5 EC) Genomics and Statistics (4 EC) Excellence in communication (1.5 EC) International Master class (1.5 EC) Science and society (1.5 EC) Elective courses (9.5) Infection immunity and tissue repair (5.5 EC) Metabolism, transport and motion (5.5 EC) Cell growth and differentiation (5.5 EC) International Master classes (3 EC) Elective courses (2 EC) Research internship (34 EC) Research internship (45 EC) Career opportunities The MMD Master s programme provides students with the qualifications to enter an international PhD programme. Our graduate students distinguish themselves through their high knowledge level and independent working attitude. About 90% of them enter a PhD programme in Nijmegen or elsewhere in the world. PhD scholarships are available for the best MMD students who can apply for their self-written research-project within the NCMLS, thereby allowing them to actively shape their own career and future. Eight students graduated in 2008, four of which cum laude. Three of these students have started their PhD studies in the Nijmegen Centre for Molecular Life Sciences institute. Two students have started their PhD in international institutes (Cambridge and Stockholm). Three students have started research jobs in industry or at (international) universities. www.ru.nl/master/ncmls-mmd 18 The NCMLS as Graduate School NCMLS

The Nijmegen Centre for Molecular Life Science (NCMLS) is the leading graduate school where The NCMLS students as Graduate follow a School tailormade research programmes shoulder to shoulder with professionals. NCMLS graduate school International PhD programme The aim of the NCMLS PhD training programme is to provide PhD students with a multifaceted education in the field of Molecular Life Sciences. NCMLS PhD students are offered an interdisciplinary training programme, containing both general and elective components. The general components are followed by all students, whereas the elective components allow a section of the NCMLS training programme to be tailor-made to complement the specific specialisation of the individual student. The training programme encourages both practical and theoretical participation in several NCMLS activities. The expected outcomes of the training programme are related to achievement of the following goals: The PhD student is capable of formulating a research problem, performing scientific research and has the communication skills to enable participation in scientific discussions at an international level. The PhD student has an overview of Molecular Life Science research and is capable of participating in multi-disciplinary research projects. The PhD student has the ability to communicate and co-operate with scientists from different disciplines close to his/her own field of research. The PhD student is able to write publications in international peerrefereed journals. Furthermore, the PhD student is able to present his/her work in the English language for an international scientific audience. Entrance requirements The training programme is accessible to PhD students from one of the research groups that are related to the graduate school. Students with a MSc degree, with a higher professional degree or with a similar degree from a foreign institute can enter the programme. Medicine, (Medical) Biology, Molecular Mechanisms of Disease, Chemistry, Physics, Mathematics, Computer Science and (Bio) Engineering are suitable preparatory fields of study. Because of the heterogeneity of the knowledge and interest of the students, a diverse education programme, in which many subjects will be taught, is provided. NCMLS courses will be awarded with the NCMLS certificate. To qualify for a NCMLS certificate, a total of 30 EC must be acquired during the 4 year PhD period, in addition to the requirements for PhD diplomas as formulated by the Radboud University. In four years, 17 EC should be earned within the general programme (Table 2). The remaining 13 ECs should be acquired through participation in elective elements. These can be either teaching elements (e.g. supervision of an undergraduate student, an oral or poster presentation at an official symposium, practical class for students, organisation of workshops) or scientific training elements (e.g. attendance of symposia and courses). Table 2: General elements, attended by all PhD candidates (1 EC = 28 hours of study, total is 17 EC) General components Short description PhD programme NCMLS Graduate course (3 EC) Two-week introduction to the NCMLS research themes, principal investigators and available state-of-the-art technologies. NCMLS Seminars (2 EC) Throughout the year, renowned international scientists provide seminars on their field of study. NCMLS Forum evenings (3 EC) NCMLS departments regularly present their research during themed Forum evenings. NCMLS Workshops (3 EC) Workshops deal with different topics (e.g. microarrays, career development, statistics immunology etc) for which NCMLS or external experts are invited. NCMLS Two-day retreat (4 EC) A yearly 2-day event in which all PhD candidates gather at Papendal (near Arnhem). PhD candidates in their final year present their work in oral presentations, other students present their work in posters. In addition, a keynote speaker is invited. NCMLS Symposium (2 EC) During this 1- or 2-day conference, lectures on the latest developments in a chosen field relevant to NCMLS are given by internationally renowned researchers. The NCMLS certificate Alongside practical training, PhD students are given the opportunity to broaden their skills through participation in specialised knowledge transfer courses. A certain number of courses are thought to be necessary in the development and education of each PhD student as independent researchers. Students that complete a full programme of Annual Report 2008 The NCMLS as Graduate School 19

NCMLS Members I: Infection, immunity and regenerative II: Metabolism, transport and motion III: Cell growth and differentiation medicine Ia: Infection and autoimmunity IIa: Energy and redox metabolism IIIa: Genetics and epigenetic pathways (Schalkwijk) (Wieringa) of disease (van Bokhoven) Berden Jo 1a Wieringa* Bé 2a Bokhoven*, van Hans 3a Berg, van den Wim 1a Barentsz Jelle 2a Brunner Han 3a Galama Joep 1a Heerschap Arend 2a Cremers Frans 3a Hermans Peter 1a Heuvel, van den Bert 2a Franke Barbara 3a Joosten Leo 1a Huynen Martijn 2a Geurts van Kessel Ad 3a Kullberg Bart Jan 1a Nijtmans Leo 2a Heesackers John 3a Kuppeveld, van Frank 1a Smeitink Jan 2a Hollander, den Anneke 3a Loo, van de Fons 1a Smits Paul 2a Hoogerbrugge Nicoline 3a Meer, van der Jos 1a Willems Peter 2a Kremer Hannie 3a Melchers Willem 1a Krieken, van Han 3a Netea Mihai 1a Logie Colin 3a Pruijn Ger 1a Lohrum Marion 3a Radstake Tim 1a Martens Gerard 3a Rij Ronald 1a Roepman Ronald 3a Sauerwein Robert 1a Schalken Jack 3a Schalkwijk* Joost 1a Schenck Annette 3a Verweij Paul 1a Schoenmakers Eric 3a Zeeuwen Patrick 1a Stunnenberg Henk 3a Veenstra Gert Jan 3a Veltman Joris 3a Ib: Immune regulation (Adema) IIb: Membrane Transport and Cell IIIb: Chemical and physical biology Dynamics (Bindels) (van Hest) Adema* Gosse 1b Bindels* René 2b Delft, van Floris 3b Boerman Otto 1b Deen Peter 2b Gielen Stan 3b Brock Roland 1b Drenth Joost 2b Hendriks Wiljan 3b Cambi Allesandra 1b Fransen Jack 2b Hest*, van Jan 3b Dolstra Harry 1b Friedl Peter 2b Leenders William 3b Figdor Carl 1b Hoenderop Joost 2b Leeuwen, van Jeroen 3b Hilbrands Luuk 1b Knoers Nine 2b Lubsen Lettie 3b Hoogerbrugge Peter 1b Koenderink Jan 2b Nolte Roeland 3b Jansen Joop 1b Kötter Rolf 2b Rowan Alan 3b Joosten Irma 1b Masereeuw Roos 2b Rutjes Floris 3b Leeuwen, van Frank 1b Russel Frans 2b Speller Sylvia 3b Oosterwijk Egbert 1b Theuvenet Lex 3b Punt Cees 1b Vriend Gert 3b Raymakers Reinier 1b Waal, de Rob 3b Reijden, van der Bert 1b Zoelen, van Joop 3b Vries, de Jolanda 1b Witte, de Theo 1b Ic: Tissue engineering and pathology (van Kuppevelt) Buma Pieter 1c Jansen John 1c Kraan, van der Peter 1c Kuppevelt*, van Toin 1c Torensma Ruurd 1c Vlag, van der Johan 1c Walboomers Frank 1c Wesseling Pieter 1c Bold: theme leader; * subtheme leader Our people from Faculty of Science 20 NCMLS Members NCMLS

NCMLS Committees 1. Research Council Research Council members Gosse Adema Rene Bindels Hans van Bokhoven Han Brunner Adrian Cohen Carl Figdor Martijn Huynen Nine Knoers Han van Krieken Jos van der Meer Ger Pruijn Floris Rutjes Joost Schalkwijk Jan Smeitink Bé Wieringa Department Tumor Immunology Physiology Human Genetics Human Genetics NCMLS Tumor Immunology CMBI Human Genetics Pathology General Internal Medicine Biomolecular Chemistry Synthetic Organic Chemistry Dermatology Pediatrics Cell Biology 2. MMD Educational Management Team MMD Educational Management Team members Gosse Adema Hans van Bokhoven Frans Cremers Martijn Huijnen Frank van Kuppeveld Marion Lohrum Clasien Oomen Marie Pohl Bé Wieringa Department Tumour Immunology Human Genetics Human Genetics CMBI Medical Microbiology Molecular Biology Institute for Education General Internal Medicine Cell Biology 3. MMD Student Selection Committee MMD Student Selection Committee members Gosse Adema Roland Brock Frans Cremers Peter Deen Wiljan Hendriks Jan van Hest Joost Hoenderop Martijn Huijnen Joop Jansen Frank van Kuppeveld Colin Logie Marion Lohrum Ronald van Rij Jo Zhou Department Tumor Immunology Biochemistry Human Genetics Physiology Cell Biology Organic chemistry Physiology CMBI Central Haematology Labs Medical Microbiology Molecular Biology Molecular Biology Medical Microbiology Human genetics Annual Report 2008 NCMLS Committees 21

NCMLS Committees 4. MMD Board of Examiners MMD Board of Examiners members Hans van Bokhoven Wiljan Hendriks Chantal Knibbeler Roos Masereeuw Ger Pruijn Johan van der Vlag Department Human Genetics Cell Biology Institute for Education Pharmacology & Toxicology Biochemistry Nephrology 5. MMD Programme Committee MMD Programme Committee members Alsya Affandi Aamir Aslam Lucas Brouwers Simon Kasper Ad Geurts van Kessel Peter van der Kraan Joost Schalkwijk Clasien Oomen Department (MMD student) (MMD student) (MMD student) (MMD student assessor) Human Genetics Rheumatology Dermatology Institute for Education 6. PhD Programme Committee PhD Programme Committee members Heleen Arts Wilbert Boelens Jeroen Geurts Bert van den Heuvel Joop Jansen Colin Logie Angelique Lokate Maaike Luesink Renoud Marijnissen Roos Masereeuw Laurens van der Meer Jeroen Middelbeek Chantal van de Schootbrugge Jenny van der Wijst Hanneke Wittgen Department Human Genetics Biomolecular Chemistry Rheumatology Paediatrics Central Haematology Labs Molecular Biology Biomolecular Chemistry Central Haematology Labs Rheumatology Pharmacology & Toxicology Central Haematology Labs Paediatrics Biomolecular Chemistry Physiology Pharmacology & Toxicology 7. Post-doctoral platform Committee Post-doc Committee members Sonja Buschow Alessandra Cambi Marieke Coenen Aneta Oziemlak Joost Schalkwijk Lisenka Vissers Department Tumour Immunology Tumour Immunology Human Genetics Central Haematology Labs Dermatology Human Genetics 22 NCMLS Committees NCMLS

NCMLS Committees 8. Subsidy Review Committee Subsidy Review Committee members Hans van Bokhoven Martijn Huynen Toin van Kuppevelt Gerard Martens Gert-Jan Veenstra Department Human Genetics CMBI Tissue Engineering Mol. Animal Physiology Molecular Biology 9. Seminar Committee Seminar Committee members Gert-Jan Veenstra Wilbert Boelens Hans van Bokhoven Ad Geurts van Kessel Werner Koopman Frank van Kuppeveld Angelique Lokate Department Molecular Biology Biomolecular Chemistry Human Genetics Human Genetics Cell Biology Med. Microbiology Biomolecular Chemistry 10. Principal Investigator Committee PI Committee members Gosse Adema Han Brunner Martijn Huynen Department Tumour Immunology Human Genetics CMBI 11. Fellowship Committee Fellowship Committee members Rene Bindels Hans van Bokhoven Ger Pruijn Joost Schalkwijk Peter Willems Department Physiology Human genetics Biomolecular Chemistry Dermatology Biochemistry Annual Report 2008 NCMLS Committees 23

24 NCMLS

Annual Report 2008 Theme 1 Infection, immunity and regenerative medicine

The Picornavirus family is a large group of small, non-enveloped RNA viruses that include important human and animal pathogens, Arjan de Jong e.g., polio-virus, rhi- Modification of host cell membranes by virus proteins This work was published in: de Jong A, de Mattia F, van Dommelen M, Lanke K, Melchers W, Willems P, van Kuppeveld F (2008). Functional analysis of picornavirus 2B proteins: Effects on calcium homeostasis and intracellular protein trafficking. J Virol 2008 82: 3782-3790. Understanding Picornavirus-induced alterations of the cell The Picornavirus family is a large group of small, non-enveloped RNA viruses that include important human and animal pathogens, e.g., poliovirus, rhinovirus, hepatitis A virus, and foot-and-mouth disease virus. Most of our knowledge of picornavirus replication and the function of individual viral proteins therein comes from studies on enteroviruses (members of one of its genera). Enteroviruses usually cause mild illnesses, however, they have also been implicated in both acute and chronic diseases like poliomyelitis, myocarditis and diabetes mellitus type 1 (juvenile diabetes). The enterovirus genome consists of positive strand RNA molecule of 7.5kb that contains a single large open reading frame, which is translated into a 220kDa precursor polyprotein. Cleavage by viral proteases generates the individual capsid and replication proteins; the latter mediate their functions directly or indirectly by modifying host cell functions and structures to create conditions required for RNA replication. Replication takes place in specialized compartments (replication complexes) present on the surface of membrane vesicles that accumulate in the cytoplasm of infected cells. Although multiple functions have been attributed to the individual viral proteins, their exact roles in RNA replication are not yet fully understood. The enterovirus 2B protein is a small (99aa) protein that contains two hydrophobic regions, of which the first is predicted to form a cationic amphipathic α-helix with characteristics of membrane-lytic polypeptides. In virus-infected cells, 2B is localized to the membrane vesicles of the replication complex. It has been implicated to play a role in vesicle accumulation, however, the mechanism(s) by which it modifies host cell membranes in order to do so are largely unknown. To understand the function(s) of enterovirus 2B, we investigated its properties in cells expressing 2B in the absence of other viral proteins. Biochemical and imaging techniques show that 2B is an integral membrane protein that forms homomultimers and is localized to ER and Golgi membranes. Expression of 2B results in depletion of Ca 2+ and H + from ER and Golgi lumen and in inhibition of protein transport through the Golgi apparatus. Both hydrophobic regions are essential for 2B properties and subcellular localization. Together, these data support our hypothesis that 2B forms pores in intracellular membranes. Moreover, retention of 2B in the ER abolished luminal Ca 2+ depletion and protein transport inhibition, identifying the Golgi apparatus as its target organelle. The nomenclature of picornavirus proteins is based on their position in the viral genome and does not necessarily imply a conservation of function between the proteins of different genera. To gain insight into the conservation of 2B functions, we investigated whether 2B proteins of other picornavirus family members, representing different picornavirus genera, alter organelle ion homeostasis and intracellular protein trafficking. We found that enterovirus and rhinovirus 2B have similar properties and most likely exert the same function(s) in RNA replication. The 2B proteins of other picornaviruses have a different subcellular localization and have little, if any, effect on Ca 2+ homeostasis and protein trafficking, suggesting that they have different function(s) in replication. Figure 1: (A) Using anti-egfp, 2B-myc was co-precipitated from cell lysates of 2B-myc and 2B-EGFP co-expressing cells and not from control lysates. ER retention of 2B by the KKAA retention signal did not affect homomultimerization. All proteins were expressed properly. (B) Luminal ER and Golgi Ca 2+ concentration was reduced by 15-20% in cells coexpressing the ER- or Golgi-targeted Ca 2+ indicator protein aequorin and 2B-EGFP, and not in EGFP-expressing control cells. ER retention of 2B abolished luminal Ca 2+ depletion. (C) Trafficking of VSV-G-GFP, a model protein that is transported via the secretory pathway to the plasma membrane, was inhibited in cells co-expressing 2B, resulting in VSV-G- GFP accumulation in the juxtanuclear Golgi area. ER retention of 2B abolished transport inhibition. Bar = 10 µm. All experiments were performed in BGM cells at 24 hours post-transfection as described in the paper. 26 Arjan de Jong Understanding Picornavirus-induced alterations of the cell NCMLS

Many biochemical processes in living cells are supported by macromolecular complexes. Enzymatic complexes involved in gene expression often are Sandy Mattijssen com- RNA metabolism This work was published in: Welting, TJM, Mattijssen, S, Peters, FMA, Van Doorn, NL, Dekkers, L, Van Venrooij, WJ, Heus, HA, Bonaf, L, and Pruijn, GJM (2008). Cartilage-hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly. Biochim. Biophys. Acta 1783: 455-466. Understanding the molecular basis for an inherited form of dwarfism RNase MRP assembly defects in cartilage-hair hypoplasia Many biochemical processes in living cells are supported by macromolecular complexes. Enzymatic complexes involved in gene expression often are composed of both RNA and protein molecules and are therefore designated ribonucleoprotein particles. The RNA component generally plays a crucial role in the catalytic process mediated by these particles. A striking example is the ribosome, which catalyses the formation of peptide bonds during protein synthesis. The ribosomal RNA is directly involved in the formation of these peptide bonds. The RNase MRP complex consists of a relatively small RNA component and 10 proteins. It is involved in the maturation of ribosomal RNA, the generation of certain RNAs in the mitochondria and the regulation of the levels of specific mrnas. RNase MRP acts as an endoribonuclease, i.e. an enzyme that cleaves substrate RNAs at specific sites. The recessively inherited disease cartilage hair hypoplasia (CHH) is caused by mutations in the gene encoding the RNA component of RNase MRP. To date, 64 different mutations have been found in the RNase MRP RNA gene in different patients. CHH patients are characterized by short stature (87-149 cm), thin hair and defective immunity. In addition, a predisposition to certain cancers, bowel disease and lung infections have been described. It is likely that all symptoms and health problems of CHH patients are resulting from a suboptimally functioning RNase MRP particle as a consequence of the mutations in its RNA component. assembly. Our data demonstrate that a number of nucleotide substitutions in this domain severely reduced the efficiency of its interaction with Rpp20 and Rpp25, two protein subunits which bind as a heterodimer to this domain. The CHH-associated 40G A substitution (the substitution of a guanosine by an adenosine at position 40), as well as the replacement of residue 47, almost completely abrogated Rpp20- and Rpp25-binding in different assays. Also other CHH-associated P3 mutations reduced the efficiency by which the RNase MRP RNA is bound by Rpp20 Rpp25. These data demonstrate that the most important residues for binding of the Rpp20 Rpp25 dimer reside in the apical stemloop of the P3 domain. Structural analyses by NMR not only showed that this region of the RNA probably folds into a pseudo-triloop structure, but also demonstrated that the 40G A substitution alters the folding of this part of the P3 domain. Taken together, these data indicate that the functional defect of RNase MRP in at least a number of CHH patients with mutations in the P3 domain is due to particle assembly defects resulting from improper folding of the RNA and less efficient protein binding. Our data are the first to provide insight into the molecular mechanism by which CHH-associated mutations affect the function of RNase MRP. Figure 1: The RNase MRP complex. The RNA component is in black and the proteins in blue. Of special note is the Rpp20 and Rpp25 dimer binding to the P3 domain of the RNA. How do these mutations affect the function of RNase MRP? We investigated the effect of mutations in the P3 domain, which is the evolutionarily most conserved part of the RNA, on protein binding, RNA folding and particle Figure 2: The pseudo-triloop structure of the apical P3 stemloop, the 40G A substitution alters the folding of this part of the P3 domain Annual Report 2008 Sandy Mattijssen Understanding the molecular basis for an inherited form of dwarfism 27

Psoriasis is a recurring skin condition that affects approximately 2-3% of the population. Onset of psoriasis can occur at any age, Patrick Zeeuwen although it is more like- Innate immunity and psoriasis This work was published in: Hollox EJ, Huffmeier U, Zeeuwen PL, Palla R, Lascorz J, Rodijk-Olthuis D, van de Kerkhof PC, Traupe H, de JG, den HM, Reis A, Armour JA, and Schalkwijk J (2008). Psoriasis is associated with increased beta-defensin genomic copy number. Nat Genet 40: 23-25. Psoriasis is associated with increased beta-defensin genomic copy number Psoriasis is a recurring skin condition that affects approximately 2-3% of the population. Onset of psoriasis can occur at any age, although it is more likely to appear between the ages of 11 and 45. The exact cause of psoriasis is unknown but is thought to be either a disorder of excessive growth and reproduction of the skin cells (keratinocytes) and/or a disorder of the immune system. Normally, a keratinocyte matures in 21 to 28 days during its passage to the surface of the skin where a constant invisible shedding of dead cells, scales, occurs. Psoriatic cells, however, are believed to turn over much quicker leading to an accumulation of visible dead cells at the skin surface. Psoriatic lesions appear as raised red patches of skin covered with silvery scales. Commonly affected areas include the scalp, elbows, knees, navel, palms, ears and groin. The cause of psoriasis is not known, but it is believed to have a genetic component. Gene expression changes in psoriasis lesions have been well documented, and strongly support an important role for TNF-α, IL-1 and Th17 cytokines in its pathogenesis. Several external factors are thought to aggravate psoriasis. These include stress, excessive alcohol consumption, and smoking. Psoriasis is a complex disease with complex underlying causes and although there are several (topical) treatments available, psoriasis still poses challenges to modern medicine. In this paper we describe a copy number variation of the beta-defensin locus. Betadefensins are small secreted antimicrobial peptides expressed by epithelia. In addition to their antimicrobial activity, beta-defensins are thought to have cytokine-like properties. Thus, either high resting levels or high induced levels of beta-defensins may be a precipitating factor after minor skin injury, infection or some other environmental trigger. This could lead to an inappropriate inflammatory response, generating the clinical symptoms typical of psoriasis (Figure 1). Healthy individuals have between 2 and 12 copies of beta-defensins per diploid genome, with a modal copy number of four in West- European populations. We analyzed the genomic copy number of the beta-defensin region on human chromosome 8 in 179 Dutch individuals with psoriasis and 272 controls and in 319 German individuals with psoriasis and 305 controls (Figure 2). Comparisons in both cohorts showed a significant association between higher genomic copy number for beta-defensin genes and risk of psoriasis. Figure 1: Lesional skin of a psoriasis patient showing the typical features of epidermal thickening and scaling, infiltration by lymphocytes and a high expression level of beta-defensin-2. Figure 2: Frequency distributions of beta-defensin genomic copy number. (a) Dutch controls and psoriasis cases. (b) German controls and psoriasis cases. 28 Patrick Zeeuwen Psoriasis is associated with increased beta-defensin genomic copy number NCMLS

Rheumatoid arthritis (RA) is an autoimmune disease manifested by chronic inflammation and cartilage and bone destruction in multiple Shahla Abdollahi-Roodsaz joints. Etiopathology of Toll-like receptors in arthritis This work was published in: Abdollahi-Roodsaz S, Joosten LA, Koenders MI, Devesa I, Roelofs MF, Radstake TR, Heuvelmans M, Akira S, Nicklin MJ, Ribeiro-Dias F, van den Berg WB (2008). Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis. J Clin Invest 118: 205-216. Understanding the role of Toll-like receptors in autoimmune T cell-mediated arthritis Rheumatoid arthritis (RA) is an autoimmune disease manifested by chronic inflammation and cartilage and bone destruction in multiple joints. Etiopathology of RA has been subjected to intensive research resulting in the knowledge that proinflammatory cytokines as well as T and B lymphocytes are implicated in the pathogenesis of RA; however, the exact factors regulating these parameters remain unclear. The discovery of Toll-like receptors (TLRs) as essential components of the immune system has introduced new candidates to the field of research on the pathogenesis of autoimmune diseases. TLRs are primarily involved in innate immune response to microbial pathogens through recognition of conserved pathogen-associated molecular patterns; however they also contribute to sterile inflammation by sensing the danger signals, i.e. endogenous molecules that are generated during tissue damage or inflammation. Activation of TLRs forms an important bridge between innate and adaptive immunity by regulating the expression of costimulatory molecules on antigen-presenting cells to drive T cell activation, and by creating a cytokine milieu to conduct the differentiation of T cells into the desired subset. Therefore, TLRs might contribute to the pathogenesis of RA by affecting both innate and adaptive immune response. We investigated the involvement of TLR2 and TLR4 in initiation and progression of arthritis using IL-1 receptor antagonist deficient (IL- 1rn -/- ) mice, which spontaneously develop an autoimmune T cell-mediated arthritis due to excessive IL-1 signaling. The initial observation that germ-free IL-1rn -/- mice do not develop arthritis at all, underscored possible involvement of TLRs in the disease process. Subsequently, IL-1rn -/- Tlr -/- mice were generated and compared to age- and gendermatched IL-1rn -/- Tlr +/+ littermates in terms of clinical, histological and immunologic parameters of arthritis. IL-1rn -/- mice lacking Tlr2 gene showed an enhancement of clinical and histopathological scores of arthritis, whereas IL-1rn -/- Tlr4 -/- mice were protected against severe disease. We showed that TLR2 downregulates the progression of arthritis through control of the function of regulatory T cells and regulation of IFNγ-producing Th1 cells (data not shown). TLR4, in contrast, contributed to more severe disease by supporting the highly pathogenic IL-23/IL-17 pathway. Synovial IL-23p19 and IL- 17 mrna expression was reduced in IL-1rn -/- Tlr4 -/- mice. Furthermore, the Th17% and IL- 17 production was substantially diminished in spleen and lymph node cells of IL-1rn -/- Tlr4 -/- mice (Figure 1). TLR4 stimulation of APC-T cell co-cultures in combination with neutralizing anti-il-1β antibodies revealed that TLR4-induced IL-17 production was mediated through IL-1. The differential and complex roles of TLR2 and TLR4 in defining the cytokine environment and T cell differentiation might have great implications for future interventions in (auto)immune disorders. The strict position of TLR4 upstream to a number of pathogenic cytokines including IL-17 provides an interesting therapeutic target for rheumatoid arthritis. Further evidence provided in this work indicated the presence of endogenous TLR4 ligands in RA synovial fluid and the involvement of TLR4 activation in spontaneous production of inflammatory cytokines by RA synovial tissue. This evidence emphasizes the clinical relevance of TLR4 activation in RA. Figure 1: (A) IL-1rn -/- Tlr4 -/- mice showed reduced histopathological characteristics of arthritis compared to IL-1rn -/- Tlr4 +/+ littermates as demonstrated in successive H&E and Safranin O-stained ankle joint sections. Open arrows indicate chondrocyte death and stealth arrows indicate bone erosion. IL-1rn -/- Tlr4 -/- mice exhibited diminished mrna expression of IL-23 p19 and IL-17A in synovial biopsies of the ankle joints (B, qpcr), lower Th17% in the spleen (C, FACS) and reduced IL-17 production by splenic T cells upon stimulation with anti-cd3/anti-cd28 (D, Luminex). Data are mean ± SEM. * P<0.05 and *** P<0.001. Annual Report 2008 Shahla Abdollahi-Roodsaz Understanding the role of Toll-like receptors in autoimmune T cell-mediated arthritis 29

Dendritic cells (DCs) are the most efficient professional antigen presenting cells (APCs). They can initiate immune responses Friederike Meyer-Wentrup against pathogens or C-type lectins This work was published in: Meyer-Wentrup F, Benitez-Ribas D, Tacken PJ, Punt CJ, Figdor CG, de Vries IJ, Adema GJ (2008). Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-alpha production. Blood 111(8): 4245-53. Understanding plasmacytoid dendritic cells Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-alpha production Dendritic cells (DCs) are the most efficient professional antigen presenting cells (APCs). They can initiate immune responses against pathogens or tumors, but can also prevent auto-immunity. DCs interpret their environment via pattern-recognition receptors (PRRs). Detection of pathogen-associated molecular patterns (PAMPs) results in production of distinct cytokines that direct T cell development. PAMPs are mostly essential for microbial survival and can therefore not easily be altered to evade immune recognition. DCs reside in lymphoid organs such as spleen and thymus, in peripheral blood and in bone marrow. Additionally, they are found at body surfaces in contact with the environment, such as skin and mucous membranes of lung and gut, from where they migrate to lymphoid tissues. In humans, DCs encompass at least two distinct subsets naturally occurring in vivo: myeloid and plasmacytoid dendritic cells (mdcs and pdcs, respectively). PDCs, also known as natural interferon (IFN)-producing cells, play an important role in the immune balance. Resting pdcs induce primarily regulatory responses, while activated pdcs secrete large amounts of IFN-α and -β. PDCs are crucial for initiating and sustaining anti-viral immune responses via NK and T cells. To identify microbial intruders they predominantly express the PRRs Tolllike receptor 7 (TLR7) and TLR9 allowing them to respond to single stranded RNA and DNA viruses. Still little is known about antigen uptake by pdcs. We have recently shown that pdcs can take up exogenous antigens and present them to CD 4+ T cells after Fcγ receptor II (FcγRII)-mediated uptake. Increasing evidence suggests that pdcs not only express TLRs for pathogen- recognition and FcγRII for uptake of opsonized antigens, but also C-type lectin receptors (CLRs). CLRs are PRRs that fulfill multiple functions within the immune system by recognizing carbohydrate moieties on foreign or (altered) self structures. CLRs on mdcs have been well characterized to combine ligand internalization with complex signaling events. Much less is known about CLR expression and function in human pdcs. In the present study we demonstrate that human pdcs express DCIR, a CLR with putative immune-inhibitory function. DCIR surface levels are reduced upon pdc maturation following TLR9 triggering. Interestingly, DCIR triggering inhibits TLR9-induced IFN-α production, while leaving DC activation unaffected. Furthermore, DCIR is readily internalized into pdcs after receptor triggering. We show that DCIR internalization is clathrin-dependent as it can be inhibited by hypertonic shock and dominant-negative dynamin. Our data demonstrate that endocytosis of the exogenous antigen KLH (keyhole limpet hemocyanin) via DCIR leads to efficient antigen presentation evoking a genuine recall response from fresh T cells isolated directly from peripheral blood of KLH-vaccinated melanoma patients. Antigen uptake via DCIR evokes a recall response as effectively as FcγRII-mediated endocytosis. The findings presented in this study indicate that targeting DCIR on pdcs not only results in efficient antigen presentation but also affects TLR9- induced IFN-α production. Our data support the concept of PRR cross-talk as a central element of signal integration by DCs. Collectively, we show that targeting DCIR can modulate human pdc function. These findings contribute to a better understanding of pdc biology and may ultimately help to treat immunemediated diseases. Figure 1: Cell surface DCIR is internalized into pdcs after receptor triggering. DCIR internalization is induced by receptor triggering with anti-dcir antibodies. PDCs were labeled with anti-dcir or anti-bdca-2 (a CLR known to be expressed by pdcs) antibodies, washed and incubated on ice with fluorochrome-conjugated secondary antibodies (blue). To visualize the cell surface pdcs were counter-stained with murine anti-mhc class I antibodies and isotype-specific fluorochrome-conjugated secondary antibodies (green). Cells were analyzed by CLSM. DCIR can be detected at the cell surface after antibody labeling at 4 C and is clearly internalized after elevating the incubation temperature to 37 C. Endocytosis of the BDCA-2 after receptor-triggering is detected as described in literature. Figure 2: Human pdcs present KLH to peripheral blood leukocytes (PBLs) after DCIR-mediated uptake. PDCs were freshly isolated and loaded with anti-dc-sign-klh and anti-dcir-klh conjugates, KLH alone or KLH together with patient-derived serum containing anti-klh antibodies (positive control, uptake via FcγRII). To block DCIR-mediated antigen uptake pdcs were also pre-incubated with anti-dcir mab prior to antigen targeting. PDCs were cultured with IL-3 and matured with CpG-C. Fresh PBLs and were added and thymidine incorporation was measured after 4 days of co-culture. DCIR targeting induces PBL proliferation clearly above background of pdcs loaded with KLH alone or anti-dc-sign-klh (*p 0.012 and **p 0.019 respectively). Proliferation can be blocked by preincubation with anti-dcir antibody (***p 0.051). 30 Friederike Meyer-Wentrup Understanding plasmacytoid dendritic cells NCMLS

The specific interference with protein-protein interactions inside a cell holds great promises, (i) in fundamental research to address the Ivo Ruttekolk role of individual Bioactive peptide-polymer conjugates This work was published in: Ruttekolk IR, Duchardt F, Fischer R, Wiesmüller KH, Rademann J, Brock R (2008). HPMA as a scaffold for the modular assembly of functional peptide polymers by native chemical ligation. Bioconjug Chem 19(10): 2081-7. Understanding the functional relevance of protein complexes in cellular signal transduction Cell-penetrating HPMA-peptide conjugates allow specific interference with protein-protein interactions The specific interference with protein-protein interactions inside a cell holds great promises, (i) in fundamental research to address the role of individual molecular interactions in signal transduction and (ii) in biomedical research for the interference with signaling pathways that are otherwise difficult to address pharmacologically. Peptides represent a highly attractive class of bioactive molecules for these purposes. Many protein-protein interactions are mediated by linear peptide motifs and the efficient disruption of protein complexes using synthetic peptides has been shown in vitro. Moreover, the activity and specificity of peptides can be easily modified by varying the amino acid sequence in combinatorial approaches as well as by rational peptide design. However, intracellular applications of peptides are complicated by the sensitivity of peptides to proteolytic break-down and by their poor cellular uptake. In our laboratory, we approach the functional analysis of protein-protein interactions in cellular signaling from several angles. Using peptide microarrays, detailed information on binding profiles of peptides for proteins in complex cell lysates is obtained (Mol. Cell Proteomics 6 (2007) 503-13). In order to achieve cellular import we have studied in detail the mechanisms by which so-called cell-penetrating peptides are taken up into cells (Traffic 8 (2007) 848-866). Here, we now joined both efforts using a polymer as a molecular link between bioactive and cell-penetrating peptides. Since both peptides are coupled as independent building blocks, both peptides are shorter and therefore easier to synthesize than one single peptide that is both biofunctional and cell-penetrating. Moreover, coupling of peptides to the polymer achieves a stabilization from proteolytic break-down, thereby increasing the activity-per-peptide. As a first model system we employed an apoptosis-enhancing peptide. This peptide corresponds to the seven N-terminal amino acid residues of the Smac protein. Smac is an important proapoptotic regulator protein that binds to the X-linked Inhibitor of Apoptosis Protein (XIAP). Thus, Smac interferes with one of the most prominent inhibitors of apoptosis and is therefore apoptosis-enhancing. The enhancement of apoptosis upon stimulation of Fas was measured by a cell viability test. The CPP nonaarginine and the fluorescentlylabeled bioactive Smac peptide were synthesized as independent building blocks and were subsequently combined on one polymeric backbone in a simple and robust one-pot synthesis protocol that does not require extensive expertise in organic chemistry. Coupling occurred to the thioester-preactivated polymer via N-terminal cysteine residues by Native Chemical Ligation, a highly chemoselective coupling procedure that excludes reactions by amino acid side chains within the bioactive peptide. As polymer, N-hydroxypropyl methacrylamide (HPMA) copolymer (28.5 kda) was chosen. This polymer is water-soluble, non-toxic, biologically inert and approved for in vivo pharmaceutical applications. The number of bioactive peptides per conjugate was determined by Fluorescence Correlation Spectroscopy (FCS) a technique based on confocal optics that requires only few nanogramms of the labeled constructs. FCS enables the measurement of concentrations and molecular sizes of fluorescently labeled molecules in the nanomolar range. We could show that the HPMA conjugate by itself was not toxic to the cells. Furthermore, uptake was strictly dependent on the presence of the cell-penetrating peptide. In summary, the conjugation of functional peptides to polymers reduced the synthetic effort, increased the biological activity of the bioactive peptide and allowed a modular assembly of different peptides on one polymeric backbone. In the meantime we have successfully extended the protocol to further bioactive peptides. We expect that this strategy will be highly valuable for gaining new insights into the function of cellular signaling networks and for target validation in preclinical drug development. Figure 1: The bioactive peptide and a cell-penetrating peptide (CPP) are combined on one polymeric backbone in a modular assembly by means of Native Chemical Ligation. The CPP renders the conjugate cell-permeable and enables a specific interference with protein-protein interactions inside the cell. In this case an apoptotic signal was significantly enhanced. Annual Report 2008 Ivo Ruttekolk Understanding the functional relevance of protein complexes in cellular signal transduction 31

Allogeneic hematopoietic stem cell transplantation (HSCT) has evolved to a specialized form of immunotherapy for treating hematological malignancies Wieger Norde like Cancer immunotherapy This work was published in: Norde WJ, Overes IM, Maas F, Vos JCM, Fredrix H, Kester MGD, van der Voort R, Jedema I, Falkenburg JHF, Schattenberg AV, de Witte TM, Dolstra H (2008). Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1-reactive cytotoxic T cells. Blood 2008, Dec 12 Understanding the mechanism of the graft-versus-leukemia response Allogeneic hematopoietic stem cell transplantation (HSCT) has evolved to a specialized form of immunotherapy for treating hematological malignancies like leukemia, malignant lymphomas, multiple myeloma and some solid tumors. The therapeutic efficacy is attributed to the graft-versus-tumor response, an immune reaction during which donorderived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. The remarkable strength of the response is best illustrated by the induction of clinical remission for residual and relapsed myeloid leukemia following post-hsct infusion of T lymphocytes from the original stem cell donor. Unfortunately, alloreactive CTL responses induced upon donor lymphocyte infusion (DLI) generally lack tumor specificity and are often accompanied by graft-versushost disease (GVHD). However, it has been demonstrated that graft-versus-leukemia (GVL) may occur without GVHD providing evidence that these alloimmune reactions can be separated from each other. One of the primary interests of our group is to identify target antigens that elicit selective GVL responses and to exploit them for specific immunotherapy after HSCT. The GVL reaction is due to the recognition of minor histocompatibility antigens (MiHA) by the donor immune system. MiHA arise from endogenous proteins in recipient cells differing from those in donor cells, because of genetic polymorphisms. Consequently, alloreactive donor T cells recognize recipient-specific MiHA as foreign antigens. MiHA that are ubiquitously expressed by recipient tissues have been implicated to induce GVL as well as GVHD. In contrast, MiHA with expression restricted to hematopoietic cells may induce GVL in the absence of GVHD. In our lab, a hematopoietic cell-restricted MiHA has been identified, designated LRH-1. This MiHA is not only expressed in normal hematopoietic cells, but also in the leukemic counterparts. Leukemia is regarded as a cancer stem cell disease, in which a population of dividing progenitor cells produce the malignant bulk of cells. Interestingly, LRH-1 expression was found in these leukemic progenitor cells. Therefore, targeting donor T cell immunity towards LRH-1 may lead to eradication of the primitive leukemia-initiating stem cells. In this report, we described the susceptibility of CD34+ leukemic progenitor cells to LRH-1 CTL-dependent lysis. Furthermore, in 43% of LRH-1 mismatched patients we found a LRH-1- specific T cell response in vivo. To determine if LRH-1-specific T cells specifically target progenitor cells and not their matured counterparts, we cultured chronic myeloid leukemia (CML) progenitor cells for one week with maturating cytokines, and performed cytotoxicity assays with these mature leukemic cells. Leukemic progenitor cells are efficiently targeted during co-culture with LRH-1-specific CTL, in contrast to matured cells which are not killed (Fig. 1A). On mrna level we could also show that levels of the MiHA mrna decreases upon maturation. In Figure 1B, we show that LRH-1-specific T cells selectively kill CML as well as acute myeloid leukemia (AML) progenitor cells within three days. Although CD34+ leukemic progenitor cells from most patients were susceptible to LRH-1 CTL-dependent lysis, we found resistant cells from one CML patient. We demonstrated that this resistance could be explained by an upregulation of the anti-apoptotic protein XIAP in these leukemic cells. Together, these data indicate that LRH-1 is an attractive target for eradication of leukemic progenitor cells. Targeted immune therapy after HSCT to specifically stimulate LRH-1-specific immune responses could increase the anti-leukemic effect and result in cure of leukemia. Figure 1: Myeloid leukemic CD34+ progenitor cells are lysed in a LRH-1 CTL-dependent manner, whereas maturated leukemic cells are not. (A) In a cytospin analysis we show that progenitor cells are killed within two days by LRH-1-specific CTL, whereas maturated leukemic cells are insensitive to LRH-1 CTL-dependent lysis. Both populations survive in the absence of T cells. (B) LRH-1 + CML and AML progenitor cells are efficiently killed after co-culture of three days with LRH-1-specific CTL (green), whereas in medium (blue) they proliferate well. HLA-specific CTL (red) were used as positive control. 32 Wieger Norde Understanding the mechanism of the graft-versus-leukemia response NCMLS

CD4+ T lymphocytes (T cells) are important mediators of the immune system in fighting invaders and tolerating self to prevent Hans Koenen autoimmunity. To en- T cell differentiation This work was published in: Hans JPM Koenen, RL Smeets, PM Vink, E van Rijssen, AMH Boots and I Joosten. Human CD25 high Foxp3 pos regulatory T-cells differentiate into IL-17 producing cells. Blood, 2008, 112(6): 2340-52 Understanding regulatory T cell differentiation Human regulatory T cell differentiation into Th-17 like cells CD4+ T lymphocytes (T cells) are important mediators of the immune system in fighting invaders and tolerating self to prevent autoimmunity. To ensure the efficient execution of these various activities, the immune system has distributed responsibility between different subsets of T helper (Th) and T regulatory cells (Treg). Treg are crucial in the control immune homeostasis and tolerance. Th-cells, by contrast, promote inflammation and immunity. The differentiation of T cells into effector T cells is characterized by the acquisition of new profiles of cytokine production. Over the past few years, there have been remarkable advances in our understanding of the regulation of T cell differentiation. The key areas of discovery have been identification of the cytokines, transcription factors and chromatin remodeling (epigenetic control) that regulate the function of distinct T helper cell subsets. Th17 cells, that produce their namesake cytokine IL-17 and are associated with the transcription factor RORγt, have evolved for host protection against extracellular bacteria and some fungi. Also, Th17 cells are implicated in many autoimmune and inflammatory pathologies. Last year it was established that human Th17 cells can differentiate from both naïve and memory CD4+ T cells when stimulated in the presence of pro-inflammatory cytokines. On top of this, in our current paper we report for the first time that human naturally occurring Treg can also differentiate into IL-17 producing cells. Human Treg can differentiate into IL-17 producing cells, when stimulated by allogeneic antigen presenting cells, especially monocytes, in the presence of IL- 2/IL-15. These Treg derived IL-17 producing cells showed high expression of the Th17- related transcription factor RORγt and were positively identified by CCR6 expression. This differentiation process was enhanced by exogenous IL-1β, IL-23, IL-21, while IL-6 or TGFβ did not affect the emergence of IL-17 producing cells. The addition of IL-1 receptor antagonist (IL-1Ra), but not anti-il-23 antibody, reduced IL-17 producing cell numbers. When a histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) was evaluated, we found a profound negative effect on the emergence of IL-17 producing cells from Treg. This implies that Treg differentiation into IL-17 producing cells depends on chromatin remodelling via histone/protein deacetylase activity. To date information on human Treg development and differentiation is limited. Importantly, we now demonstrate that human Foxp3+ Treg also possess an effector differentiation program resulting in IL-17 production. Our data suggest that epigenetic modification underlies this phenomenon. This flexibility in Treg differentiation programming may have evolved to anticipate local micro-environmental needs and serves to control the wide variety of immune responses that take place at distinct anatomical sites. Also, since with age, thymic egress of naïve cells is rapidly lost, this type of plasticity, whereby pre-defined lineage barriers are crossed, may in fact serve a more general phenomenon enabling homeostatic maintenance and optimal immune surveillance. This feature of Treg development can be exploited to facilitate vaccination strategies and tumor immunotherapy. However, on the downside it may have implications for the clinical application of ex vivo expanded Treg as part of tolerance induction strategies. Figure 1: Differentiation of CD25 high CD27 pos Treg into IL-17 producing cells. Flow cytometry of sorted CD25 high CD27 pos CD45RA neg CD4 pos Treg (left ) that were stimulated with allogeneic PBMC in the presence of IL-2 and supplemented with IL-1β, or IL-1 receptor antagonist (IL-1Ra). Density plots show intracellular IL-17 and Foxp3 expression at day 8 of the cultures after re-stimulation of the cells with PMA plus ionomycin in the presence of Brefeldin A. The percentages IL-17 positive cells are indicated at the top. Figure 2: Plasticity of regulatory T cells (Treg). In this piece of work we demonstrate that human CD4+Treg, like CD4+ naïve and memory T cells can differentiate. We have demonstrated that Treg can differentiate into IL-17 producing cells when stimulated with allogeneic antigen presenting cells (APC). The presence of IL-2/IL-15 is a prerequisite, supplementation with proinflammatory cytokines IL-1β, IL23 or IL-21 promotes this differentiation process. Studies by others have shown that differentiation of naïve and memory CD4+ T cells into IL-17 producing cells requires TGFβ plus IL-21 and IL-1β or IL-23 respectively [Yang-L, Nature 2008]. Annual Report 2008 Hans Koenen Understanding regulatory T cell differentiation 33

Dendritic cells (DCs) are highly specialized antigen presenting cells that play a central role in the activation of T cells. For this reason, Suzanne van Helden tumor-antigen loaded The dendritic cell cytoskeleton This work was published in: van Helden, S.F., M.M. Oud, B. Joosten, N. Peterse, C.G. Figdor, and F.N. van Leeuwen. 2008. PGE2-mediated podosome loss in dendritic cells is dependent on actomyosin contraction downstream of the RhoA-Rho-kinase axis. J Cell Sci. 121:1096-106. Understanding the biology of antigen-presenting cells Actomyosin contraction regulates podosome turnover and DC migration Dendritic cells (DCs) are highly specialized antigen presenting cells that play a central role in the activation of T cells. For this reason, tumor-antigen loaded DCs are used in cancer patients as a vaccine to enhance T-cell mediated anti tumor immunity. In response to antigen uptake and exposure to inflammatory stimuli, DCs undergo a remarkable transition from a tissue-resident, endocytic cell type to a highly migratory antigen presenting cell type, a process known as DC maturation. This phenotypical conversion not only involves up-regulation of MHC and costimulatory molecules, but is also accompanied by profound changes in cytoskeletal organization and cell adhesion. A prominent feature of immature DCs, which strongly interact with the extracellular matrix, is the presence of podosomes. These are highly dynamic adhesion structures consisting of an actin-dense core surrounded by a ring of cytoskeletal proteins and are known to connect integrins to the actin cytoskeleton. Although podosomes are generally considered important for migration and cell invasion, migration speeds in (podosome bearing) immature DCs are about 10-fold lower than those observed in mature DCs suggesting that in DCs they rather limit migration and play a role in cell adhesion. Moreover, high-speed migration of DCs, which is observed upon DC maturation - a prerequisite for efficient T cell activation in the patient -, is directly correlated with a loss of podosome structures. We previously showed that prostaglandin E2 (PGE 2 ), an important pro-inflammatory mediator produced during DC maturation and essential for the induction of full migratory capacity in DCs, induces podosome disassembly within minutes after stimulation. (Figure 1). Although PGE2 plays a key role in the DC maturation process, little is known about the signal-transduction pathways responsible for cytoskeletal remodeling during DC maturation. We demonstrate that PGE 2 induced podosome loss involves activation of the G protein-coupled receptors EP2 and EP4, which signal by raising camp levels. This increase in camp levels in turn affects activity of the small GTPases RhoA, Rac1 and Cdc42. important regulators of adhesion dynamics. Whereas PGE 2 stimulation leads to activation of the RhoA, decreased activities of Rac1 and Cdc42 are observed. Furthermore, PGE 2 -induced podosome dissolution involves activation of the RhoA-effector Rho kinase, as specific inhibition of this ser/thr kinase inhibits PGE 2 - induced podosome dissolution. It is well known that activation of the RhoA-Rho kinase pathway, affects adhesion dynamics by stimulation actomyosin-based contraction. Consistent with this notion, PGE 2 -induced podosome loss is accompanied by a redistribution of myosin II, while pharmacological inhibition of myosin II effectively interferes with podosome dissolution. We conclude that PGE 2 activates the RhoA-Rho kinase axis to promote actomyosin-based contraction and subsequently podosome dissolution (Figure 2). Because podosome disassembly is accompanied by de novo formation of focal adhesions, we propose that the assemby/disassembly of these two different adhesion structures is oppositely regulated by actomyosin contractility and relative activities of Rho GTPases. Together these experiments provide important new insights into how PGE 2 acts to enhance DC migration. Figure 1: Immature DCs display podosomes, which are lost upon PGE 2 stimulation while focal adhesions are formed. Immature DCs seeded on fibronectin-coated coverslips display podosomes that can be observed as actin dots (stained with phalloidin-texas Red to detect F-actin, red) surrounded by rings containing zyxin (green) (left image). Upon stimulation with PGE 2 for 5 minutes podosomes are dissolved and focal adhesions, seen as zyxin containing elongated spots (green), are formed (right image) Figure 2: Regulation of adhesion dynamics in dendritic cells by PGE 2 PGE 2 signaling, mediated by EP2 and EP4 receptors leads to elevation of the level of camp, activation of RhoA and inactivation of Rac1. PGE 2 -mediated activation of RhoA promotes Rho kinase activity and, subsequently, myosin II dependent contraction, leading to podosome dissolution and focal adhesion (FA) formation. By contrast, pathways leading to activation of Rac1 are known to promote podosome formation and induce cytoskeletal relaxation accompanied by a loss of focal adhesions. 34 Suzanne van Helden Understanding the biology of antigen-presenting cells NCMLS

Acute promyelocytic leu kemia (APL) is characterized by an excess of immature cells in the bone marrow that fail to differentiate toward Sake van Wageningen mature granulocytes. Acute promyelocytic leukemia This work was published in: Sake van Wageningen, Marleen C. Breems-de Ridder, Jeannet Nigten, Gorica Nikoloski, Claudia A. J. Erpelinck-Verschueren, Bob Löwenberg, Theo de Witte, Daniel G. Tenen, Bert A. van der Reijden, Joop H. Jansen. Gene transactivation without direct DNAbinding defines a novel gain-offunction for PML-RARα. Blood. 2008;111:1634-1643. Understanding differentiation-therapy in acute promyelocytic leukemia A gain-of-function for the PML-RARa oncogene Acute promyelocytic leukemia (APL) is characterized by an excess of immature cells in the bone marrow that fail to differentiate toward mature granulocytes. APL blasts can be forced to terminally differentiate using pharmacological doses of all-trans retinoic acid (ATRA). When treated with chemotherapy, APL patients can be cured in approximately 40% of the cases. The combination of ATRA with chemotherapy leads to a remarkably high cure rate of approximately 90%. In approximately 98% of the cases of APL chromosomal rearrangements are found that result in the fusion of the retinoic acid receptor gene (RARα) to the promyelocytic leukemia (PML) gene. The PML-RARα chimeric protein is involved both in transformation of APL cells and in differentiation in response to ATRA. Expression of the fusion protein in immature hematopoietic cells induced a maturation block at the promyelocytic stage. Inoculation of PML-RARα transduced bone marrow cells into irradiated syngenic mice resulted in the development of retinoic acid sensitive leukemia. RARα is a transcription factors that activate genes in a ligand-dependent manner. RARα binds to DNA as a heterodimer with RXR. In the absence of ligand, both RARa/RXR and PML-RARα bind corepressors such as N-Cor and SMRT and recruit histone deacetylases leading to gene silencing. In the presence of ligand, the corepressors are replaced by coactivators, leading to transcriptional activation. However, PMLRARα releases the corepressors at much higher concentrations of ligand compared with RARα. Since PML-RARα competes with unrearranged receptors for the same DNA binding sites, the presence of the fusion protein results in dominant silencing of retinoic acid receptor target genes at physiological concentrations of ligand. At higher, supraphysiological concentrations the fusion protein can still function as a transcriptional activator releasing the corepressor complex and allowing the transcription of genes that are important for granulocytic differentiation. The transcription factor inhibitors ID1 and ID2 are up-regulated upon treatment with ATRA and play a role in cell cycle arrest during APL differentiation. We investigated the mechanism by which these genes are regulated. We found that the ID1 and ID2 promoters are activated by PML-RARα but, unexpectedly, not by wildtype RARα/RXR. To test whether PML- RARα might transactivate the promoters without directly binding to the DNA, we used a PML-RARα construct from which the DNAbinding domain was deleted (PML-RARα R). PML-RARα R retained the ability to transactivate the ID1 promoter (Figure 1B). When the coiled-coil domain of PML-RARα was deleted (PML-RARα CC), it was no longer able to transactivate the ID1 promoter. As this domain is involved in protein-protein interactions, this suggested that interaction of PML- RARα with another protein was necessary for the transactivation of ID1. We found that PML- RARα activated the ID1 and ID2 promoter by binding the transcription factors Sp1 and NF-Y (Figure 1C), defining a gain-of-function for the oncoprotein. A B C Figure 1: (A) APL cells (upper panel) and non-apl cells (bottom panel) were stained with anti-pml specific antibody. PML is found in the nucleus of every cell and is localised in about 10 dots, called PML nuclear bodies. PML(-RARα) is dispersed in APL cells into smaller dots, called microspeckles. (B) Hep3B cells were transfected with an ID1 promoterluciferase reporter construct (ID1-luc) together with a control vector expressing Renilla luciferase. In addition, vectors coding for PML-RARα, the DNA-binding defective PML- RARα R mutant, and a mutant lacking the coiled-coil protein-protein interaction domain of PML (PML-RARα CC)) were co-transfected. Transactivation is expressed as arbitrary units and is corrected for transfection efficiency measured by Renilla luciferase. Mean values of 3 independent experiments are shown. (C) Dominant-negative and gain-of-function model for PML-RARα. Genes that are regulated through a retinoic acid responsive element (RARE) may be bound by PML-RARα (1). Competition with normal, unrearranged retinoid receptors results in a dominant-negative silencing of the gene by PML-RARα in the absence of ligand. Addition of high-dose retinoic acid may reverse the silencing, allowing transcription. Sp1- and NF-Y regulated genes may be targeted by PML-RARα through interaction with Sp1 (2). Tethering of PML-RARα to these promoters renders them responsive to retinoic acid, representing a gain-of-function for the PML-RARα fusion protein. Annual Report 2008 Sake van Wageningen Understanding differentiation-therapy in acute promyelocytic leukemia 35

Plasmacytoid dendritic cells (pdcs) are a major subset of dendritic cells in human peripheral blood that, upon stimulation by TLR ligands, Daniel Benitez-Ribas produce large amounts Immunobiology This work was published in: Benitez-Ribas D, Tacken P, Punt CJ, de Vries IJ, Figdor CG. Activation of human plasmacytoid dendritic cells by TLR9 impairs Fc gammarii-mediated uptake of immune complexes and presentation by MHC class II. J Immunol 2008 Oct 15; 181(8): 5219-24. Regulation of antigen presentation by human plasmacytoid dendritic cells Plasmacytoid dendritic cells (pdcs) are a major subset of dendritic cells in human peripheral blood that, upon stimulation by TLR ligands, produce large amounts of type I IFN and mature into competent antigen presenting cells. Human pdcs exploit Ag uptake receptors like CD32a or DCIR for internalization of exogenous Ags. Activation of pdc by TLR9 agonists induces strong maturation. Different classes of TLR9 agonists affect pdcs in distinct ways. CpG-A oligonucleotides (ODN) localize to early endosomes and induces IFN-α production and hardly affects the phenotype, CpG-B ODN localize to late endosomes and induces pdc maturation, and CpG-C ODN can mediate both effects in both locations. We assessed whether these TLR ligands could also affect the ability of pdcs to present exogenous Ags to T cells. We found that treatment of pdcs with CpG-C inhibited pdc uptake and presentation of immune complexes via CD32a without altering CD32a cell surface expression, phenotype, type I IFN secretion and without affecting the capacity of pdcs to induce an alloresponse. Prevention of endosomal acidification with Chloroquine restored CD32a-mediated immune complex internalization, suggesting that TLR9 stimulation was required for the observed effects of CpG-C. Analysis of different classes of CpG ligands indicated that CpG-B or CpG-C in the late endosomes inhibited CD32a-mediated Ag uptake and presentation, whereas CpG-A in the early endosomes had no effect. TLR9 agonists not only determine the type of response, i.e., type I IFN production (innate immunity) or maturation (adaptive immunity), but also directly affect Ag presentation capacity of pdcs. We hypothesize that pdcs, once activated via TLR9-ligands reaching the late endosomes, can only present initially sampled Ags and thus are protected from uptake and processing of additional potential self-ags in an immunogenic status. Specific TLR9 agonist can therefore directly modulate pdc Ag presentation by direct crosstalk with receptors expressed on the membrane. Our speculation was that down-modulation of Ag-presenting capacity upon pdc maturation, which has functional consequences, might prevent responses to self-ags, protecting against autoimmune disorders. Figure 1: Model of antigen uptake inhibition Upon CpG-C activation, pdcs initiate their transition from pre- to plasmacytoid-derived DCs. Endocytotic molecules like BDCA-2 are downregulated resulting in a decreased antigen uptake capacity. In our study we show that activation of pdcs does not lead to CD32a downregulation but the receptor is inactivated. This inactivation impairs further exogenous antigen uptake and presentation in MHC class II. The inhibitory effect using CpG-B and CpG-C can be linked to the NF-κB pathway associated with the late endosome signaling. 36 Daniel Benitez-Ribas Regulation of antigen presentation by human plasmacytoid dendritic cells NCMLS

A poor prognosis of cancer is primarily associated with metastasis of malignant cells from the primary tumor. Metastasis is a selective process Gerdy ten Dam involving Chondroitin sulfates This work was published in: Li, F*, Ten Dam*, GB, Murugan, S, Yamada, S, Hashiguchi, T, Mizumoto, S, Oguri, K, Okayama, M, Van Kuppevelt, TH, Sugahara, K. (2008). *These authors contributed equally to this work. Involvement of highly sulfated chondroitin sulfate in the metastasis of the lewis lung carcinoma cells (2008). J Biol Chem 283(49): 34924-304. Understanding the role of chondroitin sulfates in cancer Highly sulfated chondroitin sulfate sequences can inhibit cancer metastasis A poor prognosis of cancer is primarily associated with metastasis of malignant cells from the primary tumor. Metastasis is a selective process involving extravasation from the primary tumor, embolization, survival in the circulation, arrest in distant capillary beds, and extravasation into, and multiplication within the target organ. In the process of metastasis, tumor cells are involved in a series of interactions with the surrounding extracellular matrix (ECM) and with non-tumor cells such as platelets and endothelial cells. These interactions are modulated by proteoglycans (PGs), which bear the anionic polysaccharide heparan sulfate (HS) or chondroitin sulfate (CS)/dermatan sulfate (DS) side chains and are widely expressed on the cell surface and in the ECM. It has been well documented that PGs with HS side chains play important roles in metastasis. In addition to HS-PGs, CS/DS- PGs also have been implicated in the metastatic process. In this study we have focused on the role of highly sulfated structures in CS/DS chains and their involvement in the process of tumor formation. A structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)- derived cell lines with different metastatic potential, revealed a higher proportion of disulfated disaccharides (GlcA-GalNAc(4, 6-O- S)) in highly metastatic LM66-H11 cells compared to low metastatic P29 cells. In addition, immunocytochemical analysis showed that LM66-H11, rather than P29 cells, strongly expressed a highly sulfated CS epitope (CS-E), using the phage display antibody GD3G7, which specifically recognizes CS-E. This key finding prompted us to study the role of disulfated units (CS-E) in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS, and by preadministration of CS rich in E-units in a dosedependent manner. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66- H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that disulfated CS (CS-E) is involved in the metastatic process and is a potential target for the diagnosis and treatment of malignant tumors. Figure 1: Removal and blockage of highly sulfated chondroitin sulfate reduces tumor metastasis Lewis lung carcinoma cells LM66-H11 that strongly express highly sulfated chondroitin sulfate (CS-E), were treated with chondroitinase ABC (ABCase) to digest all CS (A), various CS preparation (100µg/mouse) (B), and antibody GD3G7 directed against highly sulfated CS (C). Treated cells were injected in the tail vein of the mice and number of lung foci was recorded after 21 days. Note the reduced number of lung foci (arrow) after treatment with ABCase (A), highly sulfated CS (B), and antibody GD3G7 (C). MPB49V: control antibody; CS-A CS-D: average sulfated chondroitin/dermatan sulfates; CS-E: highly sulfated chondroitin sulfate. Annual Report 2008 Gerdy ten Dam Understanding the role of chondroitin sulfates in cancer 37

Cytokines are small proteins that are responsible for the communication between different cells. Cytokines form a large family Ruurd Torensma of proteins that have Glycosaminoglycans modulate IL-4 This work was published in: den Dekker E, Grefte S, Huijs T, ten Dam GB, Versteeg EM, van den Berk LC, Bladergroen BA, van Kuppevelt TH, Figdor CG, Torensma R (2008). Monocyte Cell Surface Glycosaminoglycans Positively Modulate IL-4-Induced Differentiation toward Dendritic Cells. J Immunol 180: 3680-3688. Monocyte Cell Surface Glycosaminoglycans Positively Modulate IL-4- Induced Differentiation toward Dendritic Cells Cytokines are small proteins that are responsible for the communication between different cells. Cytokines form a large family of proteins that have overlapping functions but also effects on a single cell type. Distinct combinations of several members of the family direct the outcome for the target cells. Most cytokines are secreted by cells and end up in the extracellular space. Unhindered diffusion of the cytokines would limit their effect due to low concentration but also makes their effect less controllable. Indeed, it was demonstrated that the extracellular matrix traps cytokines. Extracellular matrix molecules represent one of the most versatile systems in living organisms. This is accomplished by sulfation of the carbohydrates of the glycoproteins on the cell surface. By modulation of the sulfate groups the specificity for cytokines is adapted. We hypothesized that specific cytokines are trapped by specific glycosaminoglycans (GAGs) in the extracellular space. A scan of monoclonal antibodies directed to different GAGs showed an organized expression pattern in bone marrow. Likewise, the development of the different blood cells from the hematopoietic stem cell appeared to be confined to distinct areas. Correlating the expression of special GAGs to developmental stages of blood cells was too tough. Therefore, we focused on the development of a monocyte towards a dendritic cell. IL-4 induces the differentiation of monocytes towards dendritic cells (DCs). If the activity of cytokines is modulated by glycosaminoglycans a spatial distribution of GAGs is to be expected. In this study we explored the effect of GAGs on the IL-4-induced differentiation of monocytes towards DCs. IL-4 dose-dependently up-regulated the expression of DC-SIGN (CD209), CD80, CD206 and CD1a. Monocytes stained positive with antibodies against heparan sulfate (HS) and chondroitin sulfate B (CSB, dermatan sulfate), but not with antibodies that recognize chondroitin sulfate A (CSA), chondroitin sulfate C (CSC) and chondroitin sulfate E (CSE). Inhibition of sulfation of monocyte/dc cell-surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing antibodies. This correlated with hampered IL-4-induced DC-differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell-surface GAGs support IL-4 activity. Furthermore, removal of cell-surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O-sulfates (CSB and heparin). Together, these data demonstrate that monocyte cellsurface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs. Figure 1a: Different blood cells develop in clusters in bone marrow. Figure 1b: Spatial expression of low sulfated glycoproteins in bone marrow. Figure 1c: Spatial distribution of GAGs and developing blood cells. The circles show erythrons. 38 Ruurd Torensma Monocyte Cell Surface Glycosaminoglycans Positively Modulate IL-4-Induced Differentiation toward NCMLS Dendritic Cells

The application of oral implants has been considered as a wellaccepted treatment modality to replace missing and lost teeth. Dimitrios Nikolidakis However, there is skep- The effect of TGF-β1 on the bone-healing around oral implants This work was published in: Nikolidakis D, Meijer GJ, Oortgiesen DA, Walboomers XF, Jansen JA (2008). The effect of a low dose of transforming growth factor beta1 (TGF-beta1) on the early bone-healing around oral implants inserted in trabecular bone. Biomaterials. Oct 4. Understanding implant osseointegration The role of TGF-β1 The application of oral implants has been considered as a well-accepted treatment modality to replace missing and lost teeth. However, there is skepticism about the use of implants in more demanding situations. For example, patients diagnosed with systemic diseases such as osteoporosis and diabetes are considered as a compromised indication for implant therapy. Additionally, the concept of immediate implant loading to reduce the discomfort, inconvenience, and anxiety of the patient remains a challenge. Furthermore, clinical experience has shown that implant failure often occurs at relatively early stages after surgery, which indicates the need to improve the initial response of the surrounding tissues to the implant. Transforming growth factor β1 (TGF-β1) has been shown to stimulate bone healing in several animal models and may influence bone response directly after implant installation. Aim of this study was to investigate the effect of a low dose of TGF-β1, on the early bone healing around oral implants placed in trabecular bone. Twenty-four cylindrical screw type implants were used and TGF-β1 in two different concentrations was applied on sixteen of them. Each animal received three implants: one Ti (control), one Ti-TGF 0.5 (Ti loaded with 0.5 µg TGFβ1), and one Ti-TGF 1.0 (Ti loaded with 1.0 µg TGF-β1). The eight animals were sacrificed at 6 weeks after implantation and implants with surrounding tissue were retrieved for histological preparation and histomorphometrical evaluation. Light microscopical analysis showed the occurrence of an intervening fibrous tissue layer around about half of the TGF-β1 loaded implants. Further, the histomorphometrical measurements revealed that the Ti implants demonstrated the highest percentage of bone-implant contact (65 ± 4%), while Ti- TGF 1.0 implants showed the lowest amount (45 ± 12%). The difference between these two groups was statistically significant. On basis of the results, it is concluded that TGF-β1 may have a negative influence on the integration of oral implants in trabecular bone during the early post-implantation healing phase. Figure 1: Histological section of Ti-TGF1.0 implant (Ti implants loaded with 1.0 µg TGF-β1) placed in trabecular bone of femoral condyle (magnification 25x). Figure 2: New bone formation along the implant surface was interrupted from fibrous tissue layer in five out of the eight Ti-TGF1.0 implants (magnification 100x). Annual Report 2008 Dimitrios Nikolidakis Understanding implant osseointegration 39

40 NCMLS

Annual Report 2008 Theme 2 Cell metabolism, transport and motion

To do work, cells need energy in the form of ATP. High and sudden energy demand is seen during cell-shape change, a process in Bé Wieringa which ATP fuels the Role of CK-B in phagocytosis This work was published in: Kuiper JWP, Pluk H, Oerlemans F, van Leeuwen FN, de Lange F, Fransen J, Wieringa B (2008). Creatine Kinase-Mediated ATP Supply Fuels Actin-Based Events in Phagocytosis. PLoS Biol. 6 (3): e51. Creatine Kinase-mediated phosphotransfer controls local ATP supply in cells (th)at work To do work, cells need energy in the form of ATP. High and sudden energy demand is seen during cell-shape change, a process in which ATP fuels the cytoskeletal machinery that drives cell-morphology alteration. How a cell organizes high-energy surges without disrupting global ATP homeostasis remains an important research question. One view proposes that ATP is heterogeneously distributed but the cytoskeletal proteins actin and myosin receive regional and preferential access to ATP. Yet this model raises another question: how is ATP funneled to these proteins from distant sources? To address some of these questions, we studied the highly localized molecular events controlling actin dynamics around phagocytic activity of macrophages. We demonstrate that actin and creatine kinase-b (CK-B; a long-known enzyme involved in ATP supply) are simultaneously recruited into the sites of action during the early phases of particle ingestion and coaccumulate at nascent phagosomes [Figure 1; only CK-B accumulation is shown]. Transfection studies with live cell imaging with XFP-tagged CK-B and β-actin revealed that this partitioning process is of transient and specific nature. Local availability of enzymatically active CK and local generation of ATP promotes on-site actin remodeling and particle capture efficiency, and thus supports successful initiation of the first phases of phagocytosis. Overexpression of a catalytic dead CK-B, or CK-specific pharmacological inhibition with cyclocreatine, causes a significant reduction of actin accumulation in the phagocytic cup area. Interestingly, this coupling between local CKactivity and actin regulation is only relevant for complement-mediated phagocytosis and not for Fc-γR-mediated, ingestion capacity of macrophages. Finally, CK-B effects on phagocytosis are most relevant already at the stage of particle adhesion, prior to phagocytic cup maturation, closure and ingestion events [Figure 2]. We predict that our findings may also shed light on how local shape dynamics is energized in other cell types during motility. Figure 1: Transient Recruitment of EYFP-Tagged CK-B to the phagocytic cup area. Time-lapse microscopy of uptake of zymosan (dead yeast particles) in RAW 264.7 cells transiently transfected with EYFP-tagged CK-B. Eight subsequent images captured at 6-s intervals over a 48-s recording period are shown, demonstrating transient accumulation of CK-B. Asterisks indicate zymosan particles binding at the cellular membrane, during the process of engulfment, and after internalization. Bar indicates 5 µm. Figure 2: CK-B Is Involved in the Initial Steps of Phagocytosis. Adhesion and uptake of FITC-labeled COZ particles in RAW 264.7 cells (A) and RAW 264.7 cells expressing catalytically-dead CK-B (C283S) (B) or wild type CK-B (C). Internalized particles appear green (or yellow, when colocalizing with F-actin), and external particles appear cyan. Bars indicate 10 µm. (D) Averages of total numbers of particles associated per cell (inside + outside) (±SEM). A single asterisk (*) indicates p < 0.005; double asterisks (**) indicate p < 0.001. 42 Bé Wieringa Creatine Kinase-mediated phosphotransfer controls local ATP supply in cells (th)at work NCMLS

Cell migration is a key event in many normal and disease processes, like embryogenesis, inflammation, cancer metastasis and angiogenesis. The Remco van Horssen formation Cell migration This work was published in: van Horssen R, Janssen E, Peters W, van de Pasch L, Te Lindert MM, van Dommelen MM, Linssen PC, Ten Hagen TL, Fransen JA, Wieringa B (2008). Modulation of cell motility by spatial repositioning of enzymatic ATP/ADP exchange capacity. J.Biol.Chem. Nov 12. Understanding local energy supply of motile cells Role of subcellular ATP during cell motility Cell migration is a key event in many normal and disease processes, like embryogenesis, inflammation, cancer metastasis and angiogenesis. The formation of subcellular protrusions and retractions during migration is driven by local actin dynamics. The dynamic (re)organization of the actin cytoskeleton is tightly controlled by dozens of actin binding, bundling and nucleation factors. Ultimately, actin polymerization and filament branching/bundling results in a dense network with characteristic localized features, that form structures like invadopodia or leading edges. Within this network, ATP-hydrolysis (an intrinsic feature of growing actin filaments) and myosin-regulated (ATPase) filament contraction, are strongly ATP-dependent. Proteins involved in actin dynamics are intensely studied, while much less research is devoted to the role of energy [i.e. ATP] supply and inhomogeneity in ATP distribution or availability. In this study we analyzed the coupling of local ATP supply to regulation of actin dynamics, using cell spreading and migration as functional read-outs. As experimental model system we used Mouse Embryonic Fibroblasts (MEFs) from Creatine Kinase-B (CKB)/Adenylate Kinase-1 (AK1) double knockouts. These MEFs were complemented with AK1 (which converts 2 ADP into ATP + AMP), coupled to different subcellular location-tags to manipulate its cellular location (focal contacts (FC) or membranes). YFP-variants were used as controls. In addition to the use of procedures for fixed targeting of AK1, we set up a heterodimer-induced system to transiently translocate AK1 to FC under conditions of constant global AK1 activity. After designing AK1-variants carrying different locations-tags (none, EVH1-domain for FC and MYR-domain for membranes), complementation of MEFs was verified and subcellular localization was shown by immunofluorescence. We determined the dynamics of FC-targeting in spreading fibroblasts using live-tirf imaging and found that FC are targeted within 20 minutes after cell seeding. For MEFs complemented with FC-targeted AK1, we found an increase in cell spreading. Using barriermigration assays (involving live-cell imaging of cells migrating in clean cell-free areas), we found that complementation with AK1 induced migration compared to YFP controls, but FC-targeting and to a lesser extent- membrane-targeting results in a further increase (Figure 1A, B). Total cellular ATP levels between the different cell lines were not changed by our manipulations. Use of transient targeting to FC within the same cell population confirmed our findings. Figure 2A shows the targeting principle, and immunofluorescent analysis (Figure 2B) demonstrated that we could achieve immediate translocation of AK1 enzyme by Rapalog addition. Induced repositioning resulted in a strong increase in migration capacity (Figure 2C), which accurately overlaps the induction found using complemented cells. We conclude that (re)positioning of the ATP/ADP exchange capacity of phosphotransfer enzymes is an important codetermining factor in the control of motility behavior of cells. Figure 1: FC-targeted AK1 expression induced migration. (A) Average migration distances in 24 hr. All AK1-complemented cells show increased cell migration (indicated with a ). EVH1-AK1 showed the highest migratory capacity. *p<0.05, ***p<0.001. (B) Morphology of migrating MEFs complemented with AK1 (upper) and EVH1-AK1 (lower) of cell-fronts taken from migration movies. Dashed lines indicate cell-fronts at T=0 hr, Bar=100 µm. Figure 2: Rapalog-induced repositioning of AK1 induced migration. (A) Principle of Rapalog-induced translocation of AK1 to FC. Upon addition of Rapalog FKBP and FRB domains heterodimerize resulting in translocation of AK1. (B) Immunofluorescence for AK1 and Vinculin (Vinc, FC-marker) before and 1 hr after Rapalog (RAPA) addition (arrows point FClocated AK1). (C) AK-1 repositioning results in increased cell migration capacity. Dashed lines indicate cell-fronts at T=0 hr, Bar=100 µm. Annual Report 2008 Remco van Horssen Understanding local energy supply of motile cells 43

The vast majority of cellular ATP is produced in mitochondria by oxidative phosphorylation (OXPHOS). The OXPHOS system consists of five Cindy Dieteren multi-subunit complex- Oxidative phosphorylation This work was published in: Dieteren CE, Willems PH, Vogel RO, Swarts HG, Fransen J, Roepman R, Crienen G, Smeitink JA, Nijtmans LG, Koopman WJ. Subunits of mitochondrial complex I exist as part of matrix- and membraneassocomplex Iated subcomplexes in living cells. J Biol Chem. 2008 283(50):34753-61. Understanding the dynamics of mitochondrial complex I assembly The vast majority of cellular ATP is produced in mitochondria by oxidative phosphorylation (OXPHOS). The OXPHOS system consists of five multi-subunit complexes in the mitochondrial inner membrane of which complex I is the largest and least well understood. The biogenesis of complex I is complicated by its large size (~1 MDa) and its bi-genomic dependency, with seven subunits encoded by the mtdna and the remainder (38) by nuclear genes. At the moment, it is still unclear how these different subunits are exactly pieced together to form the complete enzyme. Understanding this process is clinically relevant, since mutations in complex I genes are the most frequent cause of OXPHOS disorders. Our group provided in vitro evidence that complex I assembly occurs by a stepwise mechanism during which pre-fabricated modules, or assembly intermediates, are combined. This has led to a working model for complex I assembly that helps to better understand assembly defects in patients. Obviously, proper understanding of complex I assembly requires investigation of protein interactions within the dynamic environment of the living cell. We developed a novel mitochondrial fluorescence recovery after photobleaching (FRAP) method to establish the mobility of complex I subunits inside mitochondria of living cells. We reasoned that if complex I assembly proceeds through pre-assembled modules it should be possible to demonstrate differences in mobilities of individual GFP-tagged subunits. First we calibrated our FRAP method by determining the mobilities of a matrix-targeted version of GFP, a matrix-targeted tandem version of GFP, and an inner membranetargeted version of GFP. With these data we demonstrated that the size of a freely diffusing and non-interacting protein influences its mobility in the mitochondrial matrix. Furthermore, we showed that mobility of a protein in the inner membrane is much lower than an inert protein in the matrix. Subsequently, we studied different complex I subunits and established that each subunit was partially present in a virtually immobile fraction, likely reflecting their presence in fully assembled complex I. More detailed analysis of the mobile fractions revealed that subunits which are incorporated at an early stage of the assembly process, displayed mobilities in line with their presence in slowly mobile subassemblies in the matrix. Subunits entering the assembly route at a late stage, recovered as highly mobile matrix-soluble monomers, reflecting their absence in subassemblies. In contrast, the mobility of an integral membrane subunit corresponded to one or more membrane-bound subassemblies. This study provides first time evidence that a modular complex I assembly pathway is operational in living cells. A B C D Figure 1: (A) Complex I assembly model. (B) Typical images from sub-mitochondrial FRAP experiment in cells expressing matrix-soluble GFP. Following bleaching, fluorescence rapidly recovered in the FRAP region (square). (C) Average fluorescence recovery curve (symbols; mean ± SEM) for three control cell lines expressing matrix-targeted GFP ( GFP ), matrix-targeted tandem version of GFP ( GFP2 ) and inner membrane protein ANT1-linked GFP ( ANT ). (D) Example of differences in recovery between two different GFP-tagged complex I subunits, NDUFA2 (orange) and NDUFS3 (violet). Smooth green and blue curves represent the average GFP and ANT data, respectively (as depicted in C). 44 Cindy Dieteren Understanding the dynamics of mitochondrial complex I assembly NCMLS

Angiogenesis is thought to be indispensible for the development and progression of solid tumors. Unraveling of the biology of Vascular William Leenders Endothelial Growth Fac- USPIO-MRI for blood volume determination This work was published in: Claes A, Gambarota G, Hamans B, van Tellingen O, Wesseling P, Maass C, Heerschap A, Leenders W. Magnetic resonance imaging-based detection of glial brain tumors in mice after antiangiogenic treatment. Int J Cancer. 2008, 122, 1981-1986 and Gambarota G, Leenders W, Maass C, Wesseling P, van der Kogel B, van Tellingen O, Heerschap A. Characterisation of tumour vasculature in mouse brain by USPIO contrast-enhanced MRI. Br J Cancer. 2008 Jun 3;98(11):1784-9. Understanding vasculature in brain tumors Development of better MR protocols for delineation of brain tumors Angiogenesis is thought to be indispensible for the development and progression of solid tumors. Unraveling of the biology of Vascular Endothelial Growth Factor (VEGF-A) has contributed to the development of specific inhibitors of the VEGF pathways and these have now entered the clinic for treatment of a number of tumor types. The current thought is that anti-vegf therapies not only inhibit angiogenesis but also lead to normalization of tumor blood vessels, improving blood flow in the tumor whilst reducing vessel leakiness and a concomitant high interstitial pressure. Altogether, this supposedly results in better perfusion of the tumor, better distribution of co-administered chemotherapeutics in the tumor and improved survival. The occurrence of VEGF-induced angiogenesis in glioblastoma multiforme (GBM), a highly malignant brain tumor with very poor prognosis, has led to implementation of anti-angiogenic therapies for these tumors also. Patients do well on these therapies, a result of vessel normalization and concomitant reduction of edema and intracranial pressure. However, in GBM large tumor areas progress via diffuse infiltration utilizing pre-existent brain vessels with an intact blood brain barrier (BBB). This phenotype is insensitive to angiogenesis inhibition and even provides GBM with an escape mechanism from these therapies. Due to an intact BBB, these areas are difficult to detect with conventional Gd- DTPA -enhanced MR imaging. Under antiangiogenic therapy, vessels in the angiogenic GBM compartments will normalize and regain an intact BBB. Therefore, these therapies will result in overall diminished enhancement of brain tumors and a great overestimation of therapeutic efficacy. Expansion of tumors in brain without accompanying angiogenesis is expected to result in a relatively low tumor blood volume, whereas, obviously, vessel dilatation or angiogenesis in tumors would result in higher blood volumes. Based on this, we hypothesized that measurement of tumor blood volumes might aid in more accurate delineation of brain tumors, not only in untreated tumors but especially in tumors treated with angiogenesis inhibitors. To test this hypothesis we set up a novel method to measure blood volume in mouse models of brain cancer (E34 human glioma xenografts, intracerebral U87, angiogenic melanoma metastases) using ultrasmall iron oxide particles (USPIOs) which remain intravascularly for prolonged periods of time and have high impact on the transverse water proton MR relaxation rates (R2 and R2*). Specifically, the change in transverse relaxation rates after injection of USPIOs, R2 and R2*, provides an index which is proportional to microvasculature and microvasculature, respectively. R2 and R2* maps were generated and compared to standard Gd-DTPA enhanced MRI. As shown in Figure 1, Gd-DTPA-invisible E34 tumors were readily detected using USPIOs, based on the presence of dilated, yet minimally-leaky vasculature. Figure 2 shows an MRI of intracranial U87 tumors after treatment with the VEGFR2 inhibitor vandetanib. Whereas these tumors are normally readily visualized with Gd-DTPA MRI (not shown), visibility completely disappears upon anti-vegfr2 treatment (Figure 2C). However, the relatively decreased vessel density in treated tumors results in a hyperintense signal in USPIOenhanced scans in the same mouse (Figure 2D). Figure 2E shows a H&E staining of a corresponding slice. Thus, USPIO imaging appears to be a valuable tool for the delineation of brain tumors in which an intact blood brain barrier precludes conventional MR visualization. 1 2 Figure 1: Comparison of Gd-DTPA (B) and USPIO (D) imaging of cerebral E34 xenografts. A and C show pre-contrast images. E and F show that E34 contains a high density of dilated vessels (E) which are however minimally leaky, as established by staining for extravasated mouse IgG Figure 2: After vandetanib therapy, intracranial U87 tumors are not visible in Gd-DTPA enhanced MRI scans (C) but are readily detectable on USPIO T2* scans, based on a relatively low vascular volume. E shows a H&E staining of a corresponding section. Annual Report 2008 William Leenders Understanding vasculature in brain tumors 45

The maintenance of the body s Mg 2+ balance is of crucial importance for various vital cellular processes. The systemic balance of Mg 2+ Jenny vd Wijst and its intracellular TRPM6 the chanzyme This work was published in: Cao G, Thébault S, van der Wijst J, van der Kemp A, Lasonder E, Bindels RJ, Hoenderop JG. RACK1 a new auxiliary protein inhibiting TRPM6 activity via phosphorylation of the fused α-kinase domain. Current Biology 18: 168-176, 2008 Understanding the molecular regulation of renal Mg 2+ handling RACK1 binding to the Mg 2+ channel TRPM6 inhibits channel activity in an α-kinase activitydependent manner The maintenance of the body s Mg 2+ balance is of crucial importance for various vital cellular processes. The systemic balance of Mg 2+ and its intracellular concentration are determined by intestinal absorption and renal excretion. The renal distal convoluted tubule (DCT) reabsorbs ~10% of the filtered Mg 2+, and the reabsorption rate in this segment defines the final urinary Mg 2+ concentration. Mg 2+ reabsorption in DCT is active and transcellular in nature, but the molecular details and regulation of this pathway remain largely unknown. Recently, transient receptor potential melastatin 6 (TRPM6) was identified as a pivotal component in active Mg 2+ (re)absorption, mutations of which cause hypomagnesemia with secondary hypocalcemia (HSH). TRPM6 is localized along the apical membrane of DCT and intestinal cells. It combines a Mg 2+ -permeable channel, with an α-kinase domain whose function remains elusive. The aim of the present study was, therefore, to investigate the role of the α-kinase domain in TRPM6 channel activity through the identification of regulatory proteins specifically interacting with the α-kinase domain. To identify potential TRPM6-interacting proteins, two independent approaches were used, i.e., yeast two-hybrid screening of a mouse kidney cdna library with the complete C-tail of TRPM6 and the combination of a GST pull-down with the α-kinase domain of TRPM6 in mouse kidney lysate followed by Fourier transform ion cyclotron resonance mass spectrometry (FTMS). RACK1, a scaffold protein originally discovered as an adaptor for protein kinase C (PKC), was identified as a TRPM6 interacting protein in both approaches. GST pull-down binding assays were performed, which demonstrated that RACK1 interacts specifically with the TRPM6 α-kinase domain to the region between positions 1857 and 1885. Immunohistochemical stainings confirmed that RACK1 and TRPM6 are both present in renal Mg 2+ -transporting distal convoluted tubules. Patch clamp studies using human embryonic kidney 293 (HEK293) cells demonstrated that overexpression of RACK1 inhibits TRPM6 channel activity, whereas small interference (si)rna-mediated knockdown of RACK1 increases the current. The RACK1 inhibitory effect is dependent on the α-kinase activity, since inhibition was abolished in the phosphotransferase-deficient mutant (K 1804 R). Moreover, an in vitro phosphorylation assay followed by FTMS revealed a single autophosphorylation site within the α-kinase domain corresponding to the threonine residue at position 1851 (T 1851 ). The phosphorylation state of threonine 1851 is essential for the inhibitory effect of RACK1. Furthermore, TRPM6 autophosphorylation was shown to be strongly dependent on the Mg 2+ concentration and steadily increased in the physiological range of 0.1-1.0 mm Mg 2+. Importantly, threonine 1851 was crucial for the Mg 2+ sensitivity of TRPM6 autophosphorylation and channel activity. Together, the threonine 1851 phosphorylation-dependent inhibitory effect of RACK1 may play an important role in the suppression of the TRPM6 current by intracellular free Mg 2+. Finally, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA prohibited the inhibitory effect of RACK1 on TRPM6 channel activity. In the present study, we propose a unique mode of TRPM6 regulation in which the Mg 2+ influx is controlled by RACK1 through its interaction with the α-kinase and the phosphorylation state of the threonine 1851 residue. Our data contribute to a further understanding of molecular mechanisms involved in renal Mg 2+ handling and to the α-kinase dependent regulation of TRPM6 channel activity. Ackownledgements: This study was performed in collaboration with Hanka Venselaar (CMBI). Figure 1: RACK1 binding site in the predicted 3D structure model of the a-kinase domain. The tertiary structure of TRPM6 α-kinase domain was modeled by SWISS-MODEL, based on the homology between the TRPM6 α-kinase and the crystallized TRPM7 α-kinase domain. 46 Jenny vd Wijst Understanding the molecular regulation of renal Mg 2+ handling NCMLS

TRPM6 is an epithelial Mg 2+ channel fused with an α-kinase domain that plays a key role in Mg 2+ homeostasis regulation. TRPM6 Tim Xi is (transient re-ceptor ATP targets TRPM6 α-kinase domain This work was published in: Thébault S, Cao G, Venselaar H, Xi Q, Bindels RJ, Hoenderop JG. (2008). Role of the α-kinase domain in transient receptor potential melastatin 6 channel and regulation by intracellular ATP. J Biol Chem. 283 (29):19999-20007. Understanding Mg 2+ homeostasis ATP regulates TRPM6 channel activity via its α-kinase domain TRPM6 is an epithelial Mg 2+ channel fused with an α-kinase domain that plays a key role in Mg 2+ homeostasis regulation. TRPM6 (transient receptor potential melastatin 6) is the epithelial Mg 2+ -permeable ion channel that plays a critical role in Mg 2+ homeostasis. Importantly, patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal and renal Mg 2+ (re)absorption, were found to carry mutations in TRPM6. The most striking feature of TRPM6 is that this molecule combines a cation channel with an α-kinase domain. Our knowledge concerning the molecular regulation of the TRPM6 activity and the intriguing role of the atypical α-kinase is still in its infancy. Based on analysis of the protein domain prediction program SMART, we identified that the α-kinase domain in human TRPM6 is located within the carboxyl-terminal region between amino acids 1749 and 2022, in which there is the conserved putative ATP binding motif GXG(A)XXG. By binding to this ATP-binding pocket, both Na + -ATP and Mg 2+ - ATP inhibited TRPM6 currents in a dosedependent manner with a comparable IC50 of ~1.3 mm. Mutation of the conserved glycine residue (G 1955 D) in the ATP-binding pocket prevented the inhibitory effect of ATP on TRPM6 channel activity. Our data further demonstrated that intracellular free ATP, but not GTP or CTP, specifically inhibited TRPM6 channel activity. Physiological concentrations of intracellular Mg 2+ are in the range of 0.5 1.0 mm, whereas intracellular ATP is in the range of 1 6 mm. In the presence of 10 mm Na + -ATP, which buffers intracellular free Mg 2+, TRPM6 current amplitude is significantly reduced. TRPM6 channel activity is abolished when the Na + -ATP concentration is raised to 12 mm. The physiological relevance of this current inhibition is supported by the fact that it occurs at a membrane potential of 80 mv. To exclude the involvement of the chelators, Na + -ATP was perfused with strong chelators such as EDTA or HEDTA. Our results support that both Na + -and Mg 2+ -ATP exert comparable inhibitory effects on TRPM6 (IC50 of ~1.3 mm). Interestingly, the α-kinase domain, but not its activity, is essential for ATP-induced inhibition on TRPM6. This result supports the critical role for the α-kinase domain in TRPM6 regulation. The inhibitory ATP effect is abolished upon truncation of the α-kinase domain but was still present in the TRPM6 phosphotransferase-deficient mutants. Mutation of Thr1851, identified as a TRPM6 auto-phosphorylation site, did not prevent the inhibitory effect of ATP. In contrast to ATP, Na + -CTP or Na + -GTP did not affect the TRPM6 current in transfected HEK293 cells, indicating the unique role of ATP in regulating the channel. In summary, the present study provides molecular insights into the inhibitory effect of Na + -ATP on TRPM6 channel activity. The intracellular ATP effect depends on the Gly1955 residue within the TRPM6 α-kinase domain, whereas TRPM6 α-kinase activity is not required. However, it is still unknown to what extent and how ATP can modulate the transcellular Mg 2+ reabsorption in the kidney, which deserve a further investigation. In conclusion, intracellular free ATP regulates TRPM6 channel activity via its α-kinase domain independently of α-kinase activity. A B C Mg 2+ -ATP Na + -ATP [X-ATP] (mm) Figure 1: Intracellular free ATP inhibits TRPM6 channel activity via its α-kinase domain. A. The putative well conserved ATP binding motif in the TRPM6 α-kinase domain. B. Both Na + -and Mg 2+ -ATP inhibited TRPM6 current with a similar IC50. C. Mutation of the conserved glycine residue (G 1955 D) in the ATP-binding pocket prevented the inhibitory effect of ATP on TRPM6 channel activity. Annual Report 2008 Tim Xi Understanding Mg 2+ homeostasis 47

Cell invasion is a fundamental process that occurs during processes such as morphogenesis, wound healing and angiogenesis, but Katarina Wolf also in disease, such as Cell invasion This work was published in: Friedl, P. and Wolf, K. (2008). Tube travel: protease functions in individual and collective cancer invasion. Cancer Res. 68:7247-7249. Understanding proteolysis in tumor cell invasion For effective invasion, cancer cells segregate the protruding leading edge into 2 functional zones for adhesive traction and proteolytic removal of extracellular matrix Cell invasion is a fundamental process that occurs during processes such as morphogenesis, wound healing and angiogenesis, but also in disease, such as arthritis and cancer. In cancer, cell invasion is often associated with the proteolytic remodeling of extracellular matrix (ECM), thereby promoting both, tissue destruction and dissemination of tumor cells as a prerequisite for the formation of metastasis in distant organs. During cancer progression, proteases, such as serine, cysteine or matrix metalloproteases (MMP), become upregulated in both, the tumor and its environment. These enzymes cleave different substrate classes such as growth factors, cell-surface receptors, and ECM components, with secondary promigratory effects. Proteases that are expressed at the cell surface, such as membrane-type 1 (MT1)-MMP, pericellularly cleave ECM and other substrates and thereby promote cell movement in tissues. The single steps of cell migration have been well established for movement on two-dimensional (2D) ECM-coated surfaces. Cell movement in 3D tissues, however, differs from 2D migration in that the ECM represents a physical barrier towards the forward moving cell body. Since the migration of many cell types involves both, ECM-directed adhesion and proteolysis that may be carried out simultaneously at cell-ecm contact locations it was unclear how a cell invading 3D tissue degrades ECM without challenging the traction force-mediating adhesion. Our studies using a 3D type I collagen model reveal how tumor cells spatially and temporally separate adhesion for initial collagen fibre attachment and cell elongation from subsequent degradation of these fibres, and further show pericellular proteolysis as an integrated step in the process of cell migration. In order to migrate, a cell first must polarize by actin flow-mediated lamellipod or pseudopod protrusion towards certain stimuli (e.g. a chemokine). Second, integrin receptors adhere to and focalize towards collagen fibres, thereby forming focal contacts. These contacts at the tip of a leading egde (zone 1 in figure) mediate fibre bundling and transmit traction towards ECM induced by contraction of the actomyosin cytoskeleton. While thin pseudopods flow through pores and gaps present in the ECM without apparent resistance, several micrometers rearward the cell gains shape by pseudopod branching and maximally expands around the more rigid nucleus. Here, collagen fibres form belt-like compressions that deform the cell body. In a third step, the membrane-bound surface protease MT1-MMP clusters for prolongued duration (30 min or longer) at pressure zones, i.e. between extending pseudopodia or in front of the nucleus, which leads to degradation of these particular fibres (zone 2 in figure 1). Following cleavage of such fiber belts, de novo space is generated for the cell body and nucleus to migrate through. In a forth step, acto-myosin contraction leads to tension along the cellular length axis so that, fifth, adhesive bonds at the cell posterior detach and slide, leading to rear retraction and the appearance of a proteolytic track. The cyclic repetition of these five steps results in cell movement. Blocking proteolysis by small molecule inhibitors in cell invasion models has revealed that some cancer cells can overcome this loss of protease activity and trail generation by deforming the cell body and squeezing through tissue gaps and trails. Therefore, proteolytic degradation of impeding ECM structures is an optional, facultative process that enhances invasive migration which upon loss, however, can be compensated down to certain minimal pore sizes. Understanding the mechanisms of proteolytic as well as non-proteolytic cancer cell invasion and metastatic dissemination will be important in defining where, when and how protease inhibitors are effective against cancer progression, and where not. A B Figure 1: Segregation of adhesion and proteolysis in invading tumor cells. (A) HT1080 fibrosarcoma cell (blue and red) migrating in a 3D collagen lattice (gray). Proteolysis of belt-like collagen fibre constrictions is detected by COL2 C antibody (green). While at zone I the leading pseudopod tip is associated with fibre traction (white arrowheads), only at zone II belt-like proteolysis occurs. Pixel intensity of COL2 C staining signal along diagonal dotted line. Black arrowheads indicate proteolysis of collagen belts. (B) The two-zone segregation of proteolytically invading pseudopod. 48 Katarina Wolf Understanding proteolysis in tumor cell invasion NCMLS

Migration is an essential function in nearly all stages of developing and differentiating T and B lymphocytes, beginning with their Bettina Weigelin development in bone Dynamics of anti-tumor immune responses This work was published in: Friedl, P. and B. Weigelin. (2008). Interstitial leukocyte trafficking and immune function. Nat. Immunol. 9:839-848. Understanding tumor cell elimination by cytotoxic T lymphocytes Coordinated positioning and migration within the tumor tissue is essential for an efficient anti-tumor immune response Migration is an essential function in nearly all stages of developing and differentiating T and B lymphocytes, beginning with their development in bone marrow and thymus and continuing with their migration to secondary lymphoid organs and peripheral tissues. Consequently, an efficient immune response depends upon the balance of controlled migration events and adhesive interactions. The first steps of leukocyte entry into tissues, the extravasation through the vascular endothelium has been studied in detail, while the mechanisms of leukocyte navigation within and out of peripheral tissues during surveillance and effector phase are less clear. Interstitial leukocyte migration represents an integral part of immune cell function, including the search for antigen presenting cells and target cells, the scanning of receptors expressed at the cell surfaces encountered and the accumulation of signals during migration which is commonly guided by a cell type specific repertoire of expressed chemokine and adhesion receptors. Within the 3D environment of connective tissues leukocytes follow extracellular matrix scaffolds, like filamentous collagen fibers and elongated gap-like tracks that act as guidance cues for migration. Additionally and in contrast to epithelial and mesenchymal cells, leukocytes often use other cell surfaces for guided migration. Cytotoxic T lymphocytes for example migrate through interstitial spaces and across the surface of stromal and tumor cells to scan for expressed cognate antigen. This searching behaviour follows the rules of a persistent random walk, similarly to T cells in the lymph node cortex, providing an efficient scanning of the tissue (Figure 1A). Recognition of cognate antigen on target cells then leads to a temporary migration arrest with facilitating prologed cell-cell contact and the induction of effector function. Besides providing guidance cues, the tumor microenvironment may also counteract the migration of infiltrating leukocytes by as yet unresolved mechanisms, such as stop signals that may arrest the movement of tumor-infiltrating lymphocytes and thus act as tumor escape strategy. Further, the sequence of cells encountered previous to the target cell may affect effector function. As example, interactions with regulatory T cells present in the tumor microenvironment impair target cell killing but not migration or conjugation by a transforming growth factor-b-sensitive mechanism. Thus, besides immunological mechanisms such as TCR-mediated recognition and signnalling, effector function in tumors is critically dependent on coordinated leukocyte positioning and migration within the tumor tissue. Improvements in real-time intravital imaging techniques and two-photon microscopy now make it possible to visualize leukocyte migration and cellular interactions in vivo deep within intact tissues. In ongoing studies we are establishing a mouse tumor model of orthotopic intradermally grafted dual-color tumors (Figure 1B) that are amenable for direct analysis of proliferation and apoptosis. The ability to visualize when, where, and how tumor cells are killed in vivo, will improve the understanding of the dynamics of tumor cell elimination by cytotoxic T cells and how local tumor microenvironment conditions, or counteracts, an efficient immune responses. A B Figure 1: A: Intratumoral migration guided by cell matrix and cell cell contacts. B: Cytotoxic T lymphocytes invading a B16 melanoma "dual-color" spheroid in a 3D collagen matrix. Yellow, Histon-2B/EGFP; red, cytoplasmic DsRed2; blue, CTL. Inset: Polarized CTL bound to apoptotic target cell. Annual Report 2008 Bettina Weigelin Understanding tumor cell elimination by cytotoxic T lymphocytes 49

Multidrug resistance protein (MRP) 4 is a member of the MRP/ ABCC subfamily of ATPbinding cassette (ABC) transporters, which are Frans Russel capable of pumping a Drug transporters This work was published in: Russel FG, Koenderink JB, Masereeuw R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharmacol Sci. 2008 Apr;29(4):200-7. Understanding the structure and function of ABCC/MRP efflux transporters MRP4 transports drugs and signalling molecules Multidrug resistance protein (MRP) 4 is a member of the MRP/ABCC subfamily of ATPbinding cassette (ABC) transporters, which are capable of pumping a wide variety of endogenous and xenobiotic organic anionic compounds out of the cell. MRP4/ABCC4 has the remarkable ability of transporting molecules involved in cellular signalling. These include camp, cgmp, ADP, prostaglandins, leukotrienes, urate, conjugated steroids, folate, and bile acids. As a drug transporter MRP4 stands out for its particularly broad substrate specificity covering antiviral (adefovir, tenofovir, ganciclovir), antibiotic (cephalosporins), cardiovascular (loop diuretics, thiazides, angiotensin II receptor antagonists) and cytotoxic (methotrexate, 6-thioguanine, 6-mercaptopurine, topotecan) agents. MRP4 has a typical ABC transporter core structure comprising of two membrane-spanning domains (MSD) each consisting of 6 transmembrane helices (TM), with two cytosolic ATP-binding domains, which bind and hydrolyse ATP to power substrate transport. Positively charged amino acids are important for binding and transport functionality in the MRP subfamily. We built a homology model of MRP4 (Figure 1) and found that Arg375 in TM6 and Arg782 in TM8 face right into central aqueous pore and could be involved in substrate binding. The substrate interactions we found for MRP4 in vitro are complex and suggestive for regulatory binding sites that can allosterically influence the transport activity. The physiological and pharmacological importance of MRP4 has recently become apparent through the use of Mrp4-knockout mice as a model system (Figure 2). The transporter appears to be an important factor in the tissue distribution, brain penetration and toxicity towards nucleoside analogues, which are widely used in the treatment of cancer, viral infections and inflammatory diseases. MRP4 is abundantly expressed in the apical membrane of kidney proximal tubular cells, where it significantly contributes to the tubular secretion of various drugs. Although MRP4 needs to be further validated as a drug target, its specificity for signalling molecules and tissue localization suggest that specific inhibitors of this transporter may provide novel therapeutic approaches for inhibiting nociception and inflammation, preventing myocardial infarction and stroke, and improving brain penetration of antiviral nucleosides. Inducers or allosteric enhancers can be useful in the treatment of hyperuricemia, cholestasis and secretory diarrhoea. Therapeutic targeting of MRP4 will require the design of more potent and specific compounds than those currently available. Side-effects of inhibitors are expected to be limited as the Mrp4-knockout mouse does not exhibit obvious abnormalities. The putative involvement of MRP4 in various pathophysiological signalling pathways as an efflux pump of physiological substrates provides an intriguing challenge for future drug development. Figure 1: Homology model of human MRP4. The model was built using the known X-ray structures of the bacterial ABC transporter Sav1866 from S.aureus and the ATP-binding domain of human MDR1/P-glycoprotein as templates. In panel A the first half of the protein containing transmembrane helices (TM) 1 to 6 and the first ATP-binding domain are coloured. The second half of the protein is shown in grey. Panel B shows a close-up of the channel formed by the 12 transmembrane helices, looking from the extra cellular side to the inside. Arg 375 and Arg 782 are indicated and face into the pore. Figure 2: Physiological and pharmacological roles that have been derived from the Mrp4 knockout mouse model. The cell-type specific expression and subcellular localisation of MRP4 is illustrated. 50 Frans Russel Understanding the structure and function of ABCC/MRP efflux transporters NCMLS

Cyclic adenosine monophosphate (camp) is a common second messenger controlling many cellular processes. Protein kinase A Yuedan Li (PKA) is a general camp signalling This work was published in: Li Y, Konings IB, Zhao J, Price LS, de Heer E, Deen PMT. Renal expression of exchange protein directly activated by camp (Epac) 1 and 2. Am J Physiol Renal Physiol. 2008 Aug;295(2):F525-33. Mapping the renal localization of exchange protein directly activated by camp Cyclic adenosine monophosphate (camp) is a common second messenger controlling many cellular processes. Protein kinase A (PKA) is a general acceptor for camp, resulting in the phosphorylation of a large variety of cellular targets. A newly described camp-target, exchange protein directly activated by camp (Epac) 1, and its close relative Epac2 contain camp-binding domains very similar to the camp-binding domains in the regulatory subunit of PKA and activate specifically the monomeric G proteins of Ras family, Rap1 and Ras. Many actions of camp, which were thought to be mediated exclusively by PKA, are now found to be likely mediated through Epac, including integrin-mediated cell adhesion, vascular endothelial cell barrier formation, cardiac gap junction formation, mitogenactivated protein kinase (MAPK) signaling, hormone gene expression, and phospholipase C-epsilon (PLC-ε) activation. Newly published findings demonstrate that Epac-mediated actions of camp also influence Na +, K +, Ca2 + and Cl - channel function, Na + /H + and Na + /K + transporter activity, and exocytosis in multiple cell types. In the kidney, many physiological processes of ion transport and cellular proliferation are mediated via camp, classically through the activation of PKA. Interestingly, the kidney is one of the organs that show the highest levels of expression of mrnas encoding Epac1, especially in the collecting ducts, suggesting an important role of Epac in camp signalling mechanisms in the kidney. To identify which renal segments might involve Epac1 or 2 in their cellular processes, we investigated the localization of Epac1 and 2 in the rat kidney. To investigate their renal expression, antibodies specifically recognizing Epac1 and Epac2 were generated and used in rat immunofluorescence with antibodies recognizing Aquaporin-1, Tamm-Horsfall protein, Calbindin- D28K and aquaporin-2 to mark proximal tubules (PT)/thin descending limbs of Henle s loop (tdlh), thick ascending limbs of Henle s loop (TAL), distal convoluted tu bule/connecting tubule (DCT/CNT), and the collecting duct (CD) principal cells, respectively. We found that Epac1 and Epac2 were expressed at the brush border of PT cells, but were absent from tdlh cells. In the TAL, Epac1 and Epac2 were expressed throughout the cells with some confinement towards the apical membrane. In the DCT/CNT, Epac1 was confined to the apical region of the cells, whereas Epac2 was mainly expressed in the apical and basolateral regions. In the CD, a dispersed Epac1 expression was found in intercalated cells only (cortical CD), principal and intercalated cells (outer medullary CD), and mainly AQP2- negative cells in the inner medullary CD. In contrast, Epac2 expression was at the apical and basolateral membrane of cortical principal cells, dispersed and apical in the outer medullary CD, and in all cells of the inner medullary CD. A similar distribution for Epac1/2 was found in the human kidney. The observed expression in different tubular segments suggests a major role for Epac 1/2 in tubular transport physiology and cellular proliferation. Figure 1: Expression of Epac1 and 2 in the kidney inner medulla. Rat kidney sections were double-labelled for Epac1 (A, green) or Epac2 (B, green) and AQP2 (A and B, red). Images were taken by confocol laser scanning microscope and shown in a 10- and 180-fold magnification (indicated). Arrows indicate collecting duct principal cells, and arrow heads indicate collecting duct intercalated cells. Stars indicate the apical (lumen) side of the renal tubules. Figure 2: Summary of Epac1 and 2 localization in the kidney tubular cells. Epac1 and 2 localizations are listed in renal segments and cell types for rat (A) and human (B). G: glomerular cells; PT: proximal tubule cells of cortex (c) or outer medulla (o); tdlh: thin descending limb of Henle s loop cells; TAL: thick ascending limb of Henle s loop cells; DCT/ CNT: distal convoluted tubular and connecting tubular cells; CCD: cortical collecting duct cells; OMCD: outer medullary collecting duct cells; IMCD: inner medullary collecting duct cells; mcd: medullary collecting duct cells. none: no staining observed. Annual Report 2008 Yuedan Li Understanding the renal processes mediated by camp signalling 51

52 NCMLS

Annual Report 2008 Theme 3 Cell growth and differentiation

The transcription factor p63 belongs to the p53 family, and plays an important role in ectodermal development. The p63 gene encodes Tuula Rinne at least six different Developmental disorders This work was published in: Rinne T, Clements SE, Lamme E, Duijf PH, Bolat E, Meijer R, Scheffer H, Rosser E, Tan TY, McGrath JA, Schalkwijk J, Brunner HG, Zhou H, van Bokhoven H. Hum Mol Genet. 2008 Jul 1;17(13): 1968-77. Understanding molecular pathogenesis of p63-related developmental syndromes A novel translation re-initiation mechanism The transcription factor p63 belongs to the p53 family, and plays an important role in ectodermal development. The p63 gene encodes at least six different protein isoforms, due to two different promoter sites (Nterminal TA or N isoforms) and three different splicing routes (C-terminal α, β and γ isoforms) (Figure 1A), and Np63α is the most abundant isoform in most tissues. The key function of p63 in ectodermal development is underscored by animal models. In both mouse and zebrafish models, disruption of p63 expression causes severe abnormalities in epidermal development. In humans, heterozygous mutations in p63 are associated with seven distinct developmental disorders with autosomal dominant inheritance, characterized by various combinations of ectodermal dysplasia, limb malformations and orofacial clefting. These conditions have overlapping yet distinct phenotypic features and show clear genotype-phenotype correlations. Interestingly, patients with deletions of 3q27 that covers the complete p63 gene do not show any ED features, and heterozygous p63 knockout mice (p63 +/- ) do not display obvious ectodermal defects, which suggests dominant negative or gain of function mutations rather than haploinsufficiency as the pathogenic mechanism in p63-related ectodermal dysplasias. About 10% of p63-linked patients have Rapp- Hodgkin syndrome (RHS, OMIM 129400) or AEC/Hay-Wells syndrome (OMIM 106260). RHS and AEC syndromes are very similar and have been suggested to be variable manifestations of the same clinical entity. In comparison to other p63-associated disorders RHS and AEC have very severe ectodermal defects such as skin and hair defects, but do not have limb malformations. Previously, missense mutations in the 3 end of the p63 gene are associated with either RHS or AEC syndrome, and these mutations give rise to mutant p63α protein isoforms with dominant effects towards their wild type counterparts. In this study, we report four RHS/AEC-like patients with mutations (p.gln9fsx23, p.gln11x, p.gln16x) that introduce premature termination codons in the N-terminal part of the p63 protein (Figure 1B). These mutations appear to be incompatible with the current paradigms of dominant-negative/gain-offunction outcomes for other p63 mutations. Moreover it is difficult to envisage how the remaining small N-terminal polypeptide contributes to a dominant disease mechanism. Primary keratinocytes from a patient containing the p.gln11x mutation revealed a normal and aberrant p63-related protein that was just slightly smaller than the wild type p63 (Figure 1C). We show that the smaller p63 protein is produced by translation re-initiation at the next downstream methionine, causing truncation of a non-canonical transactivation domain in the N-specific isoforms. Interestingly, this new Np63 isoform is also present in the wild type keratinocytes albeit in small amounts compared to the p.gln11x patient. These data establish that the p.gln11x-mutation does not represent a nullallele leading to haploinsufficiency, but instead gives rise to an excessive amount of a novel truncated p63 isoform Np63 protein with dominant effects. Given the nature of other RHS/AEC-like syndrome mutations, we conclude that these mutations affect only the Np63α isoform and that this disruption is fundamental to explaining the clinical characteristics of these particular ectodermal dysplasia syndromes. Figure 1 A C B 54 Tuula Rinne Understanding molecular pathogenesis of p63-related developmental syndromes NCMLS

Glycosylation is one of the most complex posttranslational modifications of proteins. It plays an important role in cell migration, pathway finding and Jeroen van Reeuwijk cellular Glycosylation disorders This work was published in: Kornak U, Reynders E, Dimopoulou A, van Reeuwijk J, Fischer B, Rajab A, Budde B, Nürnberg P, Foulquier F; ARCL Debré-type Study Group, Lefeber D, Urban Z, Gruenewald S, Annaert W, Brunner HG, van Bokhoven H, Wevers R, Morava E, Matthijs G, Van Maldergem L, Mundlos S. Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2. Nat Genet. 2008 Jan;40(1):32-4. Understanding the molecular pathogenesis of glycosylation disorders Proton pump gene mutations disrupt skin, bone and nervous system development Glycosylation is one of the most complex posttranslational modifications of proteins. It plays an important role in cell migration, pathway finding and cellular connectivity, as well as localisation,interaction and function of glycoproteins. Since approximately half of the proteome consists of glycoproteins that play many different roles in the organism, it is not surprising that inborn errors of glycosylation result in a wide variety of congenital malformation syndromes. In a previous study Morava and colleagues described an association of autosomal recessive inheritance of cutis laxa (loose skin), neurological anomalies, and other growth and developmental abnormalities (ARCL type 2) with abnormal glycosylation. On the basis of these findings, we investigated glycosylation of serum proteins isolated from individuals with ARCL type 2. Serum proteins were analysed by either capillary zone electrophoresis, isofocusing of transferrin and apolipoprotein CIII, or mass spectrometry of glycans from total serum proteins. All affected individuals showed a congenital disorder of glycosylation type 2 pattern, which corresponds to a defect of N-glycosylation at the level of processing in the Golgi apparatus. In addition, half of the families showed a typical apolipoprotein CIII profile, indicative of a disturbance of O-glycosylation (Figure 1). In order to identify the genetic cause in this group of patients with ARCL type 2 and a combined defect of N- and O-linked glycosylation of serum proteins we employed a genetic linkage approach, homozygosity mapping, in patients from consanguineous parents. In 12 families we found linkage for a locus harbouring over 90 genes. Next we employed a candidate gene approach to select genes for mutation analysis. A number of factors involved in glycan biosynthesis are common to the N- and O-glycosylation process, such as Golgi trafficking and structural integrity. Based on the hypothesis that the genetic defects may result in Golgi dysfunction we selected genes for mutation analysis that encode for proteins from the Golgi proteome. In one of these genes, ATP6V0A2, we identified mutations in all linked families that are predicted to result in a loss of protein function. The ATP6V0A2 gene encodes the a2 subunit of a V0 domain of a large ATPase complex (Figure 2). This complex is described to reside in endosomes and in a compartment overlapping with the trans-golgi network. Two major functions of the V-ATPase V0 domain are (i) maintenance of the ph gradient along the secretory pathway by proton transport and (ii) the regulation of protein transport through the facilitation of vesicle fusion. Using different markers for all Golgi subcompartments and for the Golgi matrix, we did not identify any major alteration in Golgi morphology in fibroblasts from affected individuals. However, we observed a significant delay in the retrograde translocation of Golgi membranes to the endoplasmic reticulum (ER) in fibroblasts of all affected individuals. This delay in Golgi trafficking can best be explained by an impairment of vesicle fusion with the ER. It remains unclear whether this impairment of Golgi-to-ER membrane trafficking is the only cellular abnormality leading to the glycosylation defect. As the main function of the ATPase is proton transport, it is very likely that a defective ph regulation in the in the Golgi compartment will likely impair enzymatic and sorting processes that are essential for protein glycosylation. Figure 1: O-glycosylation of apolipoprotein C-III. Lane 1: iso-electric focusing pattern of a control; lane 2: apoc-iii0 profile of CDG-II patient (decreased apoc-iii2, increased apociii0 and apoc-iii1); lane 3: apoc-iii1 profile characteristic for cutis laxa patients with an ATP6V0A2 defect (decreased apoc-iii2, increased apoc-iii1). The picture also shows the O-glycan structures present on the apoc-iii isoforms. ( = GalNAc, = Gal, = sialic acid). Figure 2: Structure and function of the V-ATPase. ATP binds to and is hydrolysed by the A and B subunits of the hexameric head (orange). This generates a rotational movement of the head and the central stalk or rotor, composed of subunits D, F and d (purple), which is transferred to the proteolipid ring (subunits c and c, yellow). Subunit a (grey) and the peripheral stalks (subunits C, E, G and H, green) are static. The conformational changes generated by the rotational movement stimulate the transfer of a proton along the a-subunit and the proteolipid ring. Annual Report 2008 Jeroen van Reeuwijk Understanding the molecular pathogenesis of glycosylation disorders 55

Retinitis pigmentosa (RP) is a frequent cause of inherited blindness in the Netherlands and is characterized by progressive visual impairment, starting Rob Collin with Retinitis pigmentosa This work was published in: Collin RWJ*, Littink KW*, Klevering BJ, van den Born LI, Koenekoop RK, Zonneveld MN, Blokland EA, Strom TM, Hoyng CB, den Hollander AI, Cremers FPM (2008). Identification of a 2 Mb human ortholog of Drosophila eyes shut/spacemaker that is mutated in patients with retinitis pigmentosa. Am J Hum Genet, 83(5): 594-603. * both authors contributed equally. Understanding the genetics of inherited blindness Identification of EYS, a novel 2-Mb gene associated with retinitis pigmentosa Retinitis pigmentosa (RP) is a frequent cause of inherited blindness in the Netherlands and is characterized by progressive visual impairment, starting with night blindness and eventually resulting in complete vision loss. In the majority of cases, RP is inherited in an autosomal recessive (ar) manner. More than 20 genes have been associated with arrp, but mutations in almost all these genes separately account for no more than 1-2% of the arrp cases. The majority of recessive RP genes have been identified using a positional cloning approach in large consanguineous families with multiple affected individuals. Already more than ten years ago, this approach has also resulted in the identification of a locus predicted to harbour a recessive RP gene, namely the RP25 locus. Despite tremendous efforts of several international research groups over the last decade, the causative gene had not been identified, also hampered by the huge size (>15 Mb) of the locus and the large number of genes residing in the region. Recessive RP patients from consanguineous marriages often carry mutations homozygously, since their parents are first or second cousins. As a result, their homozygous regions (one of which contains the causative gene) are very large, up to 50 Mb or more. Due to limited migration in certain areas of the Netherlands over the last 500 years, we postulated that a reasonable amount of the Dutch recessive RP patients also carry homozygous mutations, since their parents share a common ancestor who lived 15-20 generations ago. The homozygous region flanking the mutation however will be much smaller compared to patients from consanguineous marriages, but still distinguishable from small background homozygous regions. In 2008 we genotyped 230 RP patients, mainly originating from the Netherlands, using high resolution Affymetrix 5.0 SNP arrays. This allowed us to identify significant homozygous regions, with an average size of ~4 Mb. In two Dutch siblings with RP, such a homozygous region resided within the RP25 locus. This 5-Mb region contained only five genes. Mutation analysis of two of these genes that were expressed in the retina did not reveal any mutations. Using gene prediction software tools, and subsequent RT-PCR analysis however, we were able to clone a transcript larger than 10 kb, belonging to a gene that spans more than 2 Mb of genomic DNA. The protein encoded by this gene consists of 3165 amino acids and contains multiple EGF-like and laminin A G-like domains. Evolutionary analysis of the protein showed that it is the orthologue of a Drosophila protein named eyes shut. This protein was previously shown to be essential for the development of the interrhabdomeral space of the insect eye. The human gene was named EYS, and is one of the largest human genes described thus far. Mutation analysis of EYS in the sib-pair homozygous at RP25, and in many other RP patients, revealed causative mutations in ~7% of all patients analyzed. EYS is therefore one of the most frequently mutated arrp genes. Knowing the genetic defect is of pivotal importance for a number of reasons: i) it helps calculating genetic risk for offspring, ii) it may help in providing disease prognosis and iii) it is essential for selecting proper gene / mutation-based therapeutic approaches that are beginning to be implemented in the clinic. Figure 1: Genomic structure, cdna fragments, and protein domains of human eyes shut homolog (EYS) A. Upper panel, the RP25 chromosomal region at 6p12.1-q13, the 5.0 Mb homozygous region identified in family A and the 5 known genes within the homozygous region. In the middle, the exon predictions are depicted based on RefSeq (in blue), Genescan (in black), and Ensembl (in brown), using the March 2006 UCSC genome build (hg18). Below the genomic exon-annotation and the exon structure of human EYS. The 5 - and 3 untranslated regions are indicated in black boxes; the colors of the protein-coding exons correspond with those of the protein domains in panel B. B. Protein domain structure of EYS and its Drosophila orthologue. Note the conspicuous conservation of the order of EGF- and laminin A G-like domains between human and Drosophila. The p.pro2238profsx16 frame-shift mutation truncates several EGF- and laminin A G-like domains, whereas the carboxy-terminal p.tyr3156x mutation truncates the last 10 amino acids of human EYS. EGF, epidermal growth factor domain; cbegf, calcium-binding EGF-like domain; EGF-like, EGFlike domain; LamG, laminin A G-like domain. The asterisk denotes predicted glycosaminoglycan (GAG) attachments sites. Two putative O-glycosylation sites are predicted in the human protein (Thr1268 and Thr1424). 56 Rob Collin Understanding the genetics of inherited blindness NCMLS

Synovial sarcomas are aggressive soft tissue tumors which mainly affect children and young adults and account for up to 10% of Diederik de Bruijn all human sarcomas. Synovial sarcoma This work was published in: Lubieniecka, JM, de Bruijn DRH, van Dijk AHH, Subramanian S, van de Rijn M, Poulin, N., Geurts van Kessel A, Nielsen, TO (2008). HDAC inhibitors reverse SS18-SSX mediated polycomb silencing of the tumor suppressor EGR1 in synovial sarcoma. Cancer Res. 68: 4303-4310 (2008). Understanding the molecular pathogenesis of human synovial sarcomas HDAC inhibitor-mediated reversion of oncogene-induced transcriptional deregulation Synovial sarcomas are aggressive soft tissue tumors which mainly affect children and young adults and account for up to 10% of all human sarcomas. Previously, we and others found that in over 95% of all cases, the SS18 gene is fused to either one of three highly homologous SSX genes (SSX1, SSX2 or SSX4). The resulting SS18-SSX fusion proteins act as oncoproteins. The SS18 and SSX genes encode transcriptional cofactors which are involved in activation and repression of gene transcription, respectively. The SS18 protein interacts directly with the BRM and BRG1 components of the SWI/SNF complex, a global chromatin remodeling transcriptional coactivator complex. The SSX proteins co-localize with the RING1 and BMI1 components of the polycomb group repressor complex, and associate with condensed chromatin and core histones. Thus, fusion of the SS18 and SSX proteins brings together two opposing factors affecting both gene expression and chromatin structure in an epigenetic fashion. Recently, we found that SS18-SSX fusion proteins inhibit the expression of the tumor suppressor EGR1 in primary synovial sarcomas and in cell lines expressing the SS18-SSX fusion protein. In addition, it was shown that the clinically applicable epigenetic histone deacetylase (HDAC) inhibitor romidepsin (FK228) significantly inhibits the in vitro and in vivo growth capacities of synovial sarcoma cells. To explore the relationship between SS18-SSX induced alterations in gene expression and romidepsin-induced synovial sarcoma growth inhibition, we set out to identify SS18-SSX target genes using an integrated (epi)genetic approach, and to investigate whether romidepsin may specifically counteract the SS18-SSX-mediated transcriptional (de)regulation of these target genes. By doing so, we found that the tumor suppressor EGR1 is repressed by the endogenous SS18-SSX fusion protein in the synovial sarcoma cell line SYO1 through direct binding to its promoter. This binding correlates with trimethylation of Lysine 27 of histone H3 (H3K27-M3) and recruitment of the polycomb group proteins BMI1 and EZH2 to this promoter. In addition, we found that incubation with romidepsin reverts these modifications and, concomitantly, reactivates EGR1 expression in synovial sarcoma cells. A direct involvement of the SS18-SSX fusion protein and histone deacetylases in EGR1 repression was supported by our subsequent observation that sirna-mediated knockdown of the SS18-SSX fusion protein leads to EGR1 reactivation. Our data implicate polycomb-mediated epigenetic gene repression as a mechanism of oncogenesis in synovial sarcoma and highlight a possible mechanism explaining the efficacy of a clinically applicable HDAC inhibitor in the treatment of this highly aggressive soft tissue tumor. In conclusion, we have provided evidence that the tumor suppressor EGR1 acts as a direct target of the SS18-SSX oncoprotein and is down-regulated via repressive histone modifications and recruitment of polycomb proteins to its promoter. The growth inhibitory effect of romidepsin in synovial sarcoma cells is accompanied by reactivation of EGR1 expression through removal of these repressive histone modifications and dissociation of the polycomb repressor proteins from its promoter. Our work not only provides the first evidence for a direct transcriptional target of the SS18-SSX fusion protein, but also demonstrates how the transcriptional deregulatory effects of this oncoprotein can be reversed directly by a new class of anticancer drugs. Figure 1: Endogenous SS18-SSX and the polycomb group proteins BMI1 and EZH2 are recruited to the EGR1 promoter in SYO1 synovial sarcoma cells. Binding of polycomb group proteins to the EGR1 promoter was determined by chromatin immunoprecipitation (ChIP) using antibodies specific to SS18, BMI1 and EZH2. Total chromatin (Input) and anti-rabbit IgG were used as a positive and negative control for each ChIP assay, respectively. PCR analyses were performed with primers recognizing the EGR1 promoter region. The polycomb group proteins bind to the EGR1 promoter in synovial sarcoma cells, and are removed by romidepsin (FK228) treatment. Annual Report 2008 Diederik de Bruijn Understanding the molecular pathogenesis of human synovial sarcomas 57

Age-related macular degeneration (AMD) is the most common cause of blindness in the Western world, with a prevalence of 12% Camiel Boon after 80 years of age. Drusen This work was published in: Boon CJF, Klevering BJ, Hoyng CB, Zonneveld-Vrieling MN, Nabuurs SB, Blokland EAW, Cremers FPM, den Hollander AI. (2008) Basal laminar drusen caused by compound heterozygous variants in the CFH gene. Am J Hum Genet 82: 516-523. Understanding basal laminar drusen and age-related macular degeneration Figure 1: Fluorescein angiography of basal laminar drusen, showing a stars-in-the-sky picture, corresponding with the small drusen. Age-related macular degeneration (AMD) is the most common cause of blindness in the Western world, with a prevalence of 12% after 80 years of age. The presence of macular and/or extramacular drusen is an important risk factor for the development of advanced AMD. Different clinical subtypes of drusen have been described, but all drusen appear to be similar on the level of ultrastructural and molecular composition. AMD is a multifactorial disorder and variants in several genes have shown to be associated with the disease. Strong association is seen with the Tyr402His variant and several non-coding variants in the Complement factor H (CFH) gene. The CFH protein has been detected in drusen and acts as an inhibitor of the alternative complement cascade. Basal laminar drusen refers to an earlyonset drusen phenotype, which shows a pattern of uniform small (25 to 75 µm), slightly raised, yellow subretinal nodules randomly scattered in the macula. On fluorescein angiography, a typical stars-in-the-sky picture may be observed (Figure 1). The basal laminar drusen phenotype has an even stronger association with the Tyr402His variant than AMD, with a previously described allele frequency of 70%, compared to 55% in a cohort of typical AMD patients and 34% in controls. We evaluated 30 probands diagnosed with basal laminar drusen maculopathy. The age at onset ranged from 35 to 70 years (mean, 50 years). Eight patients reported family members with macular disease or visual disturbances compatible with macular disease. We evaluated the role of CFH in the 30 probands by sequence analysis of the 22 coding exons and splice junctions. The Tyr402His variant was present in 48% of CFH alleles. More importantly, we found that 7 patients in two families (A and B) were compound heterozygous for the nonsense mutation Gln408X in CFH and the AMD risk allele Tyr402His (Figure 2). In a third family (C) we identified three individuals with drusen who carried a heterozygous variant (Arg1078Ser) on one allele, and the Tyr402His variant on the other allele (Figure 2). In patient D, we identified the Tyr402His variant homozygously, and a heterozygous variant in the splice donor site of exon 3 (c.350+6t>g) (Figure 2). A patient in a fifth family (E) carried a heterozygous amino acid variant (Arg567Gly), and was heterozygous for the Tyr402His variant (Figure 2). Our findings strongly support a recessive disease model in a subgroup of patients with basal laminar drusen. In these families, individuals develop drusen when they carry a CFH mutation on one allele and the Tyr402His variant on the other allele. We show that monogenic inheritance of CFH variants can result in basal laminar drusen in young adults. This condition can progress to maculopathy and severe vision loss later in life. Our findings indicate that basal laminar drusen and AMD belong to a spectrum of diseases associated with either monogenic or multifactorial inheritance of variants in the CFH gene. Figure 2: Molecular genetic analysis of the CFH gene in families affected with drusen maculopathy. A and B, Seven affected individuals in two families are compound heterozygous for the nonsense mutation Gln408X and the AMD risk allele Tyr402His. Brackets flanking the CFH variants of A-I.3 indicate that they were deduced through CA-marker analysis. C, Three affected individuals in a third family are compound heterozygous for the missense variant Arg1078Ser and Tyr402His. D, Patient D is homozygous for the Tyr402His variant, and in addition carries a heterozygous variant in the splice donor site of exon 3 (c.350+6t>g). E, A patient in a fifth family carries a heterozygous amino acid variant (Arg567Gly), and is heterozygous for the Tyr402His variant. Segregation analysis could not be performed. A-E Grey pedigree symbols denote patients with basal laminar drusen. The black symbols denote two females affected with AMD. Numbers in the pedigree symbols reflect current age. F, Sequences of heterozygous variants detected in the CFH gene. 58 Camiel Boon Understanding basal laminar drusen and age-related macular degeneration NCMLS

Hearing loss is a common disorder and often inherited, especially in early-onset cases. Different patterns of inheritance are seen and the Hannie Kremer disorder exhibits a very Hereditary hearing loss This work was published in: Ahmed ZM*, Masmoudi S*, Kalay E*, Belyantseva IA, Mosrati MA, Collin RW, Riazuddin S, Hmani-Aifa M, Venselaar H, Kawar MN, Tlili A, van der Zwaag B, Khan SY, Ayadi L, Riazuddin SA, Morell RJ, Griffith AJ, Charfedine I, Caylan R, Oostrik J, Karaguzel A, Ghorbel A, Riazuddin S, Friedman TB, Ayadi H, Kremer H (2008). Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in human. Nat Genet 40: 1335-40. *Equal contributions Understanding the molecular pathogenesis of hearing loss Hearing loss is a common disorder and often inherited, especially in early-onset cases. Different patterns of inheritance are seen and the disorder exhibits a very high degree of genetic heterogeneity. Identification of deafness genes has already provided important insights into the molecular processes that underlie inner ear development and function and into the pathogenesis of deafness. Identification of novel deafness genes will further add to this knowledge. A novel deafness locus, DFNB63, on chromosome 11q13 was identified in a large consanguineous family from Turkey and simultaneously by others in families from Tunesia and Pakistan which allowed fine mapping of the critical region. By sequencing of the 26 known and predicted genes in this region in the DNFB63 families, we found pathogenic mutations in four of these families in a gene called LRRC51, now LRTOMT, that had not been fully characterized. Five alternative transcripts of the gene were found to be expressed in many tissues. Curiously, the longest transcript contains two alternative open reading frames encoding two very different proteins, LRTOMT1 and LRTOMT2. Leucine-rich repeats are predicted in LRTOMT1 and in LRTOMT2, a catechol-omethyltransferase domain is predicted. One of the mutations is a splice site mutation and predicted to affect both proteins by a reading frame shift in the mrna. The three other mutations only seem to affect the LRTOMT2 protein by amino acid substitutions. Based on the crystal structure of catechol-omethyltransferase (COMT), homology modelling of the catechol-o-methyltransferase domain of LRTOMT2 was possible. This revealed that three amino substitutions affect residues of LRTOMT2 that are neighbouring in the protein and two of these are predicted to form a salt bridge and hydrogen bonds. It can be predicted that the three mutated residues are important for protein stability and that the amino acid substitutions could indirectly affect the substrate-binding region of the catechol-o-methyltransferase domain of LRTOMT2. These results contribute to our hypothesis that the hearing loss in the DFNB63 families is mainly due to defects in LRTOMT2. In mice the homologues of LRTOMT1 and LRTOMT2 are known as Lrrc51 and Tomt respectively but they are encoded by two separate genes. Analysis of these genes in primates suggest that during recent evolution there has been gradual fusion of the two genes so that in man the vestige is with two alternative reading frames of one gene. The murine TOMT and LRRC51 proteins are detected by immunohistochemistry in hair cells and supporting cells of the cochlea and the vestibular system and might be associated with the endoplasmic reticulum. LRRC51 immunoreactivity was most prominent along the basolateral wall of the outer hair cells. So far, no catechol-o-methyltransferase activity was described in hair cells. However, the conservation of the substrate-binding residues in the catechol-o-methyltransferase domain suggest that LRTOMT2 might indeed function as a methyltransferase. Taken together, the identification of the gene responsible for DFNB63 and the proteins that are encoded opened up exciting new fields for genetic and physiological studies of the inner ear. Also, our data illustrate that the annotation of the human genome is far from complete and suggest that pleiotropy and variation in phenotypes of different gene mutations may be explained by (unrecognized) alternative reading frames in a gene. Figure 1: LRTOMT has ten exons encoding multiple isoforms. The dual reading frames are colored orange and green. The two predicted translation start-codons are in exons 3 and 5. Grey boxes denote UTRs. Isoforms A to E of LRTOMT1 have one predicted transmembrane domain (TM) and two leucinerich repeats. Transcripts D` and E` are identical in sequence to D and E, respectively, but encode an entirely different protein, LRTOMT2, when translation starts in exon 5 and stops in exon 10. LRTOMT2 isoform D` has a predicted catechol-o-methyltransferase domain and a TM. Annual Report 2008 Hannie Kremer Understanding the molecular pathogenesis of hearing loss 59

The life cycle of human malaria parasite Plasmodium falciparum within the mosquito vector begins when gametocytes from infected red blood Edwin Lasonder cells Sporozoite maturation This work was published in: Lasonder E, Janse CJ, van Gemert GJ, Mair GR, Vermunt AM, Douradinha BG, van Noort V, Huynen MA, Luty AJ, Kroeze H, Khan SM, Sauerwein RW, Waters AP, Mann M, Stunnenberg HG. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity. PLoS Pathog. 2008 Oct;4(10) Understanding sporozoite development of the malaria parasite in mosquitoes Specific proteins involved at different maturation stages of P.falciparum sporozoites in mosquito midguts and salivary glands The life cycle of human malaria parasite Plasmodium falciparum within the mosquito vector begins when gametocytes from infected red blood cells are taken up in a blood meal; after forming gametes that are fertilised, the resulting zygote differentiates into a motile ookinete that traverses the midgut epithelium and transforms into an oocyst between the midgut epithelial cells and the basal lamina. The oocyst is an asexually replicating form of the parasite, which produces up to 2000-4000 sporozoites in about two weeks. Rupture of mature oocysts releases oocyst-derived sporozoites into the hemocoel of the mosquito. The movement of the hemolymph brings the oocyst-derived sporozoites in contact with the salivary glands, which they then invade. The sporozoites mature inside the salivary glands and then are stored ready for transmission to the mammalian host upon the next blood meal, where they migrate from the skin to the liver to establish an infection inside hepatocytes. Consequently, the sporozoite has to complete a number of functions and metabolic readjustments during its journey from mosquito to mammalian host. Despite all the different biological events, general morphology of the sporozoite is not visibly altered at any stage in contrast to other life cycle stages. Since sporozoites play an essential role in the first phase of a malaria infection, an understanding of its biology is of great importance in order to develop intervention methods against initial infection and consequently disease. However, only a few (less than 25) proteins have been characterized as being essential for sporozoite development and infectivity. These include several proteins that are currently under investigation as either potential subunit-vaccines or may serve in the generation of whole organism, genetically attenuated sporozoite vaccines eliminated from the Plasmodium genome. The lack of large-scale in vitro culture methods for oocysts and sporozoites has restricted high throughput protein expression studies to only mature sporozoites, which are more readily obtained from infected salivary glands. We have performed a detailed proteomic comparison of sporozoites obtained from oocysts and salivary glands which were obtained by hand-dissection of infected mosquito midguts and salivary glands. The proteome analysis was performed using essentially the same high throughput mass spectrometric analysis that we previously applied to generate the proteomes of the blood stages of P. falciparum as well as the proteomes of male and female gametocytes of P. berghei. Our analyses resulted in a proteome of oocysts (n=127), oocyst-derived sporozoites (n=450) and salivary gland sporozoites (n=477), which represent 728 individual Plasmodium proteins, of which 250 were exclusively detected in the oocyst/sporozoite stages when compared to the P. falciparum blood stage proteomes. The identification of these proteins and their relative distributions within the different proteomes suggest specific metabolic adaptations and other biological functions of the maturing sporozoite. Moreover, we analyzed the function of eight sporozoite-specific proteins identified in our proteome analyses that were annotated as hypothetical proteins, by targeted gene disruption of the orthologous genes of the rodent malaria parasite, P. berghei. We were able to demonstrate an essential and distinct role for three of these proteins in sporozoite development at different stages of maturation. A Figure 1: Phenotypic characterization of P. berghei mutants (841cl1, 843cl1, 802cl1) with disrupted genes. (A) Numbers of oocyst-derived sporozoites and salivary gland sporozoites per mosquito from day 14 till day 27 post mosquito infection. Scale bars indicate 50?m. Wild type (WT) sporozoite numbers are shown in blue bars, 841 clone (PB000251.01.0 / PFD0425w) gene disruptant sporozoite numbers in purple, 843 clone (PB402680.00.0 / MAL8P1.66) gene disruptant sporozoite numbers in yellow, and 802 clone (PB101363.00.0-PB000829.02.0- PB105739.00.0 / PF14_0435) gene disruptant sporozoite numbers are shown in pale blue. B (B) Oocysts and sporozoites of the three mutant lines. Upper panel: GFP-expressing mature oocysts at day 10 after infection. Middle panel: Representative images (phase contrast microscopy) of mature (day 12) oocyst. Sporozoite formation in mutant 841 (PB000251.01.0 / PFD0425w) is same as WT whereas in lines 843 (PB402680.00.0 / MAL8P1.66) and 802 (PB101363.00.0-PB000829.02.0-PB105739.00.0 / PF14_0435) sporozoite development is either affected (i.e. 843) or completely absent (i.e. 802). Lower panel: GFP-expressing sporozoites (released by mechanical rupture of oocysts at day 18-20). Scale bars indicate 12 um. 60 Edwin Lasonder Understanding sporozoite development of the malaria parasite in mosquitoes NCMLS

The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. Upon a variety of cellular stresses such as Leonie Smeenk DNA damage, UV radia- The p53 stress response This work was published in: Smeenk L*, van Heeringen SJ*, Koeppel M, van Driel MA, Bartels SJ, Akkers RC, Denissov S, Stunnenberg HG, Lohrum M. *These authors contributed equally. Characterization of genome-wide p53-binding sites upon stress response. Nucleic Acids Res. 2008 Jun;36(11):3639-54. Understanding the transcriptional functions of the tumor suppressor gene p53 A genome-wide identification of p53 binding sites using ChIP-on-chip The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. Upon a variety of cellular stresses such as DNA damage, UV radiation and hypoxia, p53 gets stabilized and can function as a sequence specific transcription factor. When p53 is stabilized it regulates the expression of target genes involved in growth arrest, apoptosis, DNA repair, senescence, differentiation and other responses. Many p53 target genes are currently known, e.g. identified with microarray expression profiling and at the moment it is intensively studied how p53 determines which target genes to activate or repress in a certain stress response. It is known that cofactors, p53 binding proteins and several post-translational modifications of p53 play a role in target gene selection. Besides this, there is also evidence that the p53-family members p63 and p73 can influence p53- recruitment at specific target genes. To understand how p53 determines which target genes to activate or repress in a certain stress response, it is important to have more insight in its transcriptional functions. Besides the experimentally identified p53 target genes there are also computationally predicted binding sites, but these predictions do not necessarily reflect the actual target sites bound in vivo by p53. For the selection of functional binding sites the involvement of other cellular factors, chromatin accessibility, DNA sequences surrounding the potential binding site and DNA topology have to be taken into consideration, in addition to the consensus binding sequence itself. Up to now it was thought that this p53 response element is mostly found within a few thousand base pairs of the transcriptional start site. Binding sites which differ from the classical p53 binding motif have also been reported. However, these findings are largely based on single target genes; a genome-wide analysis of binding sequences for common motifs will be very informative. To understand p53 s essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. We have identified and characterized 1546 binding sites of p53 using a genome-wide tiling ChIP-on-chip approach. Among those binding sites were known as well as novel p53 target sites which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus binding-motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. To obtain a more complete picture of the in vivo bound target genes of p53, we have also performed ChIP-on-chip analyses with two of its family members, p63 and p73. We show that a large fraction of these newly identified binding sites for p53 could also be bound by p63 and p73 in vivo. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research. Figure 1: Binding profile and expression change of four ESTs bound by p53. In the upper panel, the ChIP-on-chip data is visualized. In the lower left panel, these binding sites are confirmed by targeted ChIP for p53. Shown is the enrichment in fold over negative control (myoglobin). In the lower right panel, the expression change of the EST is shown after 24 h of 5nM Actinomycin D treatment in fold over untreated U2OS cells. Error bars represent standard deviation of three independent experiments. Figure 2: p53 motif identified de novo with MD-module, visualized using WebLogo. Annual Report 2008 Leonie Smeenk Understanding the transcriptional functions of the tumor suppressor gene p53 61

Phosphorylation of tyrosine residues within proteins represents a fast and extremely sensitive switch that cells use to control protein Wiljan Hendriks function, especially in Regulating signal regulators This work was published in: Noordman YE, Augustus ED, Schepens JTG, Chirivi RG, R os P, Pulido R, Hendriks WJAJ (2008). Multimerisation of receptor-type protein tyrosine phosphatases PTPBR7 and PTP- SL attenuates enzymatic activity. Biochim Biophys Acta Mol Cell Res 1783: 275-286. Understanding regulatory principles that act on protein tyrosine dephosphorylation Phosphorylation of tyrosine residues within proteins represents a fast and extremely sensitive switch that cells use to control protein function, especially in cellular signalling pathways. The extent of tyrosine phosphorylation is dictated by the interplay of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Much is known about the regulation of protein tyrosine kinase activity, and in fact the ligand-induced dimerisation and subsequent auto- phosphorylation of growth factor receptors, transmembrane tyrosine kinases, is a mandatory subject in cell biology courses. In contrast, the mechanisms controlling PTP enzymatic activity remain to be disclosed. Recent examples of aberrant PTP actions underlying various human diseases underscore the importance of reversibility in protein tyrosine phosphorylation and have put PTPs in the limelight as novel therapeutic drug targets. The human PTP family contains 38 phosphotyrosine-specific ( classical ) enzymes, half of which are single-pass transmembrane, receptor-type proteins. Our ignorance on potential ligands for these receptor-type PTPs (RPTPs) is hampering studies addressing the regulation of their activity. Only for RPTPζ a liganddependent dimerisation and consequent inactivation has been demonstrated, mirror-imaging receptor-type kinase regulation. A recent large-scale structural study of PTP domains provided evidence for a dimerisation-induced inactivation model for the twelve RPTPs that contain two tandem catalytic domains. Ligand binding would induce a head-to-toe arrangement of the cytosolic parts resulting in the blocking of the membrane-proximal catalytic domain s active site of one protein by the membrane-distal domain of the other, and vice versa. This model, however, is not applicable to RPTPs with single catalytic PTP domains, like the transmembrane PTPRR isoforms. We investigated whether the PTP activity of the four neuronal PTPRR isoforms is regulated via multimerisation. PTPRR isoforms, all encoded by the single-copy mouse Ptprr gene, differ in their amino-terminal sequence and, as a consequence, subcellular localisation: PTPBR7 is localised on the plasma membrane, PTPPBSγ-42 and PTPPBSγ-37 are in the cytosol, and PTP-SL is distributed at vesicular structures and the Golgi apparatus. Intriguingly, we noted that the transmembrane, receptor-type isoforms PTPBR7 and PTP-SL formed homo- and hetero-dimers constitutively, as opposed to the cytosolic PTPPBSγ isoforms that remained monomeric. We hypothesised that a heterodimer consisting of the full-length PTPBR7 and a mutant that lacks the cytosolic region would mimick an RPTP monomeric state. To measure changes in PTPBR7 specific activity upon complexing with the truncated mutant, proteins were immunopurified from transfected cell lysates and assayed using a small-molecule artificial substrate that is turned into a fluorescent product. Indeed, the monomer-mimicking heterodimer displayed a specific activity that exceeded that of PTPBR7 homodimers. Because immunoprecipitates contained an excess of PTPBR7 over the truncated mutant, this activity may be confounded by PTPBR7 homodimers and possibly monomers. Therefore we also compared the phosphatase activity of PTPBR7 with the exclusively monomeric PTPPBSγ isoforms. PTPRR monomeric isoforms were found to be twice as active as the receptor-type isoform. Since conceptually an on/off regulation by dimerisation is the most straightforward, the residual activity in PTPBR7 samples implies that about half of the PTPBR7 subunits remains monomeric in cells. Our findings add further proof to the concept that RPTP dimerisation results in the attenuation of phosphotyrosine phosphatase activity, also for the ones containing a single catalytic domain. Figure 1: Protein tyrosine phosphatase activity is reduced upon multimerisation. Left panel. Full-length PTPBR7 (BR7) containing complexes, with or without co-expressed excess truncated PTPBR7 (BR7-T), were immunopurified from cell lysates. Steady state activities were assessed in a fluorescent in vitro phosphatase activity assay and normalised for the amount of full-length PTPBR7. Results (n=3) are presented as mean values ± SEM (p<0.009). Middle panel. Schematic representation of the PTPRR isoform variants used in COS-1 cell transfection experiments and subsequent assays. On the left the signal peptide (SP), hydrophobic region (HR), kinase-interacting motif (KIM) and the catalytic phosphotyrosine phosphatase domain (PTP) are indicated. Right panel. Fulllength PTPBR7 or PTPPBSγ was immunoprecipitated, released from the beads and analysed on immunoblots and in fluorescent phosphatase activity assays. An inactive Cys-Ser mutant (BR7-CS) was used as negative control. Activities (n=4) were normalised for PTP protein content and are presented as mean values ± SEM (p<0.002). 62 Wiljan Hendriks Understanding regulatory principles that act on protein tyrosine dephosphorylation NCMLS

The (re)discovery of penicillin in 1928 by Alexander Fleming, provided humanity with a powerful tool in the fight against bacteria. Dennis Waalboer Although penicillin was Synthetic organic chemistry This work was published in: Dennis C J, Waalboer M, Schaapman C, van Delft FL., FPJT Rutjes, Angew. Chem. Int. Ed. 2008, 47: 6576-6578. High-Pressure Entry into Platencin The (re)discovery of penicillin in 1928 by Alexander Fleming, provided humanity with a powerful tool in the fight against bacteria. Although penicillin was hailed as a miracle drug, within four years the antibiotic was ineffective in certain cases. It turned out that prolonged exposure of bacteria to antibiotics could eventually lead to resistance. This phenomenon is known today as antibiotic resistance and formed the start of a continuing struggle, the search for new antibiotics. Since then many antibiotics have been discovered and numerous lives have been saved. However, as in the case of penicillin, resistance to these new antibiotics is emerging which is caused mainly by misuse such as continuous overprescription and the use of antibiotic growth promoters in animal food. The decreasing effectiveness of antibiotics combined with the fact that in the last five decades efforts in antibiotic drug discovery met with little success clearly show the need for new antibiotics. In this respect the discovery of two new antibiotics, platensimycin (1, 2006) and platencin (2, 2007) was considered a breakthrough scientists had been looking for. (Figure 1) Both compounds have an unprecedented mode of action which makes them especially attractive drug candidates. They target the fatty acid synthesis in gram-positive bacteria very efficiently with no resistance observed. Unfortunately, the rapid clearance of platensimycin and platencin from the blood in micemodel studies indicated that both compounds suffer from poor pharmacokinetics. To turn these compounds into successful drugs, access to derivatives with hopefully improved pharmacokinetic properties, is vital. One way of doing this proceeds via total synthesis. In these particular cases, both compounds possess an intriguing unprecedented cage-like core structure which makes their synthesis very challenging. Recently, we developed a formal total synthesis of platencin starting from inexpensive, commercially available (S)-( )-perillaldehyde (3). (Figure 2) In contrast to its well known isomer carvone, perillaldehyde has seen little use in total synthesis. We envisioned that a Diels-Alder reaction of (S)-( )-perillaldehyde with the Danishefsky diene (4) could be key in accessing the platencin core structure (8) in a very concise manner. First, we had to address the challenge posed by such cyclohexenes, which are notoriously unreactive towards Diels Alder reactions with Danishefsky s diene (4). Since the standard means of activation like thermal or Lewis acid activation are insufficient or incompatible, respectively, we turned our attention to high-pressure activation. To carry out reactions under a pressure of up to 15.000 bar, dedicated synthesis equipment is required, which belongs to the infrastructure of the Institute for Molecules and Materials. (Figure 3) Fortunately, the Diels Alder reaction of 3 and 4 proceeded smoothly at 15 kbar and 50 C to give, after Lewis acidic work-up, adduct 5 in 81% yield. After three straightforward steps 6 was reacted in the second key step, a SmI 2 -mediated pinacol cyclization, which gave after deprotection compound 7 in an excellent yield of 85%. With the carbon-skeleton in place, only three steps were needed to elaborate 7 to the platencin core structure 8. Since 8 is an intermediate in a previously reported total synthesis of platencin, this route represents a concise, enantiopure formal total synthesis of platencin. Currently, we are exploring the potential of this route for the succinct synthesis of derivatives. Figure 1: Structures of platensimycin (1) and platencin (2). Figure 2: Asymmetric synthesis of the platencin core 8. Figure 3: The IMM high-pressure parallel synthesis facility. Annual Report 2008 Dennis Waalboer High-Pressure Entry into Platencin 63

Annual Report 2008 Scientific publications 2008

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Scientific publications 2008 Aarntzen, E.H.J.G., Figdor, C.G., Adema, G.J., Punt, C.J.A. & Vries, I.J.M. de (2008). Dendritic cell vaccination and immune monitoring. Cancer Immunology Immunotherapy, 57(10), 1559-68. Aarts, F., Bleichrodt, R.P., Man, B de, Lomme, R, Boerman, O.C. & Hendriks, T. (2008). The effects of adjuvant experimental radioimmunotherapy and hyperthermic intraperitoneal chemotherapy on intestinal and abdominal healing after cytoreductive surgery for peritoneal carcinomatosis in the rat. Annals of Surgical Oncology, 15(11), 3299-307. Aarts, F., Bleichrodt, R.P., Oyen, WJ & Boerman, O.C. (2008). Intracavitary radioimmunotherapy to treat solid tumors. Cancer Biotherapy and Radiopharmaceuticals, 23(1), 92-107. Aarts, F., Hendriks, T., Boerman, O.C., Oyen, WJ & Bleichrodt, R.P. (2008). Hyperthermia and fibrinolytic therapy do not improve the beneficial effect of radioimmunotherapy following cytoreductive surgery in rats with peritoneal carcinomatosis of colorectal origin. Cancer Biotherapy and Radiopharmaceuticals, 23(3), 301-9. Abdollahi-Roodsaz, S., Joosten, L.A.B., Helsen, M.M.A., Walgreen, B., Lent, P.L.E.M. van, Bersselaar, L.A.M. van den, Koenders, Mieke & Berg, W.B. van den (2008). Shift from toll-like receptor 2 (TLR-2) toward TLR-4 dependency in the erosive stage of chronic streptococcal cell wall arthritis coincident with TLR-4-mediated interleukin-17 production. Arthritis and Rheumatism, 58(12), 3753-64. Abdollahi-Roodsaz, S., Joosten, L.A.B., Koenders, Mieke, Devesa, I., Roelofs, M.F., Radstake, T.R.D.J., Heuvelmans-Jacobs, M., Akira, S., Nicklin, MJ, Ribeiro-Dias, F & Berg, W.B. van den (2008). Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis. Journal of Clinical Investigation, 118(1), 205-16. Ahmed, Z.M., Masmoudi, S., Kalay, E., Belyantseva, I.A., Mosrati, M.A., Collin, R.W.J., Riazuddin, Saima, Hmani-Aifa, M., Venselaar, H., Kawar, M.N., Tlili, A., Zwaag, B. van der, Khan, S., Ayadi, L., Riazuddin, S.A., Morell, R.J., Griffith, A.J., Charfedine, I., Caylan, R., Oostrik, J., Karaguzel, A., Ghorbel, A., Riazuddin, Sheikh, Friedman, T.B., Ayadi, H. & Kremer, H. (2008). Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans. Nature Genetics, 40(11), 1335-40. Alasti, F, Sanati, MH, Behrouzifard, AH, Sadeghi, A, Brouwer, A.P.M. de, Kremer, H., Smith, RJ & Camp, G Van (2008). A novel TECTA mutation confirms the recognizable phenotype among autosomal recessive hearing impairment families. International Journal of Pediatric Otorhinolaryngology, 72(2), 249-55. Alexander, R.T., Hoenderop, J.G.J. & Bindels, R.J.M. (2008). Molecular determinants of magnesium homeostasis: insights from human disease. Journal of the American Society of Nephrology, 19(8), 1451-8. Alexander, S, Koehl, GE, Hirschberg, M, Geissler, EK & Friedl, P.H.A. (2008). Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochemistry and Cell Biology, 130(6), 1147-54. Almirza, W.H., Dernison, M.M., Peters, P.H.J., Zoelen, E.J.J. van & Theuvenet, A.P.R. (2008). Role of the prostanoid FP receptor in action potential generation and phenotypic transformation of NRK fibroblasts. Cellular Signalling, 20(11), 2022-2029. Alten, R, Gram, H., Joosten, L.A.B., Berg, W.B. van den, Sieper, J., Wassenberg, S, Burmester, G, Riel, P.L.C.M. van, Diaz-Lorente, M, Bruin, GJ, Woodworth, TG, Rordorf, C, Batard, Y, Wright, AM & Jung, T (2008). The human anti-il-1 beta monoclonal antibody ACZ885 is effective in joint inflammation models in mice and in a proof-of-concept study in patients with rheumatoid arthritis. Arthritis Research & Therapy, 10(3), R67. Altink, ME, Arias-Vasquez, A, Franke, B., Slaats-Willemse, DI, Buschgens, CJ, Lambregts-Rommelse, N.N.J., Fliers, EA, Anney, R, Brookes, KJ, Chen, W, Gill, M, Mulligan, A, Sonuga-Barke, E, Thompson, M, Sergeant, J.A., Faraone, SV, Asherson, P & Buitelaar, J.K. (2008). The dopamine receptor D4 7-repeat allele and prenatal smoking in ADHD-affected children and their unaffected siblings: no gene-environment interaction. Journal of Child Psychology and Psychiatry, 49(10), 1053-60. Ammerlaan, AC, Ararou, A, Houben, MP, Baas, F., Tijssen, CC, Teepen, JL, Wesseling, P. & Hulsebos, TJ (2008). Long-term survival and transmission of INI1-mutation via nonpenetrant males in a family with rhabdoid tumour predisposition syndrome. British Journal of Cancer, 98(2), 474-9. Ammerlaan, AC, Houben, MP, Tijssen, CC, Wesseling, P. & Hulsebos, TJ (2008). Secondary meningioma in a long-term survivor of atypical teratoid/rhabdoid tumour with a germline INI1 mutation. Childs Nervous System, 24(7), 855-7. Anney, RJ, Hawi, Z., Sheehan, K, Mulligan, A., Pinto, C, Brookes, KJ, Xu, X., Zhou, K., Franke, B., Buitelaar, J.K., Vermeulen, H.H.M., Banaschewski, T., Sonuga-Barke, E., Ebstein, R, Manor, I., Miranda, A., Mulas, F., Oades, R.D., Roeyers, H., Lambregts-Rommelse, N.N.J., Rothenberger, A., Sergeant, J.A., Steinhausen, H.C., Taylor, E., Thompson, M., Asherson, P., Faraone, S.V. & Gill, M. (2008). Parent of origin effects in attention/deficit hyperactivity disorder (ADHD): analysis of data from the international multicenter ADHD genetics (IMAGE) program. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics, 147B(8), 1495-500. Anney, RJ, Lasky-Su, J, O'Dushlaine, C, Kenny, E, Neale, BM, Mulligan, A, Franke, B., Zhou, K, Chen, W, Christiansen, H, Arias-Vasquez, A, Banaschewski, T, Buitelaar, J.K., Ebstein, R, Miranda, A, Mulas, F, Oades, RD, Roeyers, H, Rothenberger, A, Sergeant, J.A., Sonuga-Barke, E, Steinhausen, H, Asherson, P, Faraone, SV & Gill, M (2008). Conduct disorder and ADHD: evaluation of conduct problems as a categorical and quantitative trait in the international multicentre ADHD genetics study. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics, 147B(8), 1369-78. Antonius, T, Draaisma, J, Levtchenko, E.N., Knoers, N.V.A.M., Renier, W & Ravenswaaij, C van (2008). Growth charts for Wolf-Hirschhorn syndrome (0-4 years of age). European Journal of Pediatrics, 167(7), 807-10. Aral, A, Yalcin, S, Karabuda, ZC, Anil, A, Jansen, J.A. & Mutlu, Z (2008). Injectable calcium phosphate cement as a graft material for maxillary sinus augmentation: an experimental pilot study. Clinical Oral Implants Research, 19(6), 612-7. Arisan, V., Ozdemir, T., Anil, A, Jansen, J.A. & Ozer, K. (2008). Injectable Calcium Phosphate Cement as a Bone-Graft Material Around Peri-implant Dehiscence Defects: A Dog Study Source. International Journal of Oral & Maxillofacial Implants, 23(6), 1053-62. Asherson, P, Zhou, K, Anney, RJ, Franke, B., Buitelaar, J.K., Ebstein, R, Gill, M, Altink, M, Arnold, R, Boer, F, Brookes, K, Buschgens, C, Butler, L, Cambell, D, Chen, W, Christiansen, H, Feldman, L, Fleischman, K, Fliers, E, Howe-Forbes, R, Goldfarb, A, Heise, A, Gabriels, I, Johansson, L, Lubetzki, I, Marco, R, Medad, S, Minderaa, R, Mulas, F, Muller, U, Mulligan, A, Neale, B, Rijsdijk, F, Rabin, K, Lambregts-Rommelse, N.N.J., Sethna, V, Sorohan, J, Uebel, H, Psychogiou, L, Weeks, A, Barrett, R, Xu, X, Banaschewski, T, Sonuga-Barke, E, Eisenberg, J, Manor, I, Miranda, A, Oades, RD, Roeyers, H, Rothenberger, A, Sergeant, J.A., Steinhausen, HC, Taylor, E, Thompson, M & Faraone, SV (2008). A high-density SNP linkage scan with 142 combined subtype ADHD sib pairs identifies linkage regions on chromosomes 9 and 16. Molecular Psychiatry, 13(5), 514-21. Aslam, M.W., Busscher, G.F., Weiner, D.P., Gelder, R. de, Rutjes, F. & Delft, F.L. van (2008). Fully orthogonally protected 2-deoxystreptamine from kanamycin. Journal of Organic Chemistry, 73(13), 5131-5134. Asten, J.J.A. van, Cuijpers, V.M.J.I., Hulsbergen- van de Kaa, C.A., Soede-Huijbregts, C., Witjes, J.A., Verhofstad, A.A.J. & Heerschap, A. (2008). High resolution magic angle spinning NMR spectroscopy for metabolic assessment of cancer presence and Gleason score in human prostate needle biopsies. Magnetic Resonance Materials in Physics Biology and Medicine, 21(6), 435-42. Baldwin, R.J., Dam, G.B. ten, Kuppevelt, A.H.M.S.M. van, Lacaud, G, Gallagher, J.T., Kouskoff, V & Merry, C.L. (2008). A developmentally regulated heparan sulfate epitope defines a subpopulation with increased blood potential during mesodermal differentiation. Stem Cells, 26(12), 3108-18. Bauler, TJ, Hendriks, W.J.A.J. & King, PD (2008). The FERM and PDZ domain-containing protein tyrosine phosphatases, PTPN4 and PTPN3, are both dispensable for T cell receptor signal transduction. PLoS ONE, 3(12), e4014. 66 Scientific publications 2008 NCMLS

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bavel, C.C.A.W. van, Fenton, KA, Rekvig, OP, Vlag, J. van der & Berden, J.H.M. (2008). Glomerular targets of nephritogenic autoantibodies in systemic lupus erythematosus. Arthritis and Rheumatism, 58(7), 1892-9. Behring, J, Junker, R., Walboomers, X.F., Chessnut, B & Jansen, J.A. (2008). Toward guided tissue and bone regeneration: morphology, attachment, proliferation, and migration of cells cultured on collagen barrier membranes. A systematic review. Odontology, 96(1), 1-11. Belletti, B, Nicoloso, MS, Schiappacassi, M, Berton, S, Lovat, F, Wolf, K. van der, Canzonieri, V, D'Andrea, S, Zucchetto, A, Friedl, P.H.A., Colombatti, A & Baldassarre, G (2008). Stathmin activity influences sarcoma cell shape, motility, and metastatic potential. Molecular Biology of the Cell, 19(5), 2003-13. Benitez-Ribas, D., Tacken, P, Punt, C.J.A., Vries, I.J.M. de & Figdor, C.G. (2008). Activation of human plasmacytoid dendritic cells by TLR9 impairs Fc gammarii-mediated uptake of immune complexes and presentation by MHC class II. Journal of Immunology, 181(8), 5219-24. Berg, W.B. van den (2008). Cytokines and joint erosion in arthritis. Nature Clinical Practice Rheumatology, 4(2), 58-9. Berg, W.B. van den (2008). Lessons from animal models of osteoarthritis. Current Rheumatology Reports, 10(1), 26-9. Bergmann, JF, Slavenburg, S., Roomer, R, Knegt, R.J. de & Drenth, J.P.H. (2008). Rationale and design of the virological response and ribavirin dosage (VIRID) study in hepatitis. Netherlands Journal of Medicine, 66(1), 44-5. Berk, L.C.J. van den, Figdor, C.G. & Torensma, R. (2008). Mesenchymal stromal cells: tissue engineers and immune response modulators. Archivum Immunologiae et Therapiae Experimentalis, 56(5), 325-9. Berkel, S.S. van, Dirks, A.J., Meeuwissen, S.A., Pingen, D.L.L., Boerman, O.C., Laverman, P., Delft, F.L. van, Cornelissen, J.J.L.M. & Rutjes, F. (2008). Application of metal-free triazole formation in the synthesis of cyclic RGD-DTPA conjugates. Chembiochem, 9(11), 1805-1815. Berkel, SS van, Dirks, AT, Meeuwissen, S.A., Pingen, D.L.L., Boerman, O.C., Laverman, P., Delft, F.L. van, Cornelissen, JJ & Rutjes, F.P.J.T (2008). Application of metal-free triazole formation in the synthesis of cyclic RGD-DTPA conjugates. Chembiochem, 9(11), 1805-15. Berkhout, M., Roelofs, H.M.J., Morsche, R.H.M. te, Dekker, E. den, Krieken, J.H.J.M. van, Nagengast, F.M. & Peters, W.H.M. (2008). Detoxification enzyme polymorphisms are not involved in duodenal adenomatosis in familial adenomatous polyposis. British Journal of Surgery, 95(4), 499-505. Beucken, J.J.J.P van der, Plachokova, A.S., Link, D.P., Mikos, A.G. & Jansen, J.A. (2008). In vivo effects of TGF-beta 1-loaded polymeric microsphere incorporation in injectable cap cement. Tissue Engineering Part A, 14(5), 745-6. Beucken, J.J.J.P van der, Walboomers, X.F., Leeuwenburgh, S.C.G., Vos, M.R-J., Sommerdijk, N.A.J.M., Nolte, R.J.M. & Jansen, J.A. (2008). DNA-coatings: bioactive properties and effects on oseoblast-like celles. Key Engineering Materials, 361-363, 605-608. Beusink, J.B., Lokate, A.M.C., Besselink, G.A.J., Pruijn, G.J.M. & Schasfoort, R.B.M. (2008). Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays. Biosensors and Bioelectronics, 23(6), 839-844. Bleeker-Rovers, C.P. & Meer, J.W.M. van der (2008). [Diagnostic approach to fever of unknown origin]. Nederlands Tijdschrift voor Geneeskunde, 152(15), 869-73. Bleijenberg, G. & Meer, J.W.M. van der (2008). Klachten blijven na cognitieve gedragstherapie. Medisch Contact, 63(17), 737-737. Bodar, E, Netea, M.G., Jong, D.J. de, Kullberg, B.J., Joosten, L.A.B. & Meer, J.W.M. van der (2008). NOD2 engagement induces proinflammatory cytokine production, but not apoptosis, in leukocytes isolated from patients with Crohn's disease. European Cytokine Network, 19(4), 185-9. Bodde, E.W.H., Boerman, O.C., Russel, F.G.M., Mikos, A.G., Spauwen, P.H.M. & Jansen, Jarno (2008). The kinetic and biological activity of different loaded rhbmp-2 calcium phosphate cement implants in rats. Journal of Biomedical Materials Research Part A, 87(3), 780-91. Bodde, E.W.H., Spauwen, P.H.M., Mikos, A.G. & Jansen, J.A. (2008). Closing capacity of segmental radius defects in rabbits. Journal of Biomedical Materials Research Part A, 85(1), 206-17. Boer, D. den, Rip, A. & Speller, S. (2009). Scripting possible futures of nanotechnologies: a methodology which enhances reflexivity. Technology in Society. Boer, D. den, Shklyarevskii, O.I., Elemans, J.A.A.W. & Speller, S. (2008). Low-temperature dissociative adsorption of Hydrogen on W, Mo, and Ta surfaces studied with mechanically controllable break-junctions. Physical Review B, 77(16), 165423-1-165423-8. Boer, D. den, Shklyarevskii, O.I., Elemans, J.A.A.W. & Speller, S. (2008). Low-temperature dissociative adsorption of Hydrogen on W, Mo, and Ta surfaces studied with mechanically controllable break-junctions. Virtual Journal of Nanoscale Science & Technology, 2008(18, May 5), 1-8. Boerman, O.C. & Oyen, W.J.G. (2008). Multimodality probes: amphibian cars for molecular imaging. Journal of Nuclear Medicine, 49(8), 1213-4. Boerman, O.C., Stollman, T.H. & Oyen, W.J.G. (2008). Scintigraphic imaging of angiogenesis. Tijdschrift voor Nucleaire Geneeskunde, 30, 177-183. Bon, B.W.M. van, Koolen, D.A., Borgatti, R, Magee, A, Garcia-Minaur, S, Rooms, L, Reardon, W, Zollino, M, Bonaglia, MC, Gregori, M De, Novara, F, Grasso, R, Ciccone, R, Duyvenvoorde, HA van, Aalbers, AM, Guerrini, R, Fazzi, E, Nillesen, W.M., McCullough, S, Kant, SG, Marcelis, C.L.M., Pfundt, R.P., Leeuw, N. de, Smeets, D, Sistermans, E.A., Wit, JM, Hamel, B.C.J., Brunner, H.G., Kooy, F, Zuffardi, O & Vries, BB de (2008). Clinical and molecular characteristics of 1qter microdeletion syndrome: delineating a critical region for corpus callosum agenesis/hypogenesis. Journal of Medical Genetics, 45(6), 346-54. Bongaerts, GP & Heuvel, L.P.W.J. van den (2008). Microbial mito-pathogens: fact or fiction? Medical Hypotheses, 70(5), 1051-3. Bongaerts, GP & Heuvel, L.P.W.J. van den (2008). Microbial proteinase inside human cells as anti-mitochondrial activity: a new virulence factor in infectious diseases? Medical Hypotheses, 70(4), 883-5. Bongers, E.M.H.F., Wijs, I.J. de, Marcelis, C, Hoefsloot, L.H. & Knoers, N.V.A.M. (2008). Identification of entire LMX1B gene deletions in nail patella syndrome: evidence for haploinsufficiency as the main pathogenic mechanism underlying dominant inheritance in man. European Journal of Human Genetics, 16(10), 1240-4. Boon, C.J.F., Klevering, B.J., Hoyng, C.B., Zonneveld-Vrieling, MN, Nabuurs, SB, Blokland, E, Cremers, F.P.M. & Hollander, A.I. den (2008). Basal laminar drusen caused by compound heterozygous variants in the CFH gene. American Journal of Human Genetics, 82(2), 516-23. Boon, CJ, Hollander, A.I. den, Hoyng, CB, Cremers, F.P.M., Klevering, BJ & Keunen, JE (2008). The spectrum of retinal dystrophies caused by mutations in the peripherin/rds gene. Progress in Retinal and Eye Research, 27(2), 213-35. Boon, H, Bosselaar, M., Praet, SF, Blaak, EE, Saris, W.H.M., Wagenmakers, AJ, McGee, SL, Tack, C.J.J., Smits, P., Hargreaves, M & Loon, LJ van (2008). Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients. Diabetologia, 51(10), 1893-900. Boone, M. & Deen, P.M.T. (2008). Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption. Pflugers Archiv- European Journal of Physiology, 456(6), 1005-24. Boonstra, AM, Kooij, JJ, Buitelaar, J.K., Oosterlaan, J, Sergeant, J.A., Heister, J.G.A.M. & Franke, B. (2008). An exploratory study of the relationship between four candidate genes and neurocognitive performance in adult ADHD. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics, 147(3), 397-402. Boraschi, D, Alemayehu, M Abebe, Aseffa, A, Chiodi, F, Chisi, J, Prete, G. 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Haese, A, Taille, A de la, Poppel, H van, Marberger, M, Stenzl, A, Mulders, P.F.A., Huland, H, Abbou, CC, Remzi, M, Tinzl, M, Feyerabend, S, Stillebroer, AB, Gils, M.P.M.Q. & Schalken, J.A. (2008). Clinical utility of the PCA3 urine assay in European men scheduled for repeat biopsy. European Urology, 54(5), 1081-8. Hameren, R. van, Buul, A.M. van, Castriciano, M.A., Villari, V., Micali, N., Schon, P., Speller, S., Scolaro, L. Monsu, Rowan, A.E., Elemans, J.A.A.W. & Nolte, R.J.M. (2008). Supramolecular porphyrin polymers in solutation and at the solid-liquid interface. Nano Letters, 8(1), 253-259. Hamont, D. van, Bekkers, R.L.M., Massuger, L.F.A.G. & Melchers, W.J.G. (2008). Detection, management, and follow-up of pre-malignant cervical lesions and the role for human papillomavirus. Reviews in Medical Virology, 18(2), 117-32. Hamont, D. van, Bulten, J., Shirango, H, Melchers, W.J.G., Massuger, L.F.A.G. & Wilde, P.C.M. de (2008). 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202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 Hartmann, A, Quist, J, Hamm, H., Brocker, EB & Friedl, P.H.A. (2008). Transplantation of autologous keratinocyte suspension in fibrin matrix to chronic venous leg ulcers: improved long-term healing after removal of the fibrin carrier. Dermatologic Surgery, 34(7), 922-9. Hartog, A, Hougee, S, Faber, J, Sanders, A, Zuurman, C, Smit, HF, Kraan, P.M. van der, Hoijer, MA & Garssen, J (2008). The multicomponent phytopharmaceutical SKI306X inhibits in vitro cartilage degradation and the production of inflammatory mediators. Phytomedicine, 15(5), 313-20. Hatzis, P, Flier, LG van der, Driel, M.A. van, Guryev, V., Nielsen, F., Denissov, S., Nijman, IJ, Koster, J., Santo, E., Welboren, W., Versteeg, R., Cuppen, E., Wetering, M van de, Clevers, H. & Stunnenberg, H.G. (2008). Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells. 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Helden, S.F.G. van, Oud, M.M., Joosten, B, Peterse, N., Figdor, C.G. & Leeuwen, F.N. van (2008). PGE2-mediated podosome loss in dendritic cells is dependent on actomyosin contraction downstream of the RhoA-Rho-kinase axis. Journal of Cell Science, 121(Pt 7), 1096-106. Hendricksen, K., Molkenboer-Kuenen, J, Oosterwijk, E., Hulsbergen- van de Kaa, C.A. & Witjes, J.A. (2008). Evaluation of an orthotopic rat bladder urothelial cell carcinoma model by cystoscopy. BJU International, 101(7), 889-93. Hendriks, M.P., Sevaux, R.G.L. de & Hilbrands, L.B. (2008). Response to letter to the editor. Netherlands Journal of Medicine, 66(9), 397-397. Hendriks, MP, Sevaux, R.G.L. de & Hilbrands, L.B. (2008). Encapsulating peritoneal sclerosis in patients on peritoneal dialysis. Netherlands Journal of Medicine, 66(7), 269-74. Hendriks, W.J.A.J. & Stoker, AW (2008). Protein tyrosine phosphatases: sequences and beyond. FEBS Journal, 275(5), 815. Hendriks, W.J.A.J., Elson, A, Harroch, S & Stoker, AW (2008). 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Annual Report 2008 Scientific publications 2008 73

236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 Hollox, E.J., Huffmeier, U, Zeeuwen, P.L.J.M., Palla, R., Lascorz, J, Rodijk-Olthuis, D., Kerkhof, P.C.M. van de, Traupe, H, Jongh, G de, Heijer, M. den, Reis, A., Armour, J.A. & Schalkwijk, J. (2008). Psoriasis is associated with increased beta-defensin genomic copy number. Nature Genetics, 40(1), 23-5. Hoog, H.P.M. de, Vriezema, D.M., Nallani, M., Kuiper, S.M., Cornelissen, J., Rowan, A.E. & Nolte, R.J.M. (2008). Tuning the properties of PS-PIAT block copolymers and their assembly into polymersomes. Soft Matter, 4(5), 1003-1010. Hoogen, M van den & Hilbrands, L.B. (2008). Reduced exposure to calcineurin inhibitors in renal transplantation. New England Journal of Medicine, 358(23), 2519; author reply 2519-20. 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Hovinga, IC Kremer, Koopmans, M., Grootscholten, C., Wal, AM van der, Bijl, M. van der, Derksen, RH, Voskuyl, A.E., Heer, E de, Bruijn, JA, Berden, J.H.M. & Bajema, IM (2008). Pregnancy, chimerism and lupus nephritis: a multi-centre study. Lupus, 17(6), 541-7. Huits, RM, Bleeker-Rovers, C.P., Vos, F.J. & Kullberg, B.J. (2008). [Spinal epidural abscess as a complication of a finger infection]. Nederlands Tijdschrift voor Geneeskunde, 152(41), 2258; author reply 2258-9. Huls, M., Brown, CD, Windass, AS, Sayer, R, Heuvel, J.J.M.W. van den, Heemskerk, S., Russel, F.G.M. & Masereeuw, R. (2008). The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane. Kidney International, 73(2), 220-5. Huls, M., Russel, F.G.M. & Masereeuw, R. (2008). Insights into the role of bone marrow-derived stem cells in renal repair. Kidney & Blood Pressure Research, 31(2), 104-10. 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Protein profiling of B- cell lymphomas using tissue biopsies: A potential tool for small samples in pathology. Cellular Oncology, 30(1), 27-38. Jansen, E.J.R., Scheenen, W.J.J.M., Hafmans, T.G.M. & Martens, G.J.M. (2008). Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway. Biochimica et Biophysica Acta-Molecular Cell Research, 1783(12), 2301-2310. Jansen, E.J.S., Scheenen, W.J.J.M., Hafmans, T.G.M. & Martens, G.J.M. (2008). Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway. Biochimica et Biophysica Acta-Molecular Cell Research, 1783(12), 2301-10. Jansen, J.A., Mikos, A.G. & Johnson, P.C. (2008). A new focus on the methods of tissue engineering. Tissue Engineering Part C Methods, 14(1), 1. Jansen, R.G., Kuijpers-Jagtman, A.M., Kuppevelt, A.H.M.S.M. van & Hoff, J.W. Von den (2008). Collagen scaffolds implanted in the palatal mucosa. Journal of Craniofacial Surgery, 19(3), 599-608. Jansen, R.G., Kuppevelt, A.H.M.S.M. van, Daamen, W.F., Kuijpers-Jagtman, A.M. & Hoff, J.W. Von den (2008). Tissue reactions to collagen scaffolds in the oral mucosa and skin of rats: environmental and mechanical factors. Archives of Oral Biology, 53(4), 376-87. Janssen, AJ, Schuelke, M, Smeitink, J.A.M., Trijbels, FJ, Sengers, RC, Lucke, B, Wintjes, LT, Morava, E, Engelen, B.G.M. van, Smits, BW, Hol, F.A., Siers, M.H., Laak, H ter, Knaap, M.S. van der, Spronsen, FJ Van, Rodenburg, RJ & Heuvel, L.P.v.d. (2008). Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system. Annals of Neurology, 63(4), 473-81. Jawalekar, A.M., Meeuwenoord, N., Cremers, J.G.O., Overkleeft, H.S., Marel, G.A. van der, Rutjes, F. & Delft, F.L. van (2008). Conjugation of nucleosides and oligonucleotides by [3+2] cycloaddition. Journal of Organic Chemistry, 73(1), 287-290. Jdira, L.C., Overgaag, K., Gerritsen, J., Vanmaekelbergh, D., Liljeroth, P. & Speller, S. (2008). Scanning tunneling spectroscopy of arrays of CdSe quantum dots: Response of wavefunctions to local electric fields. Nano Letters, 8(11), 4014-4019. Jdira, L.C., Overgaag, K., Stiufiuc, R., Grandidier, B., Delerue, C., Speller, S. & Vanmaekelbergh, D. (2008). Linewidth of resonances in scanning tunneling spectroscopy. Physical Review B, 77(20), 205308-1-205308-11. Jiang, Z, Kullberg, B.J., Lee, H van der, Vasil, AI, Hale, JD, Mant, CT, Hancock, RE, Vasil, ML, Netea, M.G. & Hodges, RS (2008). Effects of hydrophobicity on the antifungal activity of alpha-helical antimicrobial peptides. Chemical Biology & Drug Design, 72(6), 483-95. Jonckheere, A.I., Hogeveen, M., Nijtmans, L.G.J., Brand, M.A.M. van den, Janssen, A.J.M., Diepstra, J.H.S., Brandt, FC van den, Heuvel, L.P.W.J. van den, Hol, F.A., Hofste, TG, Kapusta, L., Dillmann, U, Shamdeen, MG, Smeitink, J.A.M. & Rodenburg, R.J.T. (2008). A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy. Journal of Medical Genetics, 45(3), 129-33. Jones, P. A., Archer, T. K., Baylin, S. B., Beck, S., Berger, S., Bernstein, B. E., Carpten, J. D., Clark, S. J., Costello, J. F., Doerge, R. W., Esteller, M., Feinberg, A. P., Gingeras, T. R., Greally, J. M., Henikoff, S., Herman, J. G., Jackson-Grusby, L., Jenuwein, T., Jirtle, R. L., Kim, Y. J., Laird, P. W., Lim, B., Martienssen, R., Polyak, K., Stunnenberg, H.G., Tlsty, T. D., Tycko, B., Ushijima, T., Zhu, J. D., Pirrotta, V., Allis, C. D., Elgin, S. C., Rine, J. & Wu, C. (2008). Moving AHEAD with an international human epigenome project. Nature, 454(7205), 711-5. Jong, A.S. de, Mattia, F de, Dommelen, M.M.T., Lanke, K, Melchers, W.J.G., Willems, P.H.G.M. & Kuppeveld, F.J.M. van (2008). Functional analysis of picornavirus 2B proteins: effects on calcium homeostasis and intracellular protein trafficking. 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Anesthesiology, 109(3), 465-72. 84 Scientific publications 2008 NCMLS

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623 624 625 626 627 628 629 630 631 632 633 634 635 Zeitler, M., Fries, P. & Gielen, C.C.A.M. (2008). Biased competition through variations in amplitude of gamma-oscillations. Journal of Computational Neuroscience, 25(1), 89-107. Zhang, H., Langeslag, M., Breukels, V., Jenks, B.G., Roubos, E.W. & Scheenen, W.J. (2008). Calcium channel kinetics of melanotrope cells in Xenopus laevis depend on environmental stimulation. General and Comparative Endocrinology, 156(1), 104-12. Zhang, J., Hoogboom, J.T.V., Kouwer, P.H.J., Rowan, A.E. & Rasing, T.H.M. (2008). Uniform N-(2-Aminoethyl)(3-aminopropyl)trimethoxysilane Monolayer Growth in Water. Journal of Physical Chemistry C, 112(51), 20105-20108. Zhang, SJ, Sandstrom, M.E., Aydin, J, Westerblad, H., Wieringa, B. & Katz, A. (2008). Activation of glucose transport and AMP-activated protein kinase during muscle contraction in adenylate kinase-1 knockout mice. Acta Physiologica, 192(3), 413-20. Zhang, W., Walboomers, X.F. & Jansen, J.A. (2008). The formation of tertiary dentin after pulp capping with a calcium phosphate cement, loaded with PLGA microparticles containing TGF-beta1. Journal of Biomedical Materials Research Part A, 85(2), 439-44. Zhang, W., Walboomers, X.F., Kuppevelt, A.H.M.S.M. van, Daamen, W.F., Damme, P.A. van, Bian, Z. & Jansen, J.A. (2008). In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages. Journal of Tissue Engineering and Regenerative Medicine, 2(2-3), 117-25. Zhang, W., Walboomers, X.F., Osch, G.J.V.M. van, Dolder, J. van den & Jansen, J.A. (2008). Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow. Tissue Engineering Part A, 14(2), 285-94. Zhao, Y., Lang, G., Ito, S., Bonnet, J., Metzger, E., Sawatsubashi, S., Suzuki, E., Guezennec, X le, Stunnenberg, H.G., Krasnov, A., Georgieva, S. G., Schule, R., Takeyama, K. I., Kato, S., Tora, L. & Devys, D. (2008). A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing. Molecular Cell, 29(1), 92-101. Zhou, G., Li, Y., Xiao, W, Zhang, L., Zuo, Y., Xue, J & Jansen, J.A. (2008). Synthesis, characterization, and antibacterial activities of a novel nanohydroxyapatite/zinc oxide complex. Journal of Biomedical Materials Research Part A, 85(4), 929-37. Zhou, K, Asherson, P, Sham, P, Franke, B., Anney, RJ, Buitelaar, J.K., Ebstein, R, Gill, M, Brookes, K, Buschgens, C, Campbell, D, Chen, W, Christiansen, H, Fliers, E, Gabriels, I, Johansson, L, Marco, R, Mulas, F, Muller, U, Mulligan, A, Neale, BM, Rijsdijk, F, Lambregts-Rommelse, N.N.J., Uebel, H, Psychogiou, L, Xu, X, Banaschewski, T, Sonuga-Barke, E, Eisenberg, J, Manor, I, Miranda, A, Oades, RD, Roeyers, H, Rothenberger, A, Sergeant, J.A., Steinhausen, HC, Taylor, E, Thompson, M & Faraone, SV (2008). Linkage to chromosome 1p36 for attention-deficit/hyperactivity disorder traits in school and home settings. Biological Psychiatry, 64(7), 571-6. Zhou, K, Chen, W, Buitelaar, J.K., Banaschewski, T, Oades, RD, Franke, B., Sonuga-Barke, E, Ebstein, R, Eisenberg, J, Gill, M, Manor, I, Miranda, A, Mulas, F, Roeyers, H, Rothenberger, A, Sergeant, J.A., Steinhausen, HC, Lasky-Su, J, Taylor, E, Brookes, KJ, Xu, X, Neale, BM, Rijsdijk, F, Thompson, M, Asherson, P & Faraone, SV (2008). Genetic heterogeneity in ADHD: DAT1 gene only affects probands without CD. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics, 147B(8), 1481-7. Zhou, K, Dempfle, A, Arcos-Burgos, M, Bakker, SC, Banaschewski, T, Biederman, J, Buitelaar, J.K., Castellanos, FX, Doyle, A, Ebstein, RP, Ekholm, J, Forabosco, P, Franke, B., Freitag, C, Friedel, S, Gill, M, Hebebrand, J, Hinney, A, Jacob, C, Lesch, KP, Loo, SK, Lopera, F, McCracken, JT, McGough, JJ, Meyer, J, Mick, E, Miranda, A, Muenke, M, Mulas, F, Nelson, SF, Nguyen, TT, Oades, RD, Ogdie, MN, Palacio, JD, Pineda, D, Reif, A, Renner, TJ, Roeyers, H, Romanos, M, Rothenberger, A, Schafer, H., Sergeant, J.A., Sinke, RJ, Smalley, SL, Sonuga-Barke, E, Steinhausen, HC, Meulen, E van der, Walitza, S, Warnke, A, Lewis, CM, Faraone, SV & Asherson, P (2008). Meta-analysis of genome-wide linkage scans of attention deficit hyperactivity disorder. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics, 147B(8), 1392-8. Zilverschoon, GR, Tack, C.J.J., Joosten, L.A.B., Kullberg, B.J., Meer, J.W.M. van der & Netea, M.G. (2008). Interleukin-18 resistance in patients with obesity and type 2 diabetes mellitus. International Journal of Obesity, 32(9), 1407-14. Annual Report 2008 Scientific publications 2008 87