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Sub-themes: Genetics and epigenetic pathways of disease ( Prof. H. van Bokhoven ), chemical and physical biology (prof J. van Hest)
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.
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The last five years have seen extraordinary advances in genomics through technological developments. First and foremost, the complete sequence of the human genome and that of the major genetic model organisms (M. musculus, D. melanogaster, C. elegans and S. cerevisiae) has been elucidated. Second, techniques to hybridise experimentally isolated nucleic acids to micrometer size spots of defined DNA probes immobilised on glass chips commonly known as ‘DNA chips’ have allowed unprecedented rapid data acquisition.
Third, the development of high specificity affinity purification strategies that allow purification to homogeneity of low abundance protein complexes and subsequent analyses by liquid chromatography coupled mass spectrometry has been refined. The fourth advance occurred in bioinformatics, delivering whole suits of software to exploit, integrate and mine the avalanche of data. Renowned scientists active in the abovementioned areas of rapid developments are members of the functional genomics subtheme: Gert Vriend head of the Nijmegen Centre for molecular and biomolecular Informatics (CMBI),
several human geneticists (Bokhoven, Brunner, Cremers, van Kessel) and molecular biologists specialised in eukaryotic nuclear processes (Logie, Lohrum, Stunnenberg, Veenstra) are also members of this group. The aim of sub-theme 3a is to unravel the molecular basis of cell behaviour which emanates from the genetic and epigenetic code contained in the nucleus in the context of health and disease. In order to achieve this ambitious research goal, every one of the members of the functional genomics subtheme are engaged in technology development.
These range from single molecule studies of reconstituted model chromatin through elucidation of epigenetic marks on a genome wide level to development of novel diagnostic DNA arrays and software development.
The human geneticists use advanced microarray facility and intend to develop novel dedicated diagnostic chips (Bokhoven, Brunner Cremers). Most of the experimental scientists in subtheme 3a are employing mRNA hybridisation to microarrays to establish ab initio molecular phenotypes based on measuring mRNA in cells of interest so as to explore disease aetiology and functional consequences of reverse genetic inactivation of key nuclear factors (Brunner, Cremers, Logie, Veenstra). The Stunnenberg group has pioneered an experimental approach that couples immunoprecipitation of proteins cross-linked to their native chromosomal sites of action to DNA chips (ChIP-on-chip).
The applications of this experimental approach are manifold and it is beyond doubt that most members of the theme will benefit from this novel functional chip. For example to directly identify target promoters of transcription factors or to globally examine changes in target site occupancy as a function of time, exogenous stimuli and disease condition and to distinguish indirect from direct effects as a function of chromosome structure and intracellular signalling. Here high accuracy mass spectrometry, another strength of the sub-theme, comes in as it allows direct determination of all peptides within one sample - provided that the virtual peptides are represented in the database.
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This sub-theme constitutes an essential link between the NCMLS research interests and related themes in the Institute for Molecules and Materials (IMM) and the Cognitive Neuroscience Centre Nijmegen (CNCN) of the Radboud University Nijmegen. Structure and function of proteins and their complexes play crucial roles in virtually all NCMLS research projects. Understanding their role and interactions on a molecular level and in a cellular context is an ultimate goal of increasing importance.
A first research line within this subtheme deals with elucidation of protein structure and protein –protein interactions within cellular signalling pathways, in particular:
- Activation and deactivation mechanisms of tyrosine kinases and tyrosine phosphatases
- External control of cellular proliferation and differentiation
It aims at a fundamental understanding of the control of cell proliferation and differentiation, in combination with the development of diagnostic and therapeutic approaches and subsequent translational research in the field of cancer and age-related bone diseases. Approaches used include structural NMR analyses, microarray analyses, bioinformatical approaches, transgenic mice, proteomics, as well as TAP and RNAi techniques. Cell proliferation and differentiation are highly controlled processes, which are regulated at the level of growth factor/cytokine or steroid hormone availability, expression of the corresponding receptors, activation and deactivation of these receptors and their induced second messengers and target genes, as well as by intercellular contacts and cell-cell communication. Tyrosine kinase receptors are the main activators of cell proliferation, and tyrosine phosphatases the main deactivators of this process. Aberrant (in)activation of these regulatory molecules can result in a large number of growth related diseases, including cancer, heart diseases, psoriasis and auto-immune diseases. Emphasis is laid on the structure- function relationship of tyrosine kinase receptors of the ErbB family, and tyrosine phosphatases of the PTPRR and PTP-BL subtypes (Hendriks, Van Leeuwen). The ErbB signalling network is currently one of the primary targets for the development of anti-cancer drugs, particularly for disseminated carcinomas. In this subtheme we will study the structural and functional requirements for the selective binding of EGF-like growth factors to specific ErbB receptors, with the final aim to develop anti-ErbB antagonists with potential clinical relevance (Van Zoelen).
Furthermore, we will study the endosomal routing and degradation of internalised ErbB receptors after ubiquitination, as part of the cellular desensitisation mechanisms for growth stimuli.
Within the projects dealing with control of proliferation and differentiation, emphasis will be laid on the role of polypeptide growth factors and their receptors in stem cell differentiation, and during loss of density-dependent growth control following tumorigenic transformation and metastatic growth (prostate, glioma) (Theuvenet). Loss of cell-cell contacts will be studied in relation to E-cadherin expression in prostate tumour cells, to growth factor-induced epithelial-mesenchymal transitions in epithelial tumours, and to calcium signalling and cyclo-oxygenase upregulation in fibroblast cells (Olijve).
The second research line deals with the molecular and cellular aspects of neural development. The goal is to uncover the molecular pathways and processes that underlie normal functioning of the central nervous system (CNS), in particular in relation to sensori-motor and adaptive processes, and under pathological conditions, in particular in neurodegenerative and (neurodevelopmental) psychiatric disorders of the CNS (e.g. Alzheimer’s disease (AD), Parkinson’s disease, schizophrenia) and in brain tumours. For this purpose, basic and clinical researchers within this sub-theme participate in transdisciplinary research. More specifically, the affiliated PI at the Department of Biophysics (Gielen) investigates neuronal information processing underlying action and perception, including in Parkinson patients, by combining experimental (electrophysiological, neuroimaging and behavioural) approaches with theoretical modelling (neural networks integrating external inputs to dynamic synaptic changes). The Department of Molecular Animal Physiology (Martens) uses cell-specific transgenic approaches to elucidate the roles of neuronal proteins of unknown function (including Alzheimer’s APP-protein and Parkinsons synuclein), and brain tissues from schizophrenic patients and rats to examine the (epi)genetic basis of neurodevelopmental psychopathological disorders. The Department of Pathology ((de Waal, Wesseling) studies vascular pathology in AD and a transgenic mouse AD-model by identifying markers and amyloid-associated molecules involved in the pathogenesis of cerebrovascular amyloidosis with the ultimate goal to develop strategies for therapy.
The third research area of this sub-theme aims at optimally exploiting the potential of molecular chemistry to modify, design and mimic proteins and their building blocks with the purpose to modulate and analyse their activities and properties in the cellular environment. This is best illustrated by examples from this research area:
- Influencing the stability of peptide conformation and assembly
- Using non-proteinogenic amino acids in diagnosis and treatment of disease
- Employing unnatural, designed derivatives of glycopeptides to study and interfere with biological processes
- Blocking glycosaminoglycan synthesis with deoxysugars or other analogues
- Modification of the DNA-polymerase machinery for synthetic polymer synthesis
- Translation of biopolymer concepts into hybrid materials
- Controlled assembly of biocatalysts for cascade reactions
- Mimicking cellular synthetic processes in microenvironments
- Bio-hybrid architectures: mimicking Nature’s enzyme assemblies
- Hybrid cell systems: incorporation of synthetic components into living cells
Many of the research subjects of the sub-theme have only recently been started by young group leaders (Rutjes, Rowan, Speller, Van Hest). One of the ultimate goals is the construction of artificial cell systems, employing the approaches as mentioned above. Related to this is the application of genetically engineered enzymes in cascade catalysis, and the development of new diversity-oriented synthesis approaches for the generation of libraries of potentially bioactive small molecules. Control over structure on the molecular level will be achieved by bio-inspired materials and biorecognition processes. Biosynthetic pathways on a chip play a prominent role in these developments. Automated parallel synthesis of aminoglycosides (or other structures) and novel synthetic techniques for the assembly of complex carbohydrates and glycopeptides will be important. Similarly, integration of biocatalysis and organic synthesis for the preparation of new chemical entities with potential pharmaceutical application will be explored, as well as the use of oligonucleotides for the development of new RNA-ligands using fluorescence techniques and click chemistry.Combined with the study of properties and functions of single enzyme species by spectroscopic techniques, high resolution imaging of nucleic acids and protein complexes, force spectroscopy of specific binding, and local electrochemistry of organic molecules and proteins, this provides powerful tools to achieve the mission of the sub-theme: advancing the understanding of proteins in their cellular context by integrating recent advances in organic chemistry, physico-chemical analyses and molecular biology. The interaction with the bio-informatics group directed to protein structure (Vriend) is an invaluable asset to all of the activities within theme 3, and teem3b in particular. An essential development within the wider organisational context of the NCMLS is the strengthening of the molecular and cell biological expertise to fully exploit the current and future organic chemical and analytical research described above.
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Contact
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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
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