Transcription is the first step in the pathway of eukaryotic gene expression. Transcriptional regulation underlies cellular differentiation, development, oncogenesis, signaling and other fundamental processes. The aims of the proposed research concern the structural characterization of RNA polymerase II transcription initiation complexes. Structures will be determined by electron microscopy and image processing, electron crystallography, and X-ray crystallography. Comparisons of structures containing various components will reveal possible conformational changes underlying the transcription mechanism. Therefore structural characterization of such complexes will not only reveal information about interactions between components but also provide new insight into the mechanism of transcription initiation. In this way, “static” structural information may be combined to elucidate the mechanism of a dynamic process. The ultimate goal is the structure determination of a complete 48-protein RNA polymerase II transcription initiation complex, and thus elucidation of the mechanism of transcription initiation and its control. This experience will introduce me to a different model organism (yeast) that will allow me to explore novel molecular engineering tools. This fellowship will broaden my horizons in the research of regulation of gene expression since I will change from the pre-mRNA splicing field to the transcription field. Finally, this fellowship differs from my previous research experience in the structural study of a macromolecular complex as I will have the opportunity to combine electron microscopy with X-ray crystallography

Post-translational modifications of histones regulate the structure and activity of chromatin and are involved in many biological processes. The complexity and diversity of modifications allows epigenetic information to be stored in the modification patterns of histones prompting the hypothesis of a “histone code”. Although many modifications and corresponding functions are described, we are far from fully understanding the implication of the complete repertoire of modifications and how patterns of multiple modifications are translated into biological signals. The experiments proposed in this research project aim to identify and characterize new types of modifications and factors that recognize specific histone modification patterns. Nucleosomes containing specifically modified histones will be used for the affinity purification of interacting proteins and as substrates in a search for enzymatic activities. The function of newly identified proteins and novel modifications will subsequently be characterized in vitro and in vivo using mammalian cells and yeast as model systems. Having spent the past years studying the biochemistry of apoptotic signaling, I am now shifting my research focus towards studying how post-translational histone modifications regulate chromatin activity. Pursuing this project will give me the opportunity to gain new insights and skills and will guide me into the field of chromatin research. It should provide a better understanding of how specific histone modification patterns are converted into biological responses and is likely to be relevant for many biological processes.

Dopamine (DA) is a monoaminergic neurotransmitter involved in the regulation of central brain function such as reward, locomotion and affect. Cellular integration of DA signals has been associated with modulation of cAMP by D1 and D2 class dopamine receptors. However a new cAMP independent signaling mechanism has recently been shown to mediate some DA function in vivo while potentially being involved in the development of schizophrenia. Following D2 receptor stimulation, the proteine kinase Akt is negatively regulated following the formation of a signaling complex composed of Akt, beta-arrestin 2 and PP2A. Inactivation of Akt by DA results in the subsequent activation of its substrate GSK3 which in turns participates to the development of DA dependant behaviors. This project aims to identify targets of the Akt/GSK3 signaling pathway that can be regulated by DA. In a first part of the project, I propose to use biochemical and imaging methods to investigate the impact of DA upon ionotropic glutamate trafficking in primary striatal cultures derived from GSK3 beta knockout mice. In a second part of this project, stiatal specific GSK3 knockout mice will be derived and used along with primary cell culture models for a unbiased proteomic screen of GSK3 substrates regulated by DA. Identification of targets of the Akt/GSK3 pathway that are responsive to DA will allow to understand the role of this signaling pathway in the development of DA-associated behaviors and potentially in DA related psychiatric disorders such as schizophrenia.

Cell interiors provide the most sophisticated examples of assembly with unprecedented control over distinct shapes and sizes over multiple length scales from the nanometer to the micron scales. While assembly occurs far from equilibrium in living cells, nonetheless, many experiments conducted at equilibrium, under physiological buffer and salt conditions, result in self-assembled structures, where the average structure is similar to those occurring in vivo. The nerve cell cytoskeleton shows a rich variety of poorly understood structures, from single microtubules to networks of neurofilaments. The objective of the research project is to elucidate the nature of structures and interactions in supramolecular assemblies of neurofilaments, which are a major component of the axonal cytoskeleton in vertebrate neurons. The biopolymer assemblies will be characterized in un-oriented and oriented samples with the latter utilizing micro-channels, which confine and orient the biological filaments for improved structure studies. In my PhD research I studied a different complex and highly correlated system, which is high temperature superconductor. With the aid of the HFSP fellowship, I could switch to a multi-disciplinary biological research. In the proposed research I intend specialize in scattering, diffraction and microscopy methods which are better suited for the above studies. In recent year, it became clear that multi-disciplinary research will be a major player in upcoming science. Since my goal is to be University faculty member, I will spend my post-doctoral period to evolve from a hard condense matter physicist to a multi-disciplinary physicist.

Spinocerebellar ataxia 3 (SCA3) is the most common dominantly inherited ataxia worldwide and among the most frequent neurodegenerative diseases caused by polyglutamine expansions. Accumulating evidence suggests that protein misfolding and impairments of the ubiquitin-proteasome pathway (UPP) contribute to the pathogenesis in neurodegenerative diseases. Interestingly, ataxin-3, the disease protein mutated in SCA3, has itself been implicated in the UPP. However, the physiologic and pathologic functions of ataxin-3 remain poorly understood. I propose to characterize the pathologic mechanisms underlying SCA3 with a focus on the proposed role of ataxin-3 in the UPP. First, I will determine the role of the chaperone HSP27 in a mouse model of SCA3. Secondly, I will explore the potential neuroprotective effect of ataxin-3 in neurodegenerative diseases in the mouse. Lastly, I propose to identify proteins that interact with wildtype and mutant ataxin-3. This research will extend my previous expertise in the characterization of the signaling pathways that regulate neuronal cell death in vitro to in vivo studies of neurodegeneration using genetic mouse models. The elucidation of ataxin-3’s function will not only provide significant insight in the pathogenesis of SCA3 and other neurodegenerative disorders, but could ultimately lead to the development of therapeutic approaches to these devastating diseases. The post-doctoral experience gained during these studies in Prof. Kay Davies laboratory, will significantly contribute to my development into an independent researcher investigating the molecular mechanisms underlying the pathogenesis of human inherited neurologic diseases.

T cells are key mediators of normal and pathological immune responses that recognize antigenic peptides via the T cell receptor (TCR). Activation of T cells depends on their stable interaction with antigen presenting cells (APC) and the formation of the immunological synapse at the cell interface. In this proposal and my future research I strive to delineate the molecular mechanisms that regulate T cell / APC conjugates and their role in immune responses. The TCR triggered increase in the adhesive activity of integrins and induction of T cell polarity is crucial for the establishment of the immunological synapse but the specific signaling pathways involved are not well defined. The protein tyrosine kinases PYK2 and FAK have been implicated in the regulation of integrin-mediated adhesion in fibroblasts and were shown to be activated upon TCR and integrin stimulation in T cells. Thus, I hypothesize that PYK2 and FAK play a crucial role in T cell conjugate formation and T cell activation. I propose an integrated genetic approach using conditional knockout mice to determine the function of PYK2 and FAK in T cells. After my training in the biochemical analysis of innate immune signaling I will dedicate myself with this work to studying adaptive immune responses by microscopy and immunological model systems in vitro and in vivo. I expect that my research will contribute to the understanding of fundamental mechanisms underlying immunity and immune diseases. Therefore, this fellowship will provide me not only with novel techniques but also a new conceptual research perspective and prepare me for an independent research career in immunology.

The heart is one of the first organs to form and function during development. The crucial role of the heart in sustaining the life of complex animals has led to numerous studies focusing on how such an exquisitely engineered motor takes shape. Cardiac valves derive from endocardial cells to prevent retrograde blood flow. These cells undergo elaborate morphogenesis and several signaling pathways converge to orchestrate valve development. Their tight temporal and spatial regulation is vital. Epithelial to Mesenchymal Transition (EMT) plays a central role in the making of a valve. EMT is a fundamental biological process, involved in a number of developmental stages as well as tumor metastasis. The zebrafish heart provides a powerful in vivo system to study cardiac valve development at the cellular level as well as identifying the basic mechanisms of EMT. During my HFSP long-term fellowship, I identified numerous novel mutants involved in valve development, using a forward genetics approach. In parallel, I described the morphological changes of endocardium and myocardium during this process. The primary goal of this CDA proposal is to identify the actual genes responsible for the mutant phenotypes, providing multiple entry points to different aspects of valve development. The long-term aim of the proposed project is to find out how the cardiac valves form and function throughout the life of vertebrates to support appropriate heart performance. I propose to use a set of forward and reverse genetic approaches as well as pharmacological techniques in order to unravel genetic interactions and elucidate how gene networks cooperate to shape heart valves.

Pathogens secrete effectors that enable the infection process, leading to compatibility between host and pathogen. Little is known about the changes that lead to compatibility or how effectors cause those changes. We will study the root knot nematode, an obligate parasite of plants. Nematodes are believed to cause perturbation of host cell developmental pathways by secreting regulatory proteins from their stylet into the host cell cytoplasm. We will stimulate secretion of Meloidogyne incognita infective larvae and identify the secreted proteins by LC ESI MSMS mass spectrometry. A subset of these proteins will be tested for their ability to mimic symptoms of infection when expressed ectopically in transgenic plant roots. Plant parasitic nematodes are refractory to transfection. We will adapt the Designed Regulatory Protein (DRP) technology for use in the functional genomics of parasitic nematodes. DRPs will be made that specifically suppress the production of nematode candidate effector proteins during the infection process, and the effect on compatibility will be measured. I am particularly interested by the interaction between plants and their environment. My previous research experience allowed me to develop competence in cotton genetics. For my PhD, I worked on state transitions (an adaptation to light stress) in Chlamydomonas and Arabidopsis. Learning and applying new techniques (proteomics and DRP technology) to study biotic stress in an extraordinarily stimulating scientific community will enhance and complete my training.

The complex functions achieved by the nervous system rely on its high neuronal diversity. How this diversity is generated is the object of intense studies in several organisms. In C. elegans, the identity, connectivity and lineage of every neuron is known and thus it constitutes a good system to address this question. I propose here to use the well-characterised AIY interneuron to analyse how neuronal subtype-specific and pan-neuronal features are acquired during embryonic development. A battery of at least 40 subtype-specific genes expressed in AIY has recently been identified by the host laboratory. Those genes are directly activated in post-mitotic AIY by two homeobox transcription factors TTX-3 and CEH-10 which cooperate to bind to their promoters. A key question to understand the acquisition of AIY specific identity is now to determine how ttx-3 and ceh-10 are initially co-activated in AIY. First, I propose to address this question via a combination of genetic screen, promoter analysis and cell ablation approaches. Second, in ttx-3 or ceh-10 null mutants, AIY loses its subtype identity but retains pan-neuronal features suggesting that those two characteristics can be uncoupled. To determine how AIY acquires its pan-neuronal identity, I propose to analyse the promoters of a defined collection of pan-neuronal genes. This study should lead to a better understanding, at a cellular and promoter resolution level, of pan-neuronal and subtype specific identity acquisition. It will allow me to tackle later steps of nervous system development than during my PhD. I will learn a new model organism and add genetics to my previous embryology and molecular biology background.

Cancer is the result of a multi-step process requiring the accumulation of mutations in several genes. For most cancers, the target cells of oncogenic mutations are unknown. Adult stem cells (SCs) might be the initial target cells as they self-renew for extended periods of time, providing increased opportunity to accumulate the mutations required for cancer formation. Certain cancers contain cells with SC characteristics with high self-renewal capacities and the ability to reform the parental tumor upon transplantation. However, whether the initial oncogenic mutations arise in normal stem cells or in more differentiated cells that re-acquire stem cell-like properties remains to be determined. The demonstration that SCs are the target cells of the initial transforming events and that cancers contain cells with SC characteristics await the development of tools allowing for the isolation and characterization of normal adult SCs. For most epithelia in which cancer arises, such isolation techniques are not yet available. We have recently developed novel methods to isolate and specifically mark skin SCs, making it now possible to experimentally test the “cancer SC hypothesis” in the skin. In this project, we will test whether skin SCs are the initial target cells of oncogenic mutations, whether skin tumors contain tumor stem cells with skin SCs characteristics and whether oncogenic mutations lead preferentially to skin cancer when they arise in SC rather than in more committed cells. The career development award would offer me a unique opportunity as an independent young investigator to find the initial funding to address this important question in stem cell and cancer biology.

Caspases are a conserved family of cystein proteases considered essential for almost all forms of metazoan programmed cell death (PCD), the genetically controlled suicide of cells. Metacaspases are plant caspase-like proteins that can be divided into two subclasses: Type I and Type II metacaspases. The first contain a prodomain rich in proline and a Zn finger motif, that is the main structural feature of the negative regulator of cell death LSD1. I propose to define the functional role of a small set of proteins belonging to Type I metacaspases and to understand if these are required for plant PCD, as the caspases are necessary for metazoans. Reverse genetics will be adopted to investigate their role, in particular double and triple mutant combinations will help defining overlapping functions among the three Type I metacaspases in the control of PCD. The challenge is to understand how external stimuli feed into the regulatory systems to activate plant caspases, and how they in turn promote cell death execution. This will require techniques I only occasionaly utilised, like live-imaging with fluorescent fusion proteins or yeast-two-hybrid assay. The host laboratory will allow to enrich my scientific knowledge in order to elucidate and dissect the mechanisms of the plant metacaspases in controlling cell death in a variety of physiological and developmental contexts. Genetics, plant pathology and cell biology tools will implement the strong plant physiology and molecular biology background that I developed during the PhD in photosynthesis research. In the future I hope to exploit this new knowledges to face biological processes with a more complete and mature attitude.