A key feature of prion diseases is the recruitment of the normal cellular PrPC protein into a misfolded conformation (PrPSc). Currently no therapy exists for this fatal infectious disorder in animals or humans. I have recently discovered that short Phosphorothioate DNA (PS-DNA) can degrade PrPC and eliminate PrPSc in vitro. Here, I propose to investigate the mechanism by which PrP is degraded in the presence of PS-DNA. I will characterize the presumable binding site of PS-DNA on the PrP protein by molecular mapping, and study the possible contribution of other factors beside PS-DNA in the binding and degradation process through immunoprecipitation. To better understand the extent to which PS-DNA affects other proteins beside PrP a broad proteomics approach will be used. In addition, I will explore possible therapeutic effects of PS-DNA in an animal model of prion disease. The investigation of these questions will not only characterize a possible treatment for prion diseases and a presumably new biological phenomenon, but also help understand the function of PrP and its role in the disease process. This work will be performed in my new laboratory in Israel in collaboration with groups in the United States (S. Prusiner, S. Gygi, D. Harris). Beside the scientific achievements of this proposal, the study will establish my position as an independent researcher in the field of neurodegenerative diseases after a successful graduate and postdoctoral training period. Furthermore, I will acquire new technologies like ICAT that will help the development of my laboratory in Israel and the formation of new collaborations in Europe.

Reproductive development in plants requires the formation of multicellular, haploid gametophytes. Proper specification of cell types in the female gametophyte (or embryo sac) is necessary for attraction of the male gametophyte (pollen tube) to the embryo sac for successful double fertilization of the egg cell and central cell, and ultimately seed development. The goal of this proposal is to use inducible genetic ablation of embryo sac cells to determine how cell-to-cell communication influences cell fate in the female gametophyte and signaling between male and female gametophytes. Mutants with defective pollen tube/embryo sac signaling will also be used to further elucidate the mechanism behind this important signaling pathway. My dissertation research focused on the genetic control of leaf development in maize and tomato. In my post-doctoral position, I hope to change research directions and study reproductive development in Arabidopsis thaliana. Arabidopsis has several advantages over maize and tomato as a model system, including a fully sequenced genome, easy transformation, and a short life cycle. After completing my post-doc, I hope to continue using Arabidopsis as a genetic model and to apply what I learn about reproductive development to crop species in my own laboratory. The laboratory of Ueli Grossniklaus at the University of Zurich is an ideal place for me to begin this new direction of research, as it is a world leader in Arabidopsis gametophytic development and has many resources available to assist in the research I have proposed.

When the body encounters foreign or pathogenic antigens such as bacteria, viruses, cancer cells, or transplanted organs, it responds with a strong defensive reaction known as the immune response. During this process, specialized circulating cells including T cells, natural killer cells and mast cells turn on a variety of cell-specific genes. NFAT family (nuclear factor of activated T cells: NFATc1 to NFATc5) are key transcription factors for the immune system, and also play important roles for the heart and blood vessels and the muscular and nervous system. Through my research during PhD course, I have elucidated that NFATc1, a member of NFAT family, is specifically and highly expressed in osteoclasts, bone-resorbing cells, and a master regulator of osteoclast differentiation, suggesting the imporatance of NFAT family in the skeletal system. In this fellowship, I will clarify cell-type specific function of NFAT family using genetically-manipulated animals. The approaches of my study include: 1) generating conditional knock-out mice of NFAT family 2) examining the conditional knock-out mice and 3) identifying the specific function of NFAT family. Although NFAT family has been a target of many immunological diseases, e.g., autoimmunity and rheumatoid arthritis, it also causes severe side-effects. My study during this fellowship will elucidate cell-type-specific function of NFAT family, and this will be greatly helpful in designing specific therapeutic applications for diseases. I also strongly believe that the combination of my previous research in molecular biology with the expertise of the new laboratory will shed light on new aspects of the biological processes.

The vomeronasal organ (VNO) of mammals is activated by pheromone signals that elicit characteristic arrays of innate reproductive and social behaviors, along with neuroendocrine responses. Recent studies have shown that the VNO comprises two populations of neurons segregated within its apical and basal halves, expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. This research proposal aims at using a multidisciplinary approach to undercover the physiological role of the molecular and anatomical dichotomy in VNO organization. To unequivocally identify the whole range of the mouse behavioral and endocrine responses to pheromones, I will first analyze populations of mice derived from animal captured in the wild that will be raised in semi-natural habitat and crossed to mutants affecting VNO function. Second, I will generate two novel mutant mouse lines in which each basal and apical VNO neuronal populations are specifically genetically inactivated. These mutants backcrossed with wild-derived lines will be screened with the battery of pheromone related responses in the conditions set above, and their phenotype will be compared to that of their wild-type littermates. In different to my previous research experience which was based mainly on behavioral analysis, this project will integrate advanced molecular, genetic and behavioral approaches. Thus, I will gain new expertise in key novel research tools and conceptual approaches. This in turn should provide substantial advancement toward accomplishing my professional aspiration: to head my own research laboratory in the field of rodent sensory neuroethology, using behavioral, molecular and genetic tools.

I aim to develop computational tools to characterize nucleotide binding-proteins (NBPs) by a structural comparison of a sub-family. I am interested in how distinct structural subdomains combine to achieve complex function in a signaling pathway. Furthermore, I seek to reveal how differences in specific NBPs’ subdomains result in altered function and specificity by searching for commonalities and differences in their electrostatic and physico-chemical properties. This will enhance the detailed understanding of cellular signaling and provide a scientific basic for improved, target-specific therapeutics. My experimental background familiarized me with the significant questions facing the signal transduction field. However, I have felt limited by using ‘off the shelf’ software to complement my experimental efforts. In the Honig lab, a world leader in developing computational tools, I can learn to develop and utilize software that combine physical chemistry, bioinformatics and biophysics (notably, protein electrostatics). My experimental background and familiarity with the relevant signaling biology will enable me to develop and employ the relevant methods more effectively and better translate my conclusions to experimental applications. My current goal is to become an independent and successful researcher in the structural bioinformatic field. I wish to focus on problem-oriented research and gain insights that can direct experimental signal transduction research. My long-term goal is to return as an independent researcher to Israel and combine both worlds: explore protein families computationally and perform experimental work to prove or refute the hypotheses generated.

1. Despite its significance little is known about the evolutionary origin of multicellularity. To study possible scenarios, we intend to evolve multicellular growth in laboratory cultures of Saccharomyces cerevisiae and study the underlying molecular adaptations. We reason that multicellularity should confer selective advantages in situations which require efficient directional growth or are characterized by a heterogeneous distribution of nutrients. If cells needed to grow into a specific direction, the formation of multicellular filaments should provide them with increased directional stability and growth rate and hence be advantageous. Pioneering cells that conquer poor trophic environments could also benefit from cooperative nutrient sharing by cells within the filament that live in affluent surroundings, thereby providing a selective advantage to the clone of cells. 2. My Ph.D. work was concerned with a defined molecular-genetic problem pertaining to a particular pathway in animals. In the future I would like to understand how molecular ensembles achieve phenotypic tasks and how the behavior of genetic networks can change in evolution. 3. Firstly, I will learn more about whole-genome approaches to complex biological problems, and, secondly, I hope to tie my practical work more strongly to theoretical analysis to better understand fundamental cellular behavior. I hope to establish a simple system to experimentally study the evolution of complex traits and organismal development. I think that artificial evolution of model organisms can provide insights into the dynamic characteristics of signaling systems.

The mechanisms by which CD4 T cell help facilitates the generation of functional memory CD8 T cells are unknown. This proposal aims to determine whether Th1 and Th2 CD4 T cells are equally able to provide appropriate help to generate effective CD8 T cell memory. CD4 T cells could also facilitate memory generation by promoting complete differentiation to the memory T cell lineage. To test this hypothesis, the differentiation state of the CD8 T cells generated in the absence of CD4 T cell help will be analyzed. Finally, gene regulation is tightly regulated during lineage commitment steps. Little is known about the molecular changes that occur during CD8 lineage commitment. Memory T cells which develop in the presence and absence to CD4 T cell help will be compared to identify a molecular profile of functional versus non-functional memory CD8 T cells. My previous research was focused on signal transduction in T cells, which largely utilized mouse genetics and biochemical and cell biology analysis as experimental approaches. The described proposal is focused on questions surrounding host immunity and experimentally involves infectious models in vivo. This proposal will significantly strengthen my knowledge and expertise in cellular immunology and in vivo model systems. Furthermore, I will maintain a presence in the signal transduction/molecular immunology fields by performing novel molecular analyses on memory T cells generated in vivo. This training environment will provide groundwork for a multifaceted approach to the study in vivo immune responses during infection.

TCRalphabeta (TCRab) precursors are selected in the thymus based on TCR/MHC/self-peptide interactions and this process results in survival (positive selection) or deletion (negative selection) of the precursors. This selection pathway may not be the only one. Indeed some specialized T cells are selected upon agonist signals that otherwise delete conventional thymocytes. The aims of this project are to characterize precursors and selecting cells that participate in this novel selection process and to investigate the functional specialization T cells acquired during this selection process. This project covers novel aspects of T cell selection as well as functional aspects of immune regulation. Thymic selection is a new area for me and my future study will also examine the functional specialization acquired during agonist selection using animal disease models. This new line of research will widen significantly my knowledge and understanding of how the immune system provides protection while preventing self-destruction. I am convinced that this unique experience will prepare me for a successful and productive research career, and that my previous expertise during which I have characterized IEL precursors and differentiation pathways of these precursors in the gut will undoubtedly form a strong basis.

(1) Adenoviral vectors are powerful vectors for gene transfer into the brain. However, systemic anti-vector immune responses abolish vector-encoded transgene expression, and potentially inhibit endogenous brain gene expression. I will test the hypothesis that activated CD8+ T-lymphocytes down-regulate vector and brain transgene expression through IFN-gamma dependent non-cytolytic transcriptional inhibitory mechanisms. To test this hypothesis I will use molecular approaches and knockout animals in a novel paradigm of immune-mediated down-regulation of vector-encoded gene expression developed in Dr. Lowenstein's lab. (2 and 3) The proposed research complements my background in molecular biology and neuroscience to include the area of brain immunology and gene therapy. The research proposed here has far-reaching consequences for gene therapy, and our understanding of brain immune responses in health and disease. This project will broaden my capacity to address complex neurobiological issues in whole animals. Neuroimmunology, gene therapy, and whole animal approaches are completely novel research areas for me. Pursuing my training in a different country will provide me with experience in a different research organization, research field, and scientific approaches. Furthermore, because Dr. Lowenstein is a world leader in neuro-gene therapy and the neuroimmunology of viral vectors, this postdoctoral training will give me the opportunity to start collaborations with researchers all over the world. Also, the HFSP postdoctoral fellowship will provide me with necessary knowledge and autonomy, to allow me, in the future, to establish myself as an independent investigator in Canada.

Neurogenesis is a highly ordered multistep process in which multipotent neuronal stem cells (NSCs) first commit to a specific lineage and, following a proliferative phase, differentiate into a remarkable variety of neuronal subtypes of different morphologies and functions. Recently the Crabtree laboratory has purified a neuron-specific family of dedicated SWI/SNF-like chromatin remodeling complexes. Intriguingly, these complexes appear to undergo subunit exchange as neurons develop from proliferating paraventricular stem/progenitors to post-mitotic mature units. During my post doctoral fellowship, I would like to investigate the functions of these complexes as they evolve from neural stem cells to functionally mature neurons. Specifically, I will use proteomic methods to define and clone each of the subunits of the neural-specific complexes in the paraventricular and post-mitotic regions of the spinal cord. I will then use existing strains of mice in the laboratory and Cre-lox recombination to define the roles of the ATPase subunit Brg1 in proliferating paraventricular neural stem cells and post-mitotic differentiated neurons. Finally, I will define the role of the neural-specific repression of the BAF53a subunit gene in the post-mitotic neuronal compartment. These studies should provide critical insights into the developmental roles of ATP-dependent chromatin remodeling complexes in neurons as well as generating essential reagents for my transition to an independent researcher in this field. This work will be a significant departure from my graduate studies on hemopoiesis and introduce me to proteomic approaches to complex biological problems.

Apoptosis is central to the processes of organogenesis, tissue remodeling, cell homeostasis, and defence processes. Apoptosis is executed by members of the caspase protease family. Although many caspases have been identified, little is known about their physiological role, how they are activated and how their activation is regulated. Thus, using Drosophila melanogaster as a model system, I propose to conduct two complementary strategies to investigate the physiological function of caspases: First, I will employ the RNA interference technique to generate potent and specific gene-silencing of the seven currently known caspases to reveal their role in Drosophila development. Second, I will uncover genes essential for caspase activation and inhibition using a novel EMS mitotic recombination screen. This genetic analysis is thought to reveal new members of the machinery that execute and control apoptosis in Drosophila. The combination of genetic with molecular and biochemical techniques will increase our understanding of how apoptosis is used as a constructive tool in development and how it may function as a defence strategy against the emergence of cancer. During my previous experiences, I mainly have been trained in Drosophila Genetics and Immunology. In the receiving lab, however, I intend to extend my scientific and technical knowledge in Development, Cell Biology and Biochemistry. Apoptosis is a fundamental process regulating immune responses. Since my long-term goal is to establish myself as an independent research scientist in the field of immunology, I expect to gain important insights from working in a laboratory focusing on apoptosis.

The neural identity and connectivity are generated from multiple, combinatorial fusion events during the production of mature RNA transcripts from genomic template in a process termed alternative splicing. Malfunction of this process in the mammalian central nervous system (CNS) causes neurological diseases (e.g. Alzheimer’s), yet little is known about the mechanism. Given that CNS is well known for its cell diversity, it is important to understand where these alternative splicing events occur and being regulated. To address this, I will develop a multiplex fluoresent in situ hybridisation (FISH)-based assay for dissecting the alternative splicing pattern of transcripts at single-cell level, starting from known neuronal examples. Proteins responsible for the CNS-specific alternative splicing will be identified by gene silencing techniques while using the assay to monitor visually and quantitatively the level of alternative splicing. In parallel, I will work closely with an expert on RNA bioinformatics, Dr. Chris Burge at MIT, and use a proven method RESCUE (Relative Enhancer and Silencer Classification by Unanimous Enrichment) developed by his group to identify sequence elements within these transcripts that are responsible for CNS-specific alternative splicing and eventually correlate the regulation of these elements to the cellular context using the FISH assay. Studying RNA-related phenomenon has been my long-term interest and this fellowship allows me to transfer my existing skills in quantitative imaging and bioinformatics, which are now mainly on protein, to the RNA field, whilst learning new techniques in RNA biochemistry and gene silencing in the host laboratory.

1)Light is an important environmental factor for plant growth and development. Previous analysis has revealed that the expression of about one-third of the Arabidopsis genome are up- or down-regulated by light. However, the transcription network and cascade to achieve such a vast change in gene expression is not known. In this proposed project, I will employ both the dynamic genome expression profiling at different time points after light treatment and mathematical modeling to reveal insights about the transcription network and cascade involved. Further, I will also investigate whether chromatin domain or gene-specific DNA methylation and histone modification are involved in the light-regulated genome expression. 2)My Ph.D work is about the morphological and molecular analysis of rice tiller development through examining a single gene. However, in this proposed project, the approaches to addressing biological problem will be changed to the whole genome level and holistic appreciation. I will use a combination of genomic and mathematical modeling approaches to study the light control of Arabidopsis development. I will obtain integrated expertise in diverse disciplines such as biology, genomics, bioinformatics, and mathematics. 3)This fellowship will allow me a postdoctoral training according to my optimal design and not subjected to any specific limitation that may be otherwise linked to a specific support from a host laboratory research grant. My selected post-doctoral research will not only bring about fundamental changes in the approaches, but also introduce me to a new biological problem. These new experiences will be invaluable to my future independent research career.

My long-term interest is to study the molecular events leading to neurodegenerative diseases. Alzheimer's disease and related age dependent dementia are major diseases affecting the aging population globally. Recent studies from our laboratory uncover an enzyme, prolyl isomerase Pin1 that plays a pivotal role in guarding against age-dependent neurodegeneration of Alzheimer's disease and suggest a new concept for therapy. However, how Pin1 protects the brain from neurodegeneration is not clear. The goal of my proposal is to elucidate how Pin1 interacts with the known pathogenic factors and how their interactions affect the development of Alzheimer's disease. The results should help understand how Alzheimer's disease and related diseases develop and might eventually lead to new therapies. This postdoctoral training, if funded, would allow me to apply my knowledge and experience that I learnt from delineating the signaling mechanisms during my PhD training to neuroscience, a completely different and exciting field. Furthermore, it would allow me to learn how to study neurodegeneration using various experimental models, including mice and cultured cells. Finally, my host lab is the leading laboratory in studying Pin1 and Alzheimer's disease, in addition to Harvard Medical School, which is rich in scientific tradition and vibrant in international collaborations. These outstanding training environments would enhance my ability to approach scientific questions with original ideas and methods. After the training, I hope to set up my own research program on neurodegeneration in Singapore, which is becoming the scientific research center in South East Asia region.

Ras homologue enriched in striatum (Rhes), a new member of the Ras GTPase superfamily, has been implicated in several cellular responses, one of which is insulin secretion from pancreatic beta-cells after exposure to insulin secratogues. The molecular mechanism culminating in increased insulin secretion and the role of Rhes in this process is currently unknown. The proposed research effort seeks to define Rhes function in beta-cells by identifying Rhes effector proteins and signaling pathways as well as by mapping the subcellular localization of this signaling molecule. My research background is heavily rooted in mechanistic enzymology, so the proposed research effort differs in several aspects from my Ph.D. research. I will enter an entirely new field where I will be exposed to many new techniques and, more importantly, perspectives unique to studying complex processes within the context of the whole cell. The primary research focus of Prof. Hans Bos’ laboratory is characterization of small GTPase function therefore, I anticipate mastery of relevant techniques in a timely manner. This fellowship will promote my development as a researcher due to the scientific excellence of the host laboratory and my enthusiasm for the proposed research.

The ability to analyze global gene expression patterns using DNA microarrays has revolutionized basic research in the life sciences, however, its usefulness has been precluded by the difficulty in gene transcripts from specific tissues or cellular populations. The goal of this project is to develop a system by which to isolate mRNAs whose expression is specifically enriched in the C. elegans nervous system or neuronal sub-type populations with the intent of developing a comprehensive understanding of the unique patterns of neuronal gene expression and how these patterns are affected by thermal stimulation. The nature of this project demands the coordinated contribution of multiple research disciplines including genomics, molecular biology, physiology, and behavior. Although my previous research training in cellular neurobiology will be an asset, the current project represents a significant departure from that field, both in the nature of the questions being asked and the tools being used to address them. This fellowship will be my first formal exposure to DNA microarray technology and to molecular genetic experimental techniques. I believe this broad experience is essential for the increasingly multi-disciplinary field of neurobiology. The training opportunity available to me as a member of the laboratory of Dr. Goodman and of the greater scientific community of Stanford University is first-class, and will provide a solid foundation as I continue my scientific career in neuroscience at an academic or government institution.

1) Bidirectional signaling by Ephs and ephrins regulates a number of processes during nervous system development. In addition, ephrinB2 signaling has an important role in vascular development. In spite of the recent progress in understanding the importance and basic activation mechanisms of ephrinB signaling the critical determinants and downstream effectors mediating ephrinB2 transduced cellular responses remain to be elucidated. The objectives of this project are to understand these molecular signals and to identify mechanisms by which ephrinB2 signaling is mediated in endothelial cells and in neurons. The studies will be carried out using mutant mice which have specific mutations in ephrinB2 and display more subtle phenotypes when compared to mice completely deficient of ephrinB2 signaling. Phenotypic analyses and cellular and biochemical studies using cells isolated from these mice can facilitate a detailed study of the critical ephrinB2 signaling components involved in vascular and neuronal development. 2) In my PhD work I studied the molecular mechanisms of lymphangiogenesis – in the present work I will focus on the regulation of nervous system development by ephrins and Ephs. Part of my project involves analysis of lymphatic phenotype in an ephrinB2 mutant, which enables me to use my previous expertise. However, my main goal is to enter the field of neurobiology. 3) Several molecules regulating vascular development appear to be involved also in the regulation of nervous system development and vice versa. Therefore knowledge of both research fields may give me an ability to produce novel ideas and to carry out my future studies using both systems in a flexible way.