Neuronal variability, which is necessary for the proper assembly of neuronal connections and the correct projection to different specific targets in the brain, is, at least in part, a consequence of differential regulatory gene expression and downstream control of axon guidance molecules during development. The goal of my research is understand the mechanisms underlying neural identity and how they lead to different projection phenotypes. I plan to approach the problem using a different system and a new perspective compared to those used by other labs. While work from other groups has outlined programs of cell type specification and directives for axon growth to peripheral targets using the spinal cord motoneurons or the visual projections to the cortex as a model, I will use the mouse visual system to focus on the identity among retinal cells resulting in specific axonal projection patterns with respect to laterality at the midline. I will then extend these studies to other binary decision points through the developing nervous system, specially the patterning of bilateral organization of other sensory systems in the CNS. This work will primarily shed light on a long-standing unsolved question: how are retinal axons specified to diverge to both sides of the brain? Deciphering this question should also provide information on disruption of visual routes by genetic perturbations such as albinism. Moreover, since Zic2, which controls the uncrossed retinal projection at the chiasm, is also responsible for severe malformations as holoprosencephaly and spina bifida in humans, my future work on Zic2 related genes in retina could help in the investigation of these anomalies

The goal of my proposed project is to understand the role of molecular chaperones in the maintenance of the prion state. I will use Saccharomyces cerevisiae as a model system and focus on the yeast prion [RNQ+] maintenance modulated by Hsp70 Ssa, using various analysis methods of genetics, cell biology, molecular biology, biochemistry, and physical chemistry. Firstly, I will isolate SSA mutants defective in maintenance of the prion state in yeast, and then characterize these effects using cell biological techniques. Next, to understand the mechanism at a molecular level, I will purify these mutants and characterize them in vitro assay systems using biochemical techniques. This project will provide us both fundamental understanding of the prion propagation mechanism in a cell and thus potentially have important implications for medicine and biotechnology. I have studied physical properties of protein in vitro using physicochemical and physical techniques, whereas in my proposed project, I will study protein functions in the cell using cell biological methods. Thus, there are significant differences between my previous research and my proposed project in both research direction and method. I am eager to acquire expertise in the cell biological and genetics field, in addition to the physicochemical and biophysical skills I already have. I think that a cell biological and molecular biological approach combined with in vitro biochemical and physicochemical techniques is important for unraveling the complex functions of living organisms. The HFSP fellowship will provide me a great chance to obtain training in a new area of research and to perform combined analysis approaches.

Malaria afflicts between 300 and 500 million people annually. The Plasmodium parasites responsible for malaria have demonstrated increasing drug resistance, suggesting that in time current treatments will be rendered obsolete. Malaria is almost exclusively confined to developing countries, and has been neglected by most major pharmaceutical companies. This project is aimed at creating new inhibitors for the malarial protease plasmepsin II and evaluating their effect on the pathology of Plasmodium parasites. The inhibitors will be devised using computer-aided de novo design, and made by standard synthetic methods. Evaluation will begin with in vitro determination of their ability to inhibit plasmepsin II. Biophysical characterization of the inhibitor-enzyme interactions will be carried out using crystallography and calorimetry. Finally, the effect of such inhibitors on parasite pathology will be examined. My previous research concerned the creation and study of self-organizing container molecules, and was driven by curiosity about the physical behavior of molecules confined within nanoscopic spaces. The present research is interdisciplinary, and through it I hope to gain experience with a variety of biochemical and biological techniques: computer-aided protein modeling, protein activity assays, protein crystallography, isothermal titration calorimetry, and small animal disease models. In the next stage of my career, I hope to use this experience to carry out research at the interface of chemistry and biology.

Our ability to store new information and memories is believed to require a property of certain neurons called synaptic plasticity--the ability to specifically and dynamically alter pre-existing connections between neurons. While it is known that electrical and chemical signals underlie synaptic plasticity the precise molecular components regulating this process are largely uncharacterized. I propose to investigate how synaptic plasticity is affected by the organization of different molecules at synapses. My post-doctoral research will focus on Ras and Rap1/2, proteins which appear to have the ability to change the physical structure of neuronal connections. This research will extend my previous experience in characterizing regulators of neuronal architecture in vitro to a study investigating the relationship between synaptic plasticity and molecules which alter synaptic structure in vivo. This post-doctoral investigation will allow me to synthesize several different scopes of research including biochemical and molecular aspects I focussed on in my PhD, and will develop my expertise in live imaging, production of knockout mice, and ultimately in behavioural analyses. These studies will not only deepen our understanding of synaptic plasticity and memory formation, but could ultimately provide the insights needed to counter injuries and combat diseases that impair memory. The knowledge I anticipate gaining from these studies under the direction of Dr. Morgan Sheng at MIT will better prepare me to direct my own academic research in which I plan to investigate the complexities involved in the function and integration of cellular signalling systems within an entire organism.

1) Proposed project: This project is the first approach to genetically dissect the mechanisms of metastasis in Drosophila. To identify the genes regulating metastasis, I will perform a large-scale genetic screen using a Drosophila cancer model. I will utilize a Drosophila model of benign tumor established in Dr. Tian Xu’s laboratory, and conduct a genetic screen to identify mutants acquiring metastatic features in these tumors. I will genetically isolate responsible genes for the ‘metastasis promoting’ mutations, and dissect the signaling pathways of metastasis both genetically and molecularly. I believe that it would bring a breakthrough in this research field. 2) How it differs from my previous research experience: So far I have studied the mechanisms of cell death in mammals and Drosophila. Taking advantage of powerful genetics of Drosophila, I propose a novel approach dissecting the mechanisms of metastasis through genetics. Metastasis is achieved by several stepwise progressions of tumor cells including cell growth, invasion and cell migration. This line of biological phenomenon can be accomplished independently of cell death machinery, indicating this research field is completely different from all my previous experiences. 3) How it will serve my professional growth: Through this project, I will experience highly complicated genetics and strategies against complex biological phenomena in whole animals. In addition, the genetic approach to understand the mechanisms of metastasis will provide me an experience to study various biological features of cells and their signaling pathways that I have never studied. These aspects will strongly support my professional growth.

1) To become functionally active, newly synthesized protein chains must fold to unique three-dimensional structure, but regulated unfolding also is critical during the life cycle of many proteins. Some unfolding processes show surprising similarities. For instance, many proteins in degradation and membrane translocation are targeted for unfolding by terminal signal sequences and translational movement of the substrate protein often occurs with unfolding. Taking together the previous results, the susceptibility of proteins to unfolding by protease and translocase depends on the proteins' stability against global spontaneous unfolding and, more importantly, on their local structure adjacent to the targeting signal. I assume that the unfoldases trap local unfolding fluctuation in the part of the substrate structure adjacent to the targeting signal effectively pulling at the polypeptide chain. To test this hypothesis, I will measure both the local unfolding fluctuations adjacent to the targeting signal in substrate proteins by using NMR spectroscopy as well as the physical stability of substrate proteins against pulling in atomic force microscopy experiments. 2) & 3) I have worked on the biophysical side of molecular chaperone which assists protein folding and my host laboratory has worked on biochemical and molecular biological as well as biophysical aspects of in vivo protein unfolding. Although protein unfolding is an opposite phenomenon to protein folding, my biophysical background will allow me to make unique contribution to biologically important in vivo protein unfolding and I will broaden my research experience to include in vivo protein unfolding.

In the proposed project, I will investigate fundamental questions concerning the link between behavior and neuronal activity in the brain. In order to address these questions, I will develop novel techniques for combining of electrical and optical recording from rodent cerebellum in vivo. The activity of the cerebellar cortical network will be visualized using voltage-sensitive dyes and a high-speed cooled CCD camera. At the same time, cellular and synaptic activity will be investigated using patch-clamp recordings from single neurons combined with two-photon microscopy. Changes in the activity of cerebellar cortex induced by sensory stimulation will be captured by these techniques. I will also try to induce long-term depression at the parallel fiber and Purkinje cell synapse by applying a paradigm of classical conditioning in order to test whether simple motor learning is associated with plastic changes at the cerebellar synapses. Until now I have been working on synaptic transmission in the brain slice preparation using patch-clamp methods. The framework of the proposed project is consistent with that of my previous work in the sense that it investigates synaptic modulation using electrophysiology. However, the project also offers me a transition from the slice preparation to the intact brain in vivo, with imaging techniques paired with electrophysiology. These technical developments will allow me to relate the synaptic dynamics that I have been studying to the function of neural networks and ultimately to behavioral events. This project will therefore enable me to expand the scope of my research and gain a wider range of experience in the field of neuroscience.

The ultimate goal of this research proposal is to dissect the molecular nature and the post-transcriptional regulation of two proteins, ABI5 and its interacting partner AFP, that are critically involved in ABA signaling pathway. This work is of significance because these proteins have been shown to interact in vivo at the sites of nuclear bodies where ubiquitin-mediated proteolysis can possibly occur. An understanding of the role of regulated proteolysis in ABA signaling will shed light on the importance of similar processes in plant developmental pathways. In addition, nuclear body speckle formation has been shown to be associated with the modification of proteins by SUMO. My experiments will demonstrate if AFP is a substrate for SUMO and if this modification has functional significance. Thus, this research proposal will not only help to define an important regulatory step in the ABA response pathway, but also offer wide vistas into novel mechanisms for maintaining homeostasis during plant development. As a doctoral student, I have studied the role of histone deacetylase in rice development and investigated the improvement of transgene expression in rice. Hence, my research experiences have been restricted to biotechnology based on plant biology and agricultural science. The opportunity to study the roles of regulated proteolysis in ABA signaling in Dr Chua’s lab will expand my scientific horizon with respect to learning new concepts in cell and molecular biology and acquiring additional technical skills. These experiences will greatly enhance my development as an independent investigator upon the completion of my post-doctoral training.

Cell lineage specific transcriptional programmes are set early in development and maintained, via epigenetic mechanisms, throughout the lifetime of an organism. The failure of maintaining &#8216 cell memory&#8217 may result in cell identity deviance, deregulation of cell proliferation, altered developmental programmes and cancer. Modulation of chromatin structure appears to be a key pathway controlling transcription memory. The balanced action of chromatin regulators of the PcG and trxG groups controls the expression of homeotic as well as of most developmentally regulated genes. There is an increasing evidence that PcG/trxG genes might be involved in control of cell proliferation and cell cycle progression, including stem cell renewal and tumor progression. By combining molecular (chromatin immunoprecipitation, RNAi/ribozymes) and cytological (BrdU-incorporation, histone modifications immunodetection) approaches, the aim of this project is to get more insight into the role of PcG/trxG proteins in cell totipotency and epigenetic levels of genome reprogramming. The proposed research would significantly broaden my experimental skills, and challenge entirely new aspects of genome function, namely how epigenome reprogramming may influence cell identity definition and redefinition.

Synapses in a nervous system form specific connections between subsets of neurons. Once an axon has been guided to its target region, molecular cues and neuronal activity control the formation of synaptic contacts with only a part of the possible target cells. The goals of the proposed project are (i) to identify the cell surface molecules involved in selective synaptic targeting and (ii) to characterize their molecular activities using the fruit fly Drosophila melanogaster as a model system. The fly olfactory system represents a genetically tractable system for rapid identification of novel cell surface molecules involved in target specificity. I propose to establish a genetic screen based on fluorescent synaptic markers expressed in two olfactory receptor neurons. In a subsequent line of experiments I will apply the tandem-affinity purification-tag methodology to purify interactors of candidate cell surface proteins identified in the screen. Previously, I focused on the role of cell surface molecules in the adult nervous system using mouse genetics, biochemical assays, and electrophysiology. The proposed project aims at understanding a developmental process using completely different techniques in the fly nervous system. Thus, this fellowship provides the opportunity of training in complementary techniques, which will prepare me excellently for my long term goal to establish my own laboratory. These experiments will give novel insights into mechanisms involved in target selectivity of synapses and furthermore, given the fundamental conservation between Drosophila and higher mammals, provide candidates for evaluation in mammalian nervous system development.

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.