I have studied mouse neural development by focusing on transcription factors that regulate neurogenesis. Molecular biology and reverse genetics techniques such as gene targeting and viral misexpression were useful to clarify the function of transcription factors in embryogenesis. During the course of the above research, I have got interested in the mechanism by which the complex neuronal networks are generated. I propose a genetic analysis of the development and function of neural circuits regulating a simple behavior in zebrafish. The earliest locomotion behavior exhibited by developing zebrafish is spontaneous coiling of the trunk. There appears to be two different behaviors. The earlier coiling is independent of the brain and appears to require electrical activity of spinal neurons but not synaptic transmission. The later coiling requires input from the hindbrain and synaptic transmission within the spinal cord. I will examine the earlier coiling by ramp assay, which distinguish the mutants defective in slow coiling. The later coiling defect exhibits curled trunk by dechorionation at 30 hpf. Screening by these assays should identify coiling mutants. I will directly examine these behaviors by recording with a CCD camera. I will also examine both the morphology (immunostaining and dye labeling) and physiology (Calcium imaging and patch clamp recording) of spinal and hindbrain neurons involved in coiling behaviors. Possible simple circuits of zebrafish neuronal network would be suitable for detailed analysis of vertebrate behavior. With the above research project, I hope to extend my research career to zebrafish genetics and neurophysiology to be a more creative researcher.

1) Nonsense mediated decay (NMD) is a ubiquitous quality control mechanism ensuring correctly processed mRNA, and thereby preventing human genetic disease in many cases. Recently, several protein factors have been identified that seem to be involved in NMD, both in the nucleus during splicing and in the cytoplasm triggering enzymatic degradation of target mRNA. However, the interactions of these exon junction components (EJCs) with factors executing degradation have yet to be dissected in detail. We propose experiments that will result in characterization of these interactions, as well as identification of additional proteins that may be involved in NMD. Specifically, we will compare the NMD activity of the U1/U2 and U11/U12 spliceosomes, as these complexes have significantly different protein compositions we will make use of EJC protein constructs and affinity chromatography to identify additional proteins involved in NMD and we will use a tethered protein construct that triggers NMD, combined with RNA interference technology to selectively remove other cooperating proteins, in order to elucidate the interactions between the tethered protein and its NMD partners. 2&3) My prior research experience is in the area of biophysics (i.e. thermodynamics and calorimetry) and my projects were heavily grounded in physical chemistry. I have very minimal experience in molecular biology and no experience in research applied to human disease. As part of this project I will learn cutting-edge molecular biology techniques (including RNA interference technology) as well as working at the interface between medicine and biology, which will expand my scientific capabilities tremendously.

Information is stored in the brain via modifications of synapses -- a major goal in neuroscience is to understand the molecular mechanisms underlying such synaptic plasticity. Synaptic strength is determined in major part by the number of AMPA receptors in the postsynaptic membrane. Recent studies have shown that the AMPA receptor-binding protein GRIP and its binding partner liprin play an important role in the targeting of AMPA receptors to synapses. Although the biological function of liprin/GRIP association remains unclear to date, data from the host lab suggest that the liprin/GRIP complex is involved in the transport of variable cargo molecules in neurons.This proposal describes experiments that address the role of liprin/GRIP in protein trafficking in neurons. I propose to immunpurify and identify all the components of the liprin/GRIP complex by mass spectrometry to discover the possible cargo molecules that are transported in dendrites and axons by this complex. Concomitantly, fluorescent time lapse imaging will be used to analyze the dynamics of GFP-tagged liprin and GRIP in live neurons during development and in response to synaptic activity. These measurements will lead to a detailed analysis of the regulated dynamics of the liprin/GRIP complex. This project allows me to extend my knowledge in the mechanisms of synaptic plasticity and to acquire new technical skills (e.g. biochemistry, neuron culture) in neurobiology assay systems. Already during my graduate period I received excellent training in general molecular techniques and microscopical imaging. This training provides a basis to successfully accomplish the more ambitious goals set out in this proposal.

The proposed post-doctoral research is to characterize Arabidopsis SPA1 which is involved in PhyA signaling, especially, to identify the biochemical mechanisms of action of SPA1 in the far-red light signaling. Based on some previous data, it is postulated that one function of SPA1 is to regulate the activity of COP1, which acts to repress photomorhogenesis in the absence of light and directly interacts with SPA1, as a ubiqutin E3 ligase. To elucidate this hypothesis, I propose to carry out two series of experiments on SPA1. In the first, I will examine the effects of increasing the concentration of SPA1 on both auto-ubiqutination in vitro and determine the subcellular localization of SPA1 to investigate possible protein co-localization with COP1 in plant cells. In the second, to examine the function of SPA1 gene in Arabidopsis, SPA1 gene will be placed under the control beta-estradiol inducible promoter and introduced into wild type, phyA and cop1 mutants of Arabidopsis and the phenotype will be examined under various light conditions in the absence or the presence of the inducer. In addition, I will screen T-DNA-tagged Arabidopsis plants for SPA1 homologs mutations and examine the phenotype by the insertion of T-DNA. These experiments should dissect the role of SPA1 in PhyA signaling. As described above, the proposed post-doctoral training will allow me to use a combined genetic, molecular and biochemical approach to elucidate an important problem in plant development in response to light, the role of PhyA-signaling, although my doctoral training has mainly been in plant cell biology.

It was recently shown that the alpha subunits (Ga12 and Ga13) of the G12 subfamily of heterotrimeric G proteins directly interact with the cytoplasmic domains of several cadherins, a family of cell adhesion proteins. This interaction triggers the release of cadherin-bound beta (b)-catenin, which relocalizes to the nucleus and causes up-regulation of b-catenin mediated transcriptional activation of cell proliferation genes. In this application, it is proposed to dissect the cadherin-mediated signaling pathway of activated G12 from its other cellular signaling processes. The specific region of Ga12 that interacts with cadherin will be mapped and this information then used to develop Ga12 mutants and also peptide inhibitors, that selectively interfere with the Ga12/cadherin interaction but do not affect Ga12 binding to its other best-characterized effector, p115RhoGEF. The consequences of blocking the Ga12/cadherin interaction by these reagents on the ability of G12 to stimulate b-catenin-mediated transcriptional activation and promote cellular transformation will then be examined. My doctoral thesis work involved in vitro studies of protein folding using spectroscopic methods, primarily NMR. I have limited experience so far in cell biological work. Working in Dr Casey&#8217 s laboratory will provide me a solid foundation of cell biology in general and cellular signaling in particular. I feel that my protein chemistry background will help me enter the field of the cell biology of signaling with a wider perspective. In the future, research experience in structural as well as cell biology will allow me to approach important problems in biology with a broad array of techniques.

Human neurodegenerative diseases are characterized by the progressive loss of specific neuronal populations, and most of these diseases have been successfully modeled in flies. We propose to develop a new method to measure neuronal survival in the intact fly,using the luciferase gene.This new method has multiple advantages: it is very easy to perform and offers quantitative, in vivo monitoring of neuronal survival as a function of time.It will provide a powerful new tool to follow the toxicity of any protein on a specific neuron subpopulation at any developmental stage. Using this method,we will investigate the effect of expressing neurodegenerative disease proteins on specific neurons.We will then perform genetic screens,with the goal of identifying of genes involved in the enhancing and/or suppressing of the neurodegenerative phenotype associated with the expression of these disease proteins.The hope is to make a contribution to one of the most intriguing and unknown aspects of this problem: why the neuronal death is cell-type specific, i.e.,why a toxic protein kills specific neurons and not others.Some of these tools will also be used to study aspects of the Drosophila circadian clock. As a postdoctoral fellow I expect to gain experience in Neurobiology. This project will give me the opportunity to combine the vast array of molecular biology-tools learnt in my PhD and the very versatile system of fly genetics, and apply them to study a field in fast expansion as Neuroscience.After my postdoc, I plan to pursue an academic career in neuroscience.I expect that my experience as postdoc in the United States will enable me to be in touch with a vibrant scientific community.

Nuclear pore complexes (NPCs) are large supramolecular assemblies that provide channels of transport across the nuclear envelope (NE). Emerging evidence indicates that the NPC constituents, the nucleoporins, in addition to their role in macromolecule trafficking, also participate in nuclear architecture and are components of the mitotic apparatus. By using the early Drosophila embryo as a model, the objectives of this project are a) to determine the role of the Drosophila nucleoporins Nup107 and Nup133 in kinetochore structure and function during mitosis in living embryos, and b) to study the dynamics of these nucleoporines in NE assembly and disassembly in vivo. To address these novel questions, we will take advantage of the available technology that permits the introduction of interfering agents at precise moments of the mitotic cycle in the living embryos and of live-imaging technology that allows fluorescently tagged chromosomes and proteins to be followed in real time. During my PhD I was trained in biochemistry and studied steady-state protein function in well-defined in vitro conditions. This project will expose me to completely new approaches to study protein function at another level, in vivo and in real-time. My training in new disciplines, in genetics and cell biology, together with my biochemistry background will complement my scientific skills in approaching and tackling biological problems. Although I am moving to a new field, I will still remain in the broader area of nuclear organization, where I would like to pursue a career as an independent researcher.

My proposal is focused on the functional regulation of mouse ventral anterior homeobox (Vax) genes, Vax1 and Vax2, and on the identification of their targets. There is almost no information on the upstream signals that regulate the Vax genes and their downstream target genes, although the Vax genes are clearly important factors for ventral fate determination in the vertebrate eye. Thus, I want to identify the mechanisms underlying the activation of Vax proteins, and the genes regulated by Vax proteins. Vax2 has been detected in the cytoplasm as well as in nuclei of developing mouse eye, in contrast to the exclusively nuclear location of Vax1. I will use molecular biological and biochemical approaches to investigate the mechanism of nuclear localization of the Vax2 protein. I have performed similar research previously. The biological significance of the nuclear translocation of Vax2 will be studied in vivo using transgenic and knock-in mouse techniques. In addition, I will perform chromatin immunoprecipitation (ChIP) to identify Vax target genes. My previous training in biochemistry and cell biology will provide a synergistic complement to these studies in developmental biology and mouse genetics.

Flowers can be categorized into two major groups zygomorphic (asymmetrical e.g. pea and snapdragon) and actinomorphic (radially symmetrical e.g. tulip and cherry). This floral asymmetry is considered to be a derived condition to attract specific pollinators. It has been proposed that floral asymmetry has evolved many times independently. Recent work on Antirrhinum, which produces asymmetrical flowers, suggests that several genes, CYCLOIDEA, RADIALIS, DICHOTOMA and DIVARICATA, play an important role in generating floral asymmetry. A major goal of this proposal is to elucidate these gene interactions in Antirrhinum using genetic and molecular approaches. In addition, Arabidopsis orthologs of these genes will be examined to understand how regulatory relationship have changed or been conserved in Arabidopsis which produces symmetrical flowers. A comparative analysis of the changes or conservation in the regulatory cascade of these genes in Antirrhinum and Arabidopsis will help us to understand the evolution and diversity of flower morphology. This study of floral symmetry will be different from my thesis project. For my Ph. D. I studied ab-adaxiality and compound leaf development, and mainly focused on developmental and molecular aspect of leaf development. Here, I am planning to examine the regulatory relationship among genes (including protein works) to understand the floral asymmetry, which is new aspect to me. This postdoctoral research experience will be a great opportunity to understand the similarity and diversity of developmental processes between leaf and flower as well as among different species like tomato, Arabidopsis and Antirrhinum.

The aim of this project is to study the trigeminal ganglion system in zebrafish as a model for neural circuit formation and function in vivo. While the physiology of the differentiated trigeminal ganglion has been well studied due to its key function in pain perception, the processes that lead to its formation are poorly understood. Already during early development a population of cells is singled out to form neuronal precursors that grow out, form intricate projection patterns and differentiate into functional circuits of the trigeminal ganglia. Thus, the trigeminal ganglia offer the unique opportunity to investigate both the specification of neurons and the formation of functional circuits in vivo. In order to address these questions, I first propose to study the role of the transcription factor neurogenin in trigeminal specification and determine the developmental potential of placode- and neural crest-derived trigeminal precursors. Second, I propose a genetic screen assaying for neuronal specification, differentiation and circuit formation to extend the analysis to novel factors. In the past, such genetic approaches have delineated numerous biological processes at the molecular level. The proposed fellowship will provide fundamental training in vertebrate genetics, embryology, cell biology and neurobiology. It will complement my doctoral training in early vertebrate patterning and migration processes. Together, my experiences and the training obtained in the proposed studies will permit me, as an independent investigator, to continue my studies on trigeminal development and function with the final goal to understand the pathways leading to neuronal circuit formation.

In different environments, genetically divergent populations tune their way of life to compete successfully for resources. While variation across large phylogenetic distances is representative of macroevolution, polyphenisms within a species or population are likely to be the most abundant elements to drive evolution. Studies addressing the genetic basis of phenotypic variation within populations will therefore provide novel insights into the mechanisms of biodiversity. The central focus of this proposal is to understand the molecular relationship between sexual selection (female mate preferences) and the male body-color patterning in guppies, Poecilia reticulata. This tropical fish has been one of the most important species to study the relationship between “intra”-specific (within a species) natural variation and adaptive traits, including sexual selection, an important question in evolutionary theory. However, the molecular basis of this variation is completely unknown. This project aims to lay the foundation for understanding sexual selection in guppies as a model study for quantitative evolutionary genetics.

Lifespan in C. elegans is determined by a genetically manipulatable regulatory network. Components of the DAF-2 pathway, a major lifespan limiting pathway in this network, have been isolated and ordered, indicating that it acts, at least in part, by retaining a transcription factor called DAF-16 in the cytoplasm, preventing it from transcribing longevity genes. Yet, recent evidence suggests that the DAF-2 pathway diverges and inhibits lifespan in more than one way, implying that only the coordinated removal of at least two lifespan limiting signals will allow life extension. We designed genetic screens to identify lifespan regulatory pathways, diverging from the DAF-2 pathway, that inhibit or collaborate with the transcription factor DAF-16. Likewise, a biochemical approach will be used to identify trans-activating factors that might bind to DAF-16 directly, and regulate its function. The sufficiency and requirement of each gene to influence lifespan will be examined, together with its ability to regulate DAF-16. These studies should further our understanding of lifespan regulation. My experience in characterizing novel molecules provides me with tools to study the new longevity genes I will discover. At the same time, the proposed project will introduce me to the model organism C. elegans. The analysis of the longevity mutants and the techniques of the proposed genetic screen, require a high level of expertise. I will learn methodologies such as RNAi screening, genome profiling and Nomarski optics. I am confident that learning and applying these techniques will greatly enrich me and supply me with new and essential tools and perspectives for my future independent research.

This project aims to build upon recent developments in ultrafast multi-dimensional (MD) infrared spectroscopy to probe the fastest and most fundamental motions of proteins. By taking advantage of the host laboratory’s extensive experience in state of the art laser systems, MD spectroscopy, and protein dynamics, a novel two-color infrared (IR) echo experiment will be used to investigate the ultrafast dynamics of protein secondary structure. By combining a dual-wavelength IR system with ultrafast laser pulse shaping, it will be possible to observe and potentially control the nuclear motion ultimately responsible for protein function including energy transduction and folding pathways. Ultrafast spectroscopy is now a general field of study with broad applications to all areas of chemistry and physics. While both my previous work and this proposed project are examples of MD spectroscopy, the technology and systems under study are fundamentally different. A protein is a tightly bound network of amino acids with a well-defined equilibrium structure, whereas liquids are disordered systems with much more subtle interactions. Furthermore, I have no hands-on experience with infrared spectroscopy or proteins. My interest in biological systems is firmly rooted in the inherently multidisciplinary approach required to understand complex systems. The diversity of the Ecole Polytechnique and the Laboratoire d’Optique et Biosciences offers an ideal opportunity to develop a collaborative style of research that I can continue to apply and refine throughout my career. This project will expand my technical ability as well as my capacity to work with and learn from colleagues in many fields.

Plant stem cells are of central importance to us, since almost all food is ultimately derived from these cells. This proposal is aimed at elucidating how a network of transcription factors regulates stem cell homeostasis in the plant Arabidopsis thaliana. To this end, I will use an integrated approach of genetic, genomic, molecular and biochemical methods. Specifically I will: A) Collaborate with others to solve the structure of two key transcriptional regulators, LEAFY (LFY) and WUSCHEL (WUS). B) Carry out new genetic screens in yeast and Arabidopsis using inducible alleles as well as reporter genes. C) Investigate the mechanisms of cross regulation among stem cell promoting and inhibiting factors. D) Identify binding sites for LFY and WUS on a genome-wide scale using inducible alleles and genomic arrays. E) Identify both direct and indirect target genes of LFY and WUS by expression profiling. F) Identify redundant mechanisms of stem cell control. G) Analyze in detail the biological function of the identified factors. My ultimate goal is to understand the interaction between all relevant factors to create a functional map of stem cell regulation in Arabidopsis, including models with predictive functions. The proposed project will expand the scope of my research from floral patterning to stem cell regulation. Thus, It will further differentiate my research interest from that of my postdoctoral advisor, Detlef Weigel, to establish myself as an independent scientist. I will derive long-term benefits from the additional funding, which will allow me to establish cutting edge methodology and new interdisciplinary collaborations.

1.Proposed project The project I propose to do is a computational analysis of genes that are involved in human hereditary diseases. It is assumed that the genes found to be mutated in human disorders could have differential properties from the molecular, genetic and/or evolutionary perspective than other genes not known to be involved in human disease. Several analyses will be conducted using computational methods on the all known disease genes as a group, compared against a proper control set, and of several groups of genes involved in different types of diseases that would be properly classified and clustered according to their medical significance. 2.How it differs from your previous research experience My work until now has been focused on the molecular basis of hereditary hearing loss. I have been working on the analysis of several candidate genes for deafness in Spanish families and I have performed an expression analysis of beta-connexin genes involved in deafness in the developing mouse cochlea. The project I propose is also involving human genetics but from a completely different perspective. I will focus my work on the computational analysis of genes and genomes while keeping a human genetics perspective to take advantage of my previous research experience in the experimental medical genetics field. 3.How it will serve your professional growth The proposed project will enable me to learn aspects from a new discipline, which I consider valuable for my growth as a scientist. I will learn to use computational techniques for the analysis of biological problems and to develop new computational tools to achieve the proposed goals and to examine the hypotheses formulated.