During my PhD study, I dealt with neurological questions related to promotion of recovery from spinal cord injury (SCI) and to the mechanisms involved in CNS degeneration. Throughout this period I was overwhelmed by the capability of controlled cell-mediated immunity to determine the outcome of CNS injury. I have witnessed significant differences in functional recovery, which correlated with the timing, dosage and antigenic specificity of immune cells homing to the lesion site. Uncontrolled hyperinflammation led to exacerbation of the damaged while a benign, well-regulated response significantly improved recovery from SCI. Since CNS inflammation is generally considered harmful, the current treatment for SCI includes a high dose of immunosuppressive steroids. However, my view is that controlled-inflammation may be developed as a therapeutic strategy of harnessing the body's own healing process for the treatment of degenerative pathologies. Therefore, in my postdoctoral study I would like to lay aside neurobiology and learn more about immune regulation, applying both animal 'in vivo' models and the human 'in vitro' system. Control over the induction of tolerance and immunity, will allow treatment of autoimmune diseases without complete immunosuppression, and "safe" activation of specific immune responses towards toxic self-compounds and malignant cells, in accordance with particular tissue requirements .It is a new discipline and I will have to acquire numerous new techniques, but I believe that the combination of my current knowledge with the expertise I will obtain, will enable me to develop into a skilled researcher in the field of neuroimmunology.

1) Goal of the project is to address the role of aminophospholipid translocating ATPase (ATPase II) in neuronal functions, especially in the exo-endocytosis of synaptic vesicles (SVs). ATPase II is a member of a subfamily of P-type ATPases and is thought to play a role in the maintenance of membrane phosphatidylserine (PS) asymmetry in certain cellular organelles. There is indirect evidence that PS translocation is important for the exocytosis of secretory vesicles and genetic studies have demonstrated a functional interaction of the yeast homologue of this ATPase (Drs2) with clathrin and with the endocytic protein Pan1. ATPase II is especially rich in brain and my preliminary experiments indicate that ATPase II is specifically concentrated on SVs. The project will address the role(s) of ATPase II in neuronal function and will focus on its role in exo-endocytotic cycle of SVs. 2) My previous research experience has focused on the characterization of synaptic-like microvesicles in endocrine cells. As part of these studies I have identified vesicular glutamate transporters in vesicles of peripheral tissues. I have mainly used immunohistochemical and electronmicroscopical techniques. In this proposal, I want to clarify novel aspects of exo-endocytotic cycle of SVs, and I plan to familiarize myself with biochemical and molecular approaches. 3) I believe that broadening my technical and intellectual expertise in a top laboratory will be essential to complete my training toward an independent career in Neurobiology. I also believe that the field at the interface between lipids and membrane traffic is a promising area which is likely to blossom over the next few years.

1) A genome-wide RNAi based screen performed in the receiving laboratory identified 58 genes whose deletion leads to genome instabilities in the nematode C. elegans. My aim is to define which of these mutator genes imply a defect in cell cycle checkpoints, and specifically in p53 dependent growth suppression and apoptosis. I will first characterise the mutants phenotypically by screening for apoptotic cells. Next, a reporter system will be set up and used to directly measure p53 induction in both space and time. In the second part of the work, a new genome-wide RNAi based screen will be performed in order to identify genes whose deletion interfere with p53 induction following genomic instabilities. This should enable us to discover all the major components of the p53 cascade in C. elegans. 2) My proposed project will allow me to use a new, multicellular model: C. elegans. Studying genome stability, I will enter a new scientific question that I can build on my general cell cycle background. 3) My long-term goal is to set up a group using C.elegans to study how cell cycle regulations are integrated with development, a field basically left untouched so far. Therefore, this second post-doc in a leading C.elegans laboratory will be an invaluable chance carry out all the basic methods used with this organism and to complete my learning about genome stability. Last but not least, I believe that entering such a big research group will enable me to create a network of relationship in the C. elegans community that will be essential for my future.

In this project, nanoparticles will be developed and applied to the study of biomolecular conformation, dynamics and interactions using single-molecule spectroscopy, both in vitro and in vivo (in cells). Applications include DNA-DNA, DNA-protein and enzyme-substrate mechanisms. Furthermore, nanoparticles will be combined with biomolecules to design supramolecular assemblies for future applications in miniaturization of electronic devices below the limits of top-down approaches. The advantages of using semiconductor nanoparticles (quantum dots, QDs) in biomolecular fluorescence studies are numerous. They can be tuned in size, color, chemistry and biofunctionalization far more easily than dyes. They have high emission intensity, are extremely photostable, and are easily prepared. High spatial resolution can be further enhanced close to that using near field techniques (~10-50 nm) using colocalization methods – a method unique to QDs. These ultrasensitive, multicolor methods will allow one to elucidate details that are averaged out in ensemble measurements, but have so far been unattainable using current fluorophores. Moreover, metallic nanoparticles will also be employed for single molecule Raman spectroscopy (SERS). Most of my experience to date has been in the field of bulk biophysical spectroscopy. I have elucidated a number of important scientific findings using these methods, but it became clear that some details are averaged out in these ensembles. The proposed research will help alleviate many of these limitations. I plan to obtain a permanent position in academia following this research and continue to further develop the important field of nanobiotechnology.

Phytochromes, plant photoreceptors tracking the red and far-red light wavelengths, possibly perform dual role as they were found to partition between the cytosol and the nucleus in a light dependent manner. The proposed project addresses the questions of how phytochromes are retained in the cytosol and how partitioning is regulated. The functional NLS (nuclear localisation sequence) will be mapped and it will be investigated whether retention of phytochromes in the cytosol is due to intramolecular, light-dependent masking of the functional NLS or whether proteins binding to phytochromes prevent translocation to the nucleus. Light dependent nuclear translocation of phytochromes will also be analysed in insect cells transiently transformed with up to eight expression constructs. Thus it will be attempted to reconstitute light induced gene expression in this non-plant system. During my postdoctorate I would like to extend my knowledge on genetics, signal perception and transduction as well as on transcriptional regulation and circadian rhythms in plants. Therefore I decided to switch fields and join one of the leading laboratories dealing with the effects of light on phytochrome function. In Professor Schäfer’s laboratory in Freiburg I will become familiar with numerous new methods and techniques such as transient gene expression in Drosophila Schneider cells, in vivo spectroscopy and FRET (fluorescence resonance energy transfer). Especially exciting is the interdisciplinary aspect of this project dealing with the impact of the physical properties of light such as wavelength, fluence-rate, periodicity and direction on the plant biological system.

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.