Mice infected with low numbers of wild type (wt), but not of SecA2-deficient intracellular bacteria Listeria monocytogenes (LM), develop CD8+ T cell-mediated long-lasting immunity, and are resistant to secondary infection with high numbers of wt LM. SecA2 is an auxiliary secretion system of Gram-positive pathogenic bacteria which controls the secretion at least fifteen bacterial proteins. Although not protected, mice immunized with SecA2- LM mount robust secondary CD8+ T cell responses. Also, dendritic cells (DC) are required to prime T cells. Based on this, we hypothesized that one of the SecA2-secreted protein triggers an unknown intracytosolic pattern recognition receptor necessary for protective CD8+ T cells differentiation. Our first aim will assess whether immunization with DC coexpressing a SecA2-secreted protein and an LM antigenic peptide mount antibacterial protective memory responses. Our second aim will determine which intracytosolic receptor is targeted by this bacterial protein. Long-term approaches will involve knocking-in this receptor in vivo and study how it impacts protective memory induction. Our third aim will focus on real-time dynamics of bacteria, bacteria-infected cells and T cell activation using biphotonic microscopy. Specifically, we will test whether SecA2- or wt LM-primed CD8+ T cells undergo distinct dynamic phases during priming. During my HFSP-sponsored postdoctoral period, I improved my CV and was granted an Assistant Professorship in France. Since then, I built-up my research system and accumulated new data. A CDA would further secure my position and funding for the coming years as well as support my carrier long-term perspectives.

Cohesion between sister chromatids is essential for the faithful inheritance of chromosomes through cell division. Cohesin, the protein complex responsible for tethering sister chromatids was extensively characterized in yeast cells by genetic and biochemical methods. Together these data led to a comprehensive hypothesis of the cohesin cycle: the loading of the ring-shaped complex onto chromatin in G1, the establishment of cohesion in S and the release by cutting of the cohesin ring in M phase (Uhlmann, 2004, Exp Cell Res 296, 80-85). However, the biochemical assays utilized in the past lack the spatial and temporal resolution required to directly prove or refute the proposed mechanism and to generate new mechanistic models. I will therefore establish an experimental system combining biochemical and quantitative imaging methods to directly visualize the functional dynamics of cohesin on chromatin in Xenopus egg extracts as well as in cultured mammalian cells. I will develop fluorescence based reporters, to assay the local composition, conformation and dynamics of cohesin complexes. With these tools, I will focus on two major questions. First, how, when in the cell cycle and at which chromosomal locations does cohesin loading occur? And second, how do cohesin complexes establish sister chromatid tethering during replication? These experiments will advance the mechanistic understanding of the essential function of the cohesin complex. In addition, as the majority of previous work was carried out in yeasts, my studies in vertebrate systems will supply important information about the conservation of the mechanism of sister chromatid cohesion during evolution.

During the last decades biology has decomposed cellular systems into genetic, functional and molecular networks. It has become evident that these networks consists of components with specific functions (e.g. proteins and genes). This has generated a considerable amount of knowledge and new hypotheses. The idea presented here is to test part of this knowledge by building, through reverse engineering, new synthetic biological systems from the known components. Two projects are proposed: 1. A synthetic biology project, aimed at creating new modular signaling proteins: Proteins in signaling networks are modular in nature. I propose that the modularity of these proteins are now so well understood and comprehensively cataloged that we can begin to use the modules as building blocks in new synthetic modular systems and proteins. Designing and successfully introducing synthetic proteins into cellular signaling networks would provide us with a powerful research tool with many applications, such as development of biosensors, protein drugs and rewiring of biological pathways. 2. Modular decomposition of protein interaction networks, aimed at deriving network topology and dynamics: Deriving the topology and dynamics of protein-protein interaction networks in cellular signaling systems under different conditions (e.g. DNA damage response and disease states) will be useful for the understanding of robustness, complexity and checkpoint mechanisms of biological systems. I will perform integrative studies, based on synthetic biology, proteomic assays and bioinformatics techniques, of the topology and dynamics of both biological and engineered synthetic signaling protein interaction networks.

Nearly 100 years ago, a phenomenon termed heterosis was observed when hybrid plants outperformed their inbred parents for growth and vigor. Heterosis has been exploited in plant and animal food production, and it was a major contributor to the agricultural boom known as the ‘Green Revolution’. But heterosis has a more fundamental biological significance in the evolution of plant and animal species by providing a heterozygote advantage in naturally occurring hybrids. It has been shown that heterosis is a genome-wide phenomenon whose complex phenotypic effects involve the action of many genes. But the specific genes controlling heterosis remain unknown. Research in the laboratory of Dr. Dani Zamir at Hebrew University in Israel uses tomato as a model system to decipher the molecular basis of multigenic complex traits that exhibit a continuous range of phenotypes, as opposed to those that fall within discrete classes. My expertise lies in using molecular analysis to address the genome-wide regulation of gene expression through changes in chromatin (epigenetic) states. I intend to combine my knowledge with that of Dr. Zamir’s group, where I will develop new skills in quantitative genetics, statistics and bioinformatics. This combination of tools will enable me to trace heterosis to individual genes and study their molecular effects in a genomic context. This broad training will make me proficient in all aspects of analyzing complex traits, which I believe will be an exciting and leading area of research in the future.

The inter-cellular transfer of regulatory transcription factors and mRNAs has become a new paradigm in plant biology in recent years. The transcription factor KNOTTED1 (KN1) was the first such example of a developmental signal that traffics from cell to cell. Its conserved DNA binding domain, the homeodomain, is sufficient to mediate its own transfer from cell to cell in vivo, as well as promoting transfer of the KN1 mRNA. The molecular details and significance of this interaction as well as the significance of KN1 mRNA movement remain elusive. I therefore propose to investigate these questions by: (i) determining the sequences required for the interaction between KN1 and its mRNA, (ii) uncovering the molecular mechanisms of KN1/mRNA intra- and inter-cellular trafficking. (iii) Analyzing the physiological significance of KN1/mRNA trafficking in plant development. This interdisciplinary project aims to study the molecular mechanisms of a new paradigm in intercellular communication: the direct transfer of mRNA and transcription factors from cell to cell. My previous research experience has focused on evolutionary plant molecular biology and genetics. This project will train me in plant cell biology and biochemistry. Finally this project will provide the starting point for me to become an independent investigator.

During nervous system development, neurons have exquisite ability in selecting their synaptic partners among other adjacent cells and forming synapses only onto discrete and stereotyped subcellular regions of these targets. The molecular mechanism of such specificity is largely unknown. In C. elegans a genetic screen for mutants with defects in synaptic specificity has identified SYG-1 and SYG-2, two proteins of the Immunoglobulin superfamily that are both localized at synapses. In both syg-1 and syg-2 mutants, egg-laying motor neuron HSNL fails to recognize its synaptic partners and forms ectopic synapses onto abnormal targets, although its axon trajectory is normal. Interestingly the ectopic synapses in the syg mutants are still formed onto a selective group of targets, suggesting that synaptic specificity is altered but not eliminated in the absence of SYG-1/SYG-2. These data raise the possibility that synapses are formed as a result of synaptic competition between hierarchical potential targets. As a post-doctoral fellow in Dr Shen’s lab I propose to test this model using complementary cellular, molecular and genetic approaches in C. elegans. I will perform time-lapse experiments to ask if synaptic competition occurs during normal development. I will further test whether wild type synapses are required for suppression of ectopic vesicle clustering, by ablation and rescue experiments. Finally I will identify the factors downstream SYG-1/SYG-2 involved in synaptic competition using different genetic screens. In parallel I will develop trans-synaptic markers that will allow functional studies on synaptic specificity and neuronal networks development in C. elegans.

Thrombocytopenia, or low platelet count, is a serious problem in hematological and oncological clinical practice. The best mouse model for this disease is the Mpl-/- mouse, which lacks the receptor for thrombopoietin, and has only 10% of normal platelet levels. Recently, ENU mutagenesis-forward genetic screens have been used in the mouse to investigate gene-phenotype relationships. Thus far, these screens have almost exclusively used wild-type mice as a starting point. In contrast, we plan a large-scale forward genetic screen based on Mpl-/- mice. By screening the offspring of ENU-treated mice for elevated platelet counts we aim to find dominant suppressors of thrombocytopenia. These mutants will be identified and characterised biochemically. Like most small molecule therapeutics, the majority of ENU induced mutations lead to loss of function of proteins. Thus, in addition to the primary aim of significantly advancing our knowledge of platelet biology, these studies may identify new drug targets for the treatment of thrombocytopenia. During my PhD, I have used gene targeting technology or ‘reverse genetics’ to study signalling downstream of insulin/growth factor receptors. While this approach has been invaluable in investigating countless biological systems, it requires prior knowledge of the gene of interest. In contrast, a forward genetic screen selects for mutations in genes that have a function in the biological process of interest. A fellowship will allow me to acquire experience in murine forward genetic screening. I believe this, in combination with the techniques in reverse genetics learned during my PhD, will place me in a powerful position in the future.

The posterior parietal cortex (PPC) has been implicated in sensorimotor integration. Many of its specialized areas have increased firing rates during working memory in delayed-response tasks, but little is known about the neural mechanism that gives rise to the sustained activity. It has been suggested that oscillatory activity in the gamma frequency band (> 30 Hz) could be significant. The current proposal is intended to test this idea in human sensorimotor integration, using magnetoencephalography (MEG). More specifically we will ask whether gamma oscillations play a role in the encoding, maintenance, and updating of spatial working memories for eye and reaching movements. The research will yield significant new results and will have profound consequences for how we think about the computational physiology of sensorimotor control. This CDA makes it possible to develop a new research line, with novel research projects, as described in this proposal. The CDA will enable me to gain expertise with a relatively new neuroimaging technique that would be adequate to ensure future government funding for my research.

Courtship behaviors are highly specialized, innate behaviors essential for propagation in higher animals. Pheromone recognition is the most important sense for this process in many animals, including many insects. Recently, a candidate pheromone receptor with a specific function in male courtship was identified in Drosophila, providing a precedent for a single pheromone receptor gene with a specific behavioral function. Moreover, the gene is closely related to seven additional genes, and it is likely that some or all of them function as additional pheromone receptors. I plan to characterize expression of four members of this pheromone receptor gene family, and I shall determine their specific roles in courtship behavior. At present, little is known about the relationship of individual pheromone receptors and mating behaviors they control. Thus, my studies will establish a novel catalogue of innate behaviors, which are controlled by specific pheromone receptors. Moreover, these investigations will shed light onto the neurobiological and molecular processes of complex behaviors, and they will generate new tools for future studies on their neural circuitry. I have previously studied chloroplast RNA editing, a completely different topic than the one proposed here. However, I have recently become very interested in the general area of neurobiology, with a specific interest in processes that control innate or learned behaviors. Even though this new field is quite different from my previous work as a graduate student, my expertise in molecular techniques is readily applicable to the characterization of pheromone driven processes.

The structural and functional complexity of nerve cells remains one of the biggest mysteries of Neurosciences. How do neurons develop from a cellular sphere into a rich and complex cell? Once developed, how are neurons maintained in the highly dynamic and changing environment of the brain? These questions lie at the heart of brain research and, yet, remain poorly understood. Here, we propose to combine two-photon microscopy and lentivirus gene technology to develop an experimental model for studying dendritic development and synaptogenesis, in vivo. Specifically, we will study a population of neurons in the adult brain that continues to develop well into adulthood. This unique neuronal population is a pool of progenitor cells lining the lateral ventricle, which migrate into the olfactory bulb and differentiate into functional inhibitory neurons of the local circuitry. Using lentivirus transduction we will genetically tag and manipulate the newborn neurons. Using in vivo imaging we will study these neurons as they develop and integrate into functional networks. These experiments will provide new insights into the mechanism of dendritic and synaptic development as well synaptic maintenance in the intact mammalian brain. The CDA will enable our lab to acquire the necessary independence with regard to the genetic manipulations of this experimental system. Since synaptic development is a fundamental topic in the Neuroscience field, other areas of research such as physiology, molecular biology and behavior are necessary for a comprehensive understanding of this system. Thus, the CDA will prompt collaborations of my lab with other researchers in other fields and countries.

The aim of my proposal is to understand the fundamental role of chromatin modifications on RNA Polymerase II (RNAPII) regulation. To do so, I will use the retrotransposon Ty1 in S. cerevisiae as a biological model. The promoter of Ty1 is incredibly complicated with many features characteristic of higher eukaryotes and understanding its regulation provides fundamental information on transcription control. Among these aspects, Ty1 is subject to a unique transcriptional silencing by cosuppression mechanism. Transcriptional cosuppression involves RNA interference, histone or DNA methylation. Interestingly, S. cerevisiae exhibits none of characterized features identified in other organisms and Ty1 cosuppression may represent a novel regulation involving RNAPII, RNA maturation and chromatin remodeling. To understand this Ty1 specific transcription control, I will use 2 major approaches. Firstly, I will characterize Ty1 transcriptional dormancy using a range of chromatin techniques (Chromatin Immunoprecipitation coupled with real-time PCR analysis, Transcriptional Run-On, Chromosome Conformation Capture and chromatin analysis). Secondly, I will identify genetically factors affecting Ty1 transcriptional cosuppression. The use of recent and accurate techniques to detect chromatin changes and RNAPII dynamic is very demanding in financial supports. HFSP CDA will dramatically help me to set up an appropriate and competitive laboratory environment to develop this project and organize a collaboration network through laboratories with different and complementary expertise. Finally, this Award will enable me to begin building a team which is essential both technically and intellectually.