My research will focus on the development of telencephalic neurons that use GABA as their neurotransmitter. I will study the role of a family of gene regulators, the Dlx homeobox transcription factors, whose expression coincides with the location of GABAergic neurons in the forebrain. Using an array of molecular, cellular and bioinformatics tools I hope to provide insights into the role of Dlx genes in governing regional specification and differentiation in the telencephalon. My studies, combined with the enormous potential of stem cells, could be useful to genetically engineer of GABAergic neurons that could be used for transplantation in humans. Ultimately, my work could further our understanding of the mechanisms underlying human neurodevelopmental disorders such as schizophrenia and autism. During my post-graduate studies, I have acquired an expertise into the molecular mechanisms that orchestrate gene transcription and intracellular signal transduction using a wide array of molecular biology and biochemistry techniques. Although very rewarding, my studies so far have been centered on the molecular/in vitro aspect of biology. During my post-doctoral training, I intend to take a different approach and focus more on the physiological and cellular aspects of the fascinating biological system that is brain development. I strongly feel that this training, during which I will get familiar with developmental and cellular biology techniques, will perfectly complement my current expertise and provide me with the multidisciplinary tools necessary to succeed as an independent scientist in the neuroscience community.

Control of T cell generation is essential for the development immune response and maintenance of the self-integrity. Weak interaction of the CD4 T cell receptor (TCR) with self-peptides bound to MHC class II molecule is required for selection of CD4 T cell in the thymus and their long-term maintenance at the periphery. Using transgenic model for a TCR specific for an ovalbumin peptide bound by the I-Ab MHC molecule, I propose to study the adaptability of the developing and peripheral CD4 T cells to a skewing of self-peptide repertoire in mice. More precisely the molecular mechanisms of this T cells adaptability will be analyzed. During theses studies I hope to identify several key players in this process and to establish their hierarchy in, the interaction with each other and with other components of the regulatory molecular network. This project will provide a better understanding of plasticity of T cell-response and maintaining of the tolerance to the self-peptides. During my Ph.D. studies I worked in field of the virology. Measles virus pathology study stimulated my interest for the immunology. The HFS fellowship should provide me the possibility to perform my postdoctoral training in an excellent immunological laboratory where I could explore the immune system using fundamental approaches. The experimental design and the methodology are completely different from those used in my previous research. The strong molecular expertise in T cell research of the Rudensky group is essential to complete my scientific training. Altogether, my Ph.D. experience followed by this postdoctoral training will bring me a strong background in both virology and molecular immunology.

This work proposes to study the molecular mechanisms by which the microtubule and actin cytoskeletal structures regulate each other in the establishment of cell polarity, using both Schizosaccharomyces pombe and Drosophila as model systems. Fission yeast are rod-shaped cells that grow exclusively from the cell tips. Growth sites are enriched for actin structures, and their position is defined by the microtubules which grow along the length of the cell, and deposit specific markers such as tea1p at the cell ends. Tea1p co-fractionates with high molecular weight complexes containing the actin-binding protein bud6p and for3p, a member of the formin family of actin filament nucleators. These complexes may therefore represent a molecular link between the polarised organisation of the microtubules and the actin structures at growth sites. I plan to investigate the molecular composition of these complexes through biochemical purification of associated proteins, and to study their regulation during the cell cycle by generating conditional mutants in tea1, bud6 and for3. I will investigate how they direct localised actin assembly by studying their actin filament nucleating capability in vitro and their site of activity in vivo. To determine whether analogous mechanisms regulate formins in animal cells, I will analyse their function in polarised Drosophila cells. During this project, I will acquire biochemical, genetic and cytological expertise in fission yeast. Together with my previous Drosophila training, this will enable me to establish an independent research program investigating conserved mechanisms of cell polarisation using two complementary model systems.

Cell guidance has only begun to be studied in vivo for few years, and the migrating border cell in drosophila provide a powerful model to genetically dissect the molecular mechanisms involved in this process. I propose to realise a new genetic screen to identify genes that act downstream of the PVR and EGFR guidance receptors in two steps. Fist I will construct gene-trap lines mobilising the recently described PiggyBac element. Then, mitotic mutant clones of border cells, positively labelled through the use of the MARCM system, will be performed in each of these lines and their guidance phenotype directly observed in dissected ovaries. This screen will allow me to select particular lines to focus on. Epistasis analysis and biochemical studies will then lead to the comprehension of the role these selected genes play in the guidance process. Moreover, I will test the involvement in border cell guidance of candidate genes, selected because of the motility function that is assigned to their mammalian and C.elegans homologous. The experimental biology approach I have used up to now allowed me to analyse cell interactions in terms of signals, but the cellular response could hardly be addressed because of the few genetic tools available in zebrafish and the complexity of the studied systems. In the outstanding scientific environment of the EMBL, I will undoubtedly improve my skills in genetics and cellular biology, and should then be able to lead my own group once back in France.

I propose to use Affymetrix microarray analysis to identify candidate genes downstream of the posterior HoxA and HoxD genes, which are involved in digit organization and specifying the number of digits. The main phase of my proposed research centers on analyzing a subset of these candidate genes in further detail using classical mouse genetics and conditional mutant mouse models. Previously, I studied the molecular mechanisms that contribute to the generation of circadian rhythms in Drosophila. I was able to gain a significant amount of knowledge in general molecular biology, functional genomics, and Drosophila genetics. Therefore, I decided to target my postdoctoral training in the field of mouse developmental biology and to learn how to use techniques in mouse genetics to build models that address issues in human development. I am extremely new to the mouse model system, which is precisely why I proposed to begin my experience with a technique that I am familiar with and which I will introduce in Dr. Duboule’s lab, namely microarrays. I hope this strategy will allow me to accomplish my primary goal of learning the intricacies of mouse genetics and developmental biology without sacrificing my desire to make significant contributions to the field early in my postdoctoral tenure. I know this experience will help me to become a first rate mouse geneticist and allow me to gain the knowledge and skills that will allow me to function independently in managing my own lab in the future. At the same time, training in another country is a once in a lifetime opportunity to interact and share philosophies with scientists I would otherwise never have the opportunity to meet.

Small proteolipids have essential roles in the regulation of several membrane transporters. In yeast, three small proteolipids have been purified from the plasma membrane. Preliminary data have shown that one of these is involved in ion homeostasis, while the other two are involved in regulation of plasma membrane proton-pumping ATPase activity. At least two possible modes of regulation can be envisaged: the proteolipids directly regulate proton or ion transport activity in the plasma membrane, or they regulate membrane trafficking events. We propose to use genetic, biochemical and structural approaches to clarify the mechanisms by which they regulate ion homeostasis. A screen for synthetic lethality with loss of function of one of the proteolipids will be performed to identified its genetic partners. The purification of this proteolipid in the native state, using mild detergents, will then allow the identification of the proteins with which it interacts. Purified transporters and proteolipids will be co-reconstituted into liposomes to assess their activity and mode of regulation. In addition, the purified proteolipids will undergo structural analysis through international collaborations with laboratories specialized in structure determination of membrane proteins. The study of these small proteolipids will allow us to examine different aspects of cell biology, namely ion homeostasis and membrane trafficking and will be crucial for a complete understanding of the physiological and molecular mechanisms involved in these processes. A CDA would be a great help in establishing an independent and competitive research group in this competitive field.

The small GTPase Rab5 is essential in endocytosis. It controls vesicle formation at the plasma membrane,fusion with early endosomes and microtubule-dependent motility of these organelles. The group of M. Zerial purified a large number of cytosolic Rab5 effectors, demonstrating that the Rab5 multifunctional activity depends on its ability to recruit them on the membrane where it localizes.The effectors so far characterized function in a cooperative fashion and are restricted to a specific sub-domain of the early endosome.Beside studies in cultured cells, the function of Rab proteins and their effectors in an organism and during development has been poorly investigated. The aim of my project is to study the physiological role of two Rab5 effectors in vivo. I will focus on Rabenosyn-5, which regulates two consecutive steps of endocytic trafficking (endocytosis and recycling), and a novel, yet un-characterized 5-protein complex. I chose these effectors since preliminary data in mammalian cells suggest that they localize to distinct endosomes or sub-compartments of early endosomes. As model system I will use Drosophila, since it has been successfully employed to study the role of endocytosis in many developmental processes. I plan to determine the intracellular localization and function of these effectors in endocytic trafficking and wing development. The project will be performed in collaboration with M. González-Gaitán and S. Eaton. It consistently differs from my previous research experience: it regards a different aspect of intracellular trafficking and moreover,with Drosophila genetics, I will learn completely new methods and techniques on a powerful developmental system.

In C3 plants, CO2 is fixed by RubisCO. At the atmospheric CO2 concentration, oxygenase reaction of RubisCO, an energy-wasting process, is inevitable. In C4 plants, CO2 is fixed to form a C4 compound, which is transported to the bundle sheath cells (BSC), where it is decarboxylated by malic enzyme. Resulting release of CO2 maintains the CO2 concentration high enough to eliminate the oxygenase reaction. Since the malic enzyme reaction also provides NADPH, ATP is preferentially required from the light reaction in BSC. A physiological question how the ATP/NADPH production ratio is controlled has not been answered at a molecular level. I have studied molecular genetics and recently identified a gene responsible for cyclic electron flow around photosystem I in C3 plants. Highly probably, C4 photosynthesis utilizes such cyclic electron pathways around PS I in BSC, but molecular component have not been described so far. Recently, C4-like photosynthesis was discovered in stem and petiole cells of C3 plants and it seems possible that C3 plants can also manage tissue-specifically C4 photosynthesis. I focus on the control of ATP/NADPH production during C4 photosynthesis, by extending my research background from genetics to physiology and biophysics. For this purpose biophysical techniques (photoacoustic, fluorescence imaging), only available in a few laboratories including the host laboratory, will be used to monitor in vivo electron flow activity in a C4 plant (maize) as well as in specialized cells of a C3 plant (tobacco) able of C4 photosynthesis. I feel extremely important to extend my research area by establishing a bridge between current molecular genetics and physiology.

The objective of this proposal is to study the complex mechanisms of protein folding. I will develop and use single-molecule fluorescence methodology to follow conformational dynamics, at a time scale of nanoseconds to seconds, of individual fluorescent-labeled proteins. Using this method, I will be able to study protein folding in a way that is not possible with any other known method. For example, I will be able to trace sub-population or to measure fast dynamics. These, and more, are very important in order to gain better understanding of protein folding mechanisms. As a model system, I will study the protein chymotrypsin inhibitor 2 (CI2). Later I will shift to more complex proteins, relevant to biology. In my M.Sc and Ph.D. work I studied some of the basic physical properties of DNA building blocks, such as relative stability of different structures, dynamics, energy and lifetimes of electronically excited states. This was done in the gas phase, in which the molecules are isolated. In addition, the molecule was relatively small (150-500 AMU). In the propose research I will emphasize on the biological aspect of the protein folding. I intend to study much bigger molecule, such as protein (several thousands AMU), and I will do so in their real biological environment. The idea is to develop methods for solving, and to solve, specific protein folding mechanism with strong linkage to biochemistry. This fellowship will allow me to obtain training in a new research field of protein folding. And by implement my knowledge and experience gain in previous work, I will be able to study the complex function and behavior of proteins and to expand my skill into biochemistry.

Parkinson's Disease (PD) results from the loss of dopaminergic (DA) midbrain cells. My proposed project endeavors to use stem cells to engineer DA neurons by exposing them to particular inductive signals. I aim also to engineer neural stem cells (NSC) that are capable of delivering neuroprotective molecules to DA neurons (eg. growth factors). The final aim of the project is to implant the NSC derived DA neurons as well as the 'neuroprotective cells' into an animal model of PD. I will then examine the ability of the transplanted cells to restore DA levels and behavior. In PD, sprouting is one of the compensatory mechanisms that slows the progression of the disease. My previous research examined factors that regulate this sprouting response. The project examined the role of dopamine receptors, cytokines and glia in regulating terminal arbor size. This previous work increases our knowledge of PD and largely involved neuroanatomical techniques. The proposed research examines ways to treat PD using stem cell therapies and employs a number of new molecular biology techniques. The ability to carry out the proposed research at the Karolinska Institute would enable me to broaden my knowledge of neuroscience as well as learn a number of new techniques involving molecular biology (and a number of techniques not currently available in Australia). Dr Arenas is a world leader in stem cell research and his laboratory provides the ideal environment to learn and develop these new skills. Long term I endeavor to engineer stem cells that will regulate axonal growth in PD using knowledge from my previous research that has highlighted the importance of DA receptors, cytokines and glia.

My aim is to delineate the molecular mechanisms governing centrosome biogenesis in higher eukaryotes, a process culminating in the formation of the bipolar mitotic spindle. To do this, I will combine the strengths of two experimental systems, C. elegans for functional genomics, and X. laevis for biochemical mechanism. The Hyman lab has recently completed an RNAi screen of the entire C. elegans genome for genes involved in cell division. This will permit me to analyze a genome set of genes for the specific process of centrosome formation by identifying those required for spindle assembly. The proteins encoded by these genes will be characterized in vivo in C. elegans using GFP technology, immunofluorescence microscopy and mass spectrometry after immunoprecipitation. Xenopus being much more amenable to biochemical analysis, the function of the X. laevis homologues will then be further characterized in vitro using well established centrosome duplication/maturation assays. This project will differ from my Ph.D research in two ways. First, I will focus on a different organelle, the centrosome, instead of the Golgi apparatus. Secondly, I will use different experimental systems, C. elegans and X laevis. This, combined, with the skills I have already acquired in other experimental systems such as S. cerevisiae, C. albicans, T. gondii and mammalian cells will provide me with increased versatility in selecting which model system is best suited to answer various cell biological questions. In exchange, I will provide the Hyman lab with my personal experience on organelle biogenesis (Golgi and endosomes) and state of the art electron and fluorescence microscopy imaging techniques.

CD8 T cells provide a complex means of defense against pathogens. Following infection, pathogen–specific CD8 T cells proliferate and gain the capacity to enter peripheral tissues. There, they selectively destroy pathogen–infected cells and generate a local inflammation. So far, CD8 T cells have been investigated in blood and lymphoid organs, but technical limitations have hampered their analysis in peripheral tissues. Very recently, the development of non-invasive molecular imaging technologies has allowed the visualization of cell populations in vivo. My objective consists to track for the first time pathogen–specific CD8 T cells in living mice by molecular imaging. Trafficking and homing of CD8 T cells to infected tissues or tumor sites will be investigated. This project will be performed at the Center for Molecular Imaging Research (CMIR – www.mgh-cmir.org), Harvard medical School, allowing valuable and interdisciplinary scientific research. The planed fellowship represents an outstanding possibility to learn high-resolution in vivo imaging in a mouse model with a very strong scientific support. Furthermore, the application of molecular imaging techniques are expected to have soon a profound impact in numerous fields, including immunology, biotechnology, gene therapy and molecular medicine. Thus, I will gain the necessary experience and competence in disciplines such as immunology and molecular imaging, as well as promote new collaborations in the purpose to further establish my own research group.

The small GTPase Ran is a key regulator of nuclear function. Local concentration of RanGTP generated around chromatin acts as a signal for chromatin induced microtubule assembly(6). Two Ran targets involved in spindle assembly have been identified. TPX2 and NuMa(7-12). How TPX2 interacts and cooperates with importin alpha, and tubulin, to give rise to the nucleation of microtubules is presently not determined. Ran is also involved in aspects of spindle assembly that do not involve its known mitotic targets(2). A critical next step is therefore understanding in vivo the regulation of the proteins involved and identifying further key regulators of chromatin induced spindle assembly. I propose a research project to: 1. Study in vivo the TPX2-importin alpha, and TPX2-tubulin interaction. 2. Biochemically identify Ran targets that play a role in chromatin induced spindle assembly. 3. Study the function, interactions and regulation of the newly identified proteins in vitro and in vivo. The training that I have gained in cell engineering, live cell imaging, and multi-dimensional image analysis has given me the background necessary for the in vivo analysis of TPX2-importin alpha and TPX2-tubulin dymanics. The biochemical identification of new targets of Ran using Xenopus as a model system, is a research project that clearly differs from my previous research experience. Dr. Mattaj's laboratory in EMBL Heidelberg is equipped with the latest light microscopy facilities and has the expertise in the biochemical analysis of Ran function using the Xenopus system, from which I hope to benefit both in pursuing this project and in developing a broader scientific training.

Soluble enzymes, such as the modularly structured nonribosomal peptide synthetases (NRPS), efficiently catalyze the formation of many useful natural products. Attaining the same efficiency from synthetic mimics is enormously challenging. The core catalytic transformations performed by NRPS are: (i) polymer chain elongation through programmed inter-modular aminoacyl-transfer and (ii) chain functional group modification. Although several synthetic acyl-transfer catalysts have been reported, as yet there is no system able to faithfully mimic the structural modularity and programmability of NRPS. Thus, my project seeks to understand the NRPS structure-function relationship at the molecular level and to design de novo modular peptide constructs capable of catalyzing programmable aminoacyl-transfer reactions. To date I have worked in the fields of neurobiology and organic chemistry. By contrast, the proposed research is in the new field of enzymo-mimetics, which lies at the intersection of chemistry, computer science, molecular biology and bioengineering. Whereas in my PhD studies I tried to develop new anti-cancer drugs and to explore their mechanisms of action, the proposed research investigates the molecular structure-function relationship in NRPS and aims to construct new peptides that faithfully mimic it. The fellowship will serve my professional growth by enabling me to gain training in the new field with a leading laboratory that employs a multidisciplinary approach. I consider this project to be a promising starting point for me to become an independent researcher in my future academic career.

Many biological processes are carried out by macromolecular assemblies, which are transiently formed and may be composed of components participating in multiple processes. The E. coli MutS and MutL proteins and their prokaryotic and eukaryotic homologues, which recognize mismatched base pairs, are essential for mismatch repair, prevention of non-homologous recombination and DNA-damage mediated cell death. At different stages of the mismatch repair process alone, the MutS and MutL proteins interact with endonuclease, helicase, exonuclease and DNA polymerase. To understand how these proteins interact with different partners to mediate specific biological functions in different pathways, I plan to study the three-dimensional structures of MutS and MutL in complex with UvrD, a downstream helicase involved in mismatch repair, and with PCNA, a replication processivity factor, which coordinates DNA replication with repair. I will focus on making stable ternary and quaternary protein-DNA complexes and characterizing the dynamic nature of these complexes by both X-ray crystallography and, if necessary, single molecule microscopy. Up to date I have been working with single bacterial enzymes and the study of macromolecular assemblies is a completely new field for me. Nevertheless, I consider that my background in molecular biology, biochemistry and crystallography together with the high quality of Dr. Wei Yang´s laboratory situates me in an excellent position to complete this challenging project with success. This fellowship will consolidate my research career in the structural biology field, and will give me the necessary skills to develop a project on macromolecular complexes.