Chromosome segregation ensures the spatial separation of sister chromosomes into daughter progeny prior to cell division. In eukaryotic cells sister chromosomes are anchored to the spindle apparatus during mitosis by specialized protein complexes known as kinetochores that assemble on centromeric DNA and act as a bridge between chromosomes and spindle microtubules. Bacteria lack a conspicuous mitotic apparatus, and no equivalent of a kinetochore that anchors the chromosome to a cellular structure is known. Nonetheless, during the developmental process of sporulation in Bacillus subtilis sister chromosomes are remodeled into a single elongated, serpentine-like axial filament, that extends from pole to pole with the origin regions anchored to the extreme opposite ends of the cell. A division septum then forms near one pole of the cell and traps the origin-proximal region of one chromosome in the smaller compartment. The goal of this proposed research is to understand the genetic and the molecular basis for the changes in the chromosome structure upon entry into the developmental process of sporulation. Moreover, this proposal seeks to address how remodeling of the chromosome is coordinated with polar division. We recently discovered a developmental protein RacA that is responsible for the formation of the axial filament and for anchoring replication origin regions of the chromosome to the cell poles. Cells lacking RacA do not form an axial filament and frequently produce progeny cells without DNA. RacA, the first known component of the axial filament, will serve as a foundation for the proposed investigation.

Terminal differentiation of cells is characterized by the acquisition of the specialized phenotype coincidental with cell cycle arrest. The aim of this proposal is the delineation of protein-protein interactions that distinguish processes of terminal differentiation, cell cycle arrest and tumorigenesis. To achieve this goal I will combine mouse genetic approach with Tandem Affinity Purification (TAP) technology to identify protein complexes formed in vivo by the basic region-leucine zipper protein C/EBPa essential for differentiation of various tissue types. Using knockin mice expressing C/EBPa-TAP fusion protein or its functional mutants I will analyze their proteomes in vivo to get a mechanistic insight into the role of transcription factors in complex biological processes such as differentiation and growth arrest in multiple tissue types. I will also address the function of novel interactors by mouse genetic approach. I received an extensive experience in protein functional analysis in vitro and in cellular systems the mouse model-based project will allow me to address biological questions at the organism level. I will obtain indispensable knowledge of mouse genetics and biology, learn to generate and analyze multiple mouse strains, while continuing my strong interest in the mechanisms of transcriptional control of normal and aberrant developmental processes.

1)Approximately 200 21-22-nt-long RNAs, termed microRNAs (miRNAs), have been identified in metazoa. Many miRNAs are evolutionarily and also probably functionally conserved. miRNAs are part of ribonucleoprotein complexes, miRNPs, which are related to RISC complexes containing short interfering RNAs (siRNAs). Ago family and few other proteins were identified as components of these complexes. miRNPs and siRNPs guide inhibition of gene expression by translation inhibition and degradation of target mRNAs, respectively. Some evidence exists that mi/siRNPs may be associated with ribosomes. Expression of many miRNAs is temporally or spatially regulated during development and cellular differentiation but mechanistic details of miRNA function and mRNAs targeted by most miRNAs are not known. miRNAs exhibit only limited mRNA complementarity, which can not be predicted by bioinformatics. Hence, identification of miRNA targets is an important challenge. In the proposed work I want to explore several approaches to identify mRNAs targeted by miRNAs expressed in mammals. Using tools available in the host lab I also want to identify miRNAs participating in cellular differentiation and cell cycle regulation. 2&3)My previous work was focused to establish the mechanism of tRNA import in Leishmania mitochondria and was instrumental to prove the stepwise nature of tRNA import through mitochondrial membranes. Unlike the present proposal it was not related to the role of RNA in gene regulation. Training in miRNA metabolism in metazoan would greatly help to build my career as a RNA biologist.

The purpose of this proposal is to develop a general method, which will allow for solving of the quaternary structure of large physiologically relevant complexes by NMR spectroscopy. Using novel selective labeling strategies with cross-correlated and liquid crystal NMR technologies, intermolecular distance and orientation information will be measured in biomolecular systems beyond 100 kDa. Such long-range information, inaccessible using standard NMR methods, will be used to determine the relative position of each subunit within the active complex. The methodology described above will be applied to study the quaternary structure of human kinetochore, an essential multiprotein complex that assembles on centromeric DNA. Relative position and orientation of each subunit of a prekinetochore will be first determined in order to obtain a structural basis for mechanisms involved in genetic stability during mitosis. Then this prekinetochore fold will be used to monitor its interaction with identified specific mitotic factors in order to build a quaternary structure model of functional human kinetochore. These studies will allow us to obtain information on the mechanism by which kinetochores attach to spindle microtubules and to know how this kinetochore-microtubules attachment is regulated. This innovative strategy will permit to exceed the size limitations of standard NMR structural studies, and will complement existing X-ray crystallography and cryo-electron microscopic methods. This new tool for structural biology will open a new field of applications for biologists.

In the visual system, a point to point map of the visual world is formed within visual centers in the brain because connections from the retina to the brain are topographically organized. It is unclear, however, whether such a ‚retinotopic map’ exists on the much smaller scale of the dendritic tree of a visual neuron. A visual neuron responds selectively to special features of a visual stimulus, which define its‚receptive field’. It is not known, however, how the distribution of synaptic inputs on the dendritic tree contributes to this selective behavior. As a first step to elucidate the underlying mechanisms, I propose to investigate the spatial distribution of visually driven dendritic activity in relation to the spatial pattern of the visual stimulus. This will serve as a basis for further studies, which will address the role of activity-dependent changes of synaptic inputs in the maturation of receptive field properties. These studies will be done in the retinotectal system of the tadpole, using powerful techniques such as two-photon microscopy, patterned visual stimulation and patch-clamp recordings in vivo. With this project I will dedicate myself to a new and promising field in neuroscience in an exciting intellectual environment. Previously trained in physics and synaptic physiology, I will now move to visual signal processing in a developing organism. This will give me the opportunity to apply my experimental skills to qualitatively different questions, and I expect to vastly broaden my scientific experience both conceptually and technically. This is likely to put me into a position to do independent, innovative research after completion of this fellowship.

1) Transport of proteins through the Golgi apparatus is characterized by the formation of vesicles from Golgi membranes. This vesiculation process is mediated by G beta gamma and its downstream target, protein kinase D1 (PKD1), which is a cytosolic serine/threonine kinase with multiple domains. It is recruited to the trans-Golgi network (TGN) and regulates the formation of transport vesicles that are on their way to the plasma membrane in mammalian cells. My project will consist in identifying the binding properties of each of the individual subdomains of the PKD to the Golgi membranes. Moreover, I intend to study the regulation of the vesicles release from the TGN. These are some of the key issues I aim to address during my post-doctoral position, in order to better understand how this fundamental process of protein transport is regulated. 2) This fellowship experience will differ from my previous research experience which was mainly based on the molecular mechanisms underlying the intracellular activities of the basic Fibroblast Growth Factor. Indeed, I am going to work on a cellular biology project and particularly on the regulation of protein secretion from the Golgi membranes. 3) This project will allow me to perfect my knowledge in cellular biology since the laboratory of V. Malhotra is considered to be in the top few working in the area of membrane transport and dynamics. The work I will carry out is highly significant for a mechanistic understanding of the process of transport carrier formation. My findings will help me establish an independent carrier in the european community and thus bring into this community a novel and a challenging topic of research.

Certain phage (viruses of bacteria) switch between distinct commensal and aggressive modes of exploitation, and are known as ‘temperate’. The molecular genetic mechanisms driving these changes are understood in detail, whereas the evolutionary pressures shaping and fine-tuning these mechanisms have remained largely unstudied. The prevailing functional hypothesis is that phage follow a probabilistic ‘bet hedging’ strategy, stochastically mixing lysis and lysogeny to ensure continued transmission by one mode or the other. In contrast, we conjecture that temperate phage have evolved mechanisms to sense and respond appropriately to the condition of their current and prospective hosts. Mechanisms of condition-dependent phage exploitation are well understood and multiple, but their evolutionary significance is unknown. We propose to address the evolution of condition-dependent phage responses using a combination of population dynamic and molecular genetic mathematical modelling, computer simulation and microbial experimentation. As many bacterial pathogens rely on temperate phages for toxin production, this study promises implications for the public health management of opportunistic bacterial infections. My background is in theoretical evolutionary ecology, thus this project will introduce me to both experimental and theoretical molecular biology, in the context of bacteria/phage. I view experience in these new fields as essential to my goal of integrating functional and mechanistic understanding of infectious diseases. The Bull and Meyers labs have abundant resources and expertise in all aspects of the proposed project, and thus offer an ideal environment for this research

The embryonic mammalian telencephalon gives rise to both the dorsal (cortex and hippocampus) and ventral (basal ganglia) domains of the adult brain and as such represents the developmental substrate underlying higher brain function. The source of the cortical neurons relates to their function in the mature network: pyramidal neurons are generated within the pallial ventricular zone and migrate radially, whereas the majority of interneurons are generated subpallially and tangentially migrate to their final positions. Identification of genes involved in the specification of these neurons has facilitated our understanding of the processes that leads to brain formation. However due to the perinatal lethality associated with the loss of these genes, their function has not been related to phenotypes in the adult. This study aims to use recent advances in genetics combined with electrophysiology to unravel the role of these key genes. First, genes will be used as specific markers to determine the connection between the gene expression in progenitor cells and their mature firing properties. Second, we will undertake conditional mosaic loss of function analyses to determine the contribution of patterning genes to adult network properties. Together this will provide a link between the interneurons underlying cortical function and the patterning genes that generate them. This project represents a departure from my previous research in that it involves combining genetics with cortical physiology. I aim to learn a broad range of transgenic techniques that will enable me to use a multidisciplinary approach to understanding neural networks.

Plants are the foundation of most ecosystems, modify and stabilize climates and, produce oxygen through photosynthesis. Long distance transport in plants is accomplished by a vascular system composed of two different tissues: xylem, that transports water and solutes from roots to aerial parts, and phloem, that distributes photo-assimilates after photosynthesis. Despite the number of studies describing vascular anatomy, xylem-phloem patterning has been little investigated. The elegant simplicity of the Arabidopsis vascular system will be used as a model system to unravel the molecular bases of vascular patterning. Brassinosteroids (BRs) affect several plant developmental processes, such as stem elongation, leaf development and photomorphogenesis, and xylem differentiation. The identification of two novel BR receptors, BRL1 and BRL3 that function in vascular development constitutes the bases for the proposed research. Using the vascular specific expression of these receptors as molecular markers, an activation tagging screening will be conducted to identify gain-of-function mutants showing vascular patterning defects. The role of BRs in vascular cell specification will be investigated by modulating steroid synthesis in specific vascular cells by driving a BR biosynthetic enzyme under the control of vascular specific promoters. Furthermore, vascular cells will be deprived of BRs by reducing the mRNA levels of this enzyme by RNAi driven by vascular specific promoters. Finally, an integrative mathematical model will be proposed for the radial/collateral patterns of vascular bundles and how BRs contributes to this pattern.