The overall structural organization of the nervous system is mostly bilaterally symmetric. However, deviations from this left/right symmetry are observed in for example different patterns of gene expression in otherwise symmetrical structures. In C. elegans, an interesting example of asymmetric gene expression is the chemosensory neurons ASER and ASEL. These neurons asymmetrically express 3 putative receptors, which correlates with different chemosensory activities. Several transcription factors have been shown by my host laboratory to work in a regulatory cascade that leads to the asymmetric expression of the receptors, however, these transcription factors have already an asymmetric activity. The aim of my project is to identify the initial signalling pathway that leads to the asymmetries observed in the L/R ASE neurons. To determine the molecular nature of the pathway I am presently undertaking a genetic screen. At this point I have already retrieved 16 asymmetry mutant. These mutants will be characterized and their epistatic relationship determined to examine their role in the process of asymmetry generation. I expect that the identification of neural asymmetry-inducing mechanisms will provide major insights into brain patterning. My proposed work will allow me to learn C. elegans genetics as well as how to address neuronal development issues. This will be a complete new field and approach for me and will complement my training in cell biology and biochemistry that I had during my PhD. In the future as an independent researcher I would like to continue investigating brain patterning complementing the genetic studies with cell biology and biochemistry techniques.

1) PROJECT: This project aims to study the conformational changes undergone by the cytosolic chaperonin CCT upon nucleotide binding and to measure the forces behind these changes. These forces may play an important role in forcing the substrate molecules to fold inside the chaperonin. A unique aspect of the studies proposed here involves the characterization of this system using methods of single molecule analysis. These methods have several advantages over those that utilise statistical ensembles of molecules because, unlike the latter, single molecule experiments make it possible to follow the molecular trajectory of the reaction and the signals are not confused by the simultaneous presence of multiple intermediates of the reaction. Moreover, single molecule manipulation methods make it possible to recognise and directly measure the forces generated during specific steps in a reaction, and even apply external force to alter the rate of these force-generating steps. 2) HOW IT DIFFERS FROM PREVIOUS RESEARCH EXPERIENCE: My previous research has been developed in the frame of applied physics, theoretical and focused on fundamental optical problems related with wave propagation, near field optics and optical forces. 3) PROFESSIONAL GROWTH: This project offers me the opportunity to work in a biological problem and extend my previous background to the derived applications in biology. It will also involve interpreting and handling with real assays, complementing my theoretical point of view with that of the experiments. In particular, I will train on novel biophysical techniques in single-molecule analysis, namely, optical tweezers and single-molecule fluorescence microscopy.

A living organism is a highly evolved system made up from a large number of interwoven molecular networks. These networks primarily involve proteins which enable and control every chemical process that takes place in the cell. I propose to study the behaviour of proteins in molecular simulations. The big challenge of such simulations is the so called time-scale gap. The folding time of a protein is in the range from milliseconds to seconds, whereas the longest molecular simulation of a protein is in the range of microseconds. I propose to use experimental data as a constrain in molecular simulations to overcome this time-scale gap. The ultimate goal of my research plan is to gain insight of the failures of cellular control and regulation processes that can lead to neurodegenerative diseases. This research will involve (i) the study of the folding and misfolding of proteins, (ii) the interaction of a misfolded protein with a cell membrane, and (iii) the binding of a protein with the receptor of a cell nucleus. In order to achieve these goals I will have to move into a new research area. I am a computational physicist with an extensive experience on crystal nucleation of colloidal suspensions. My proposed research involves computer simulations of complex biological systems. To carry out this research it is essential to get a deep understanding of the biological systems as well as to understand the experimental opportunities needed as input for the simulations. The support of this fellowship would give me the opportunity to become a biophysicist with a deep understanding of both physics and biology.

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.