My proposal focuses on understanding the mechanisms that contribute to self-renewal of stem cells and cancer stem cells using stem cell subsets of the human haematopoietic system in normal and transformed situations. Although this is a fundamental question in stem cell biology and for cancer therapy, the mechanisms governing stem cell self-renewal are only partially understood. I propose to identify the genes involved in self-renewal of normal and cancer stem cells by genetic, genomic and immunological approaches. Moreover, I suggest here to characterize for the first time the target genes of the Notch, Shh and Wnt/b-catenin pathways and to use this information to define the interconnections between these pathways in normal and transformed stem cells. The identification of the key regulatory pathways that promote proliferation of leukemic cells is likely to facilitate the development of new diagnostic tools and therapeutics that target these dysregulated self-renewing processes. My future work differs from my Ph.D studies in that I will be studying processes that contribute to self-renewal and cancer in contrast to questions of tissue rejection. I feel that my choice will be optimal for pursuing an academic career. The change of field will allow me to mature as a scientist, expanding my horizons with regard to biological processes and experimental techniques. The dedication of Dr. Weissman and his excellent group, and the ability to carry out intelligent and sophisticated experiments will undoubtedly provide me the ideal environment for my post-graduate training.

Cells respond to environmental changes by regulating the expression of specific genes. This process involves binding or interaction of regulatory proteins to the genome, resulting in either enhancement or repression of the transcription of genes. We propose to develop a novel method for mapping regulatory proteins to both their cognate DNA binding site and to their interacting protein partners. The method is based on specifically tagging stretched DNA and proteins with luminescent quantum dots, and analyzing them using a combination of AFM and a unique optical imaging technique. The high precision of this technique will enable precise localization of the transcriptional complexes within the genome, and will provide significantly improved data for computational prediction. The success of this technique is an important step towards the understanding of how cellular activity is modulated at the level of transcription. In the last few years I have been developing tools for addressing fundamental problems in nano-science and nano-optics. My desire is to take these tools and study a “real” biological problem. This project gives me the opportunity to be engaged in research that will have a more direct impact on our basic understanding of life with a prospect of improving it. It perfectly bridges my acquired skills and my desired ones, combining familiar technologies like high resolution imaging and AFM with methods from molecular biology and biochemistry like hybridization and protein chemistry. Apart from the hands on experience in these new fields, I will gain knowledge and understanding of biological processes, opening pathways for independent research in nano-bio science

Developmental processes proceeds reliably despite a wide range of variation that may impinge on their underlying molecular mechanisms. These include genetic heterogeneity, environmental variability and the underlying stochasticity of biochemical reactions. My PhD research focused on theoretical analysis of the robustness of developmental patterning networks in Drosophila to changes in gene dosage. In the proposed project I aim at experimental characterization of the effects of stochastic variation on sporulation of Bacillus subtilis. This elaborate process of a resistant spore formation includes many well characterized stages, both on the morphological and the genetic levels. It is used as a simple model for elucidating basic aspects of Developmental processes. A key event during this process is the asymmetric compartmentalization of the bacteria and the subsequent expression of different genes in each compartment. This step happens on a restricted and precise timing despite a large underlying variation in the system's response as revealed by examination of wild-type cells and relevant mutants. Using time-lapse microscopy and image analysis tools I will continuously track single bacterium and observe their sporulation by recording the temporal evolution of morphological markers and fluorescently-tagged molecules. This will enable me to extract statistical behavior on the colony level and analyze temporal correlations between the different components. By comparing the behavior of wild-type and mutant bacteria, I will elucidate the role of developmental checkpoints and cell-state switches on the cells ability to buffer noise during this developmental process.

Shigella invades epithelial cells of the human colon. Internalization of Shigella via macropinocytosis requires the secretion of several effector proteins through a type III apparatus. Following secretion the two major effectors, invasion plasmid antigens B and C (IpaB and IpaC) assemble into a multiprotein complex and integrate into the host cell membrane to allow translocation of other type III substrates into the host cell. Here, the structure and dynamics of the secreted IpaB/C complex will be studied in model or host cell membranes to clarify its role as a translocator and in signaling to host cells. First, I will investigate the topology of the IpaB/C complex in model membranes using genetically tagged fusions of IpaB or IpaC. Atomic force microscopy and electron microscopy will reveal the stoichiometry and structure of the IpaB/C complex. Secondly, I will establish an in vivo assay to study the dynamics of IpaB and IpaC during bacterial entry using tetracysteine tagged Ipas. Thirdly, in vivo fluorescence resonance energy transfer (FRET) and high resolution fluorescence microscopy will be used to analyze interactions between the IpaB/C complex and host targets or components of the type III apparatus, as well as the hierarchy and kinetics of secretion of type III substrates during invasion. With this fellowship, I will develop approaches to reveal the role of the IpaB/C complex during the early events of Shigella infection. This is a dramatic shift from my previous studies on the nuclear pore complex. My new experimental tools can be applied very generally to study bacterial-host interactions, and will be fundamental for me to succeed as independent researcher.

Biosynthesis of polypeptide chains at the ribosome is coupled with their folding into the native tertiary structure. The folding process often requires the assistance of molecular chaperones to proceed efficiently. In bacteria, the ribosome-associated chaperone Trigger Factor and the soluble heat shock protein DnaK directly bind to nascent polypeptide chains and are thought to assist co-translational folding. How the interaction of the chaperones with the nascent chain affects the folding events is unclear. Do individual domains of a polypeptide immediately fold when the nascent chain exits the ribosomal tunnel, or is folding postponed through the action of chaperones until the complete amino acid sequence has been produced and released from the ribosome? We propose to address this question in experiments involving in vitro transcription-translation and proton-deuteron exchange measurements. In particular, we will focus on i) determining the effects of Trigger Factor and DnaK on the folding of substrates in solution, independently of translation, ii) investigating the mechanism of chaperone-assisted co-translational folding of nascent chains, and iii) characterizing the functional cooperation of Trigger Factor and DnaK in assisting folding of newly synthesized proteins at the ribosome. This research program will expand my experience with chaperone systems, and provide training in techniques of molecular biology, biochemistry and biophysics, to build on my NMR expertise. Moreover, the broad range of expertise available in the laboratory will not only provide support for the experimental work, but also enable my training in designing creative interdisciplinary experiments.

I will study the role of PP2A, ser/thr protein phosphatases, in the establishment of cell polarity in C.elegans embryo. In a genome-wide RNAi screen, inhibition of three different subunits of PP2A caused defects in mitotic spindle localisation. Moreover, RNAi of one PP2A subunit results in abnormal localisation of PAR-2 indicating that PP2A might act to establish and transduce polarity information. For the first step, I plan to characterise the effects of PP2A knock-down on the organisation of the mitotic spindle and on the localisation of different PAR proteins, both of which are key events in establishing embryonic cell polarity. Secondly, by generating specific antibodies, I will investigate whether the three different PP2A subunits are associated in the same complex. If so, I aim to study the localisation and the dynamics of this complex during early cell divisions. Finally, I will carry out a new RNAi screen and a two-hybrid screen to identify new proteins involved in the cell polarity pathway that depend on PP2A activity. This project is a logical extension of my interest and training in the mechanisms that control polarity establishment in culture cells, with a change from molecular and cell biological approaches to genetic and developmental biology in a whole organism. From my post-doctoral experience, I will acquire expertise in genome-wide RNAi develop in Ahringer's laboratory to identify gene function in C. elegans and in the excellent nematode embryo model for dissection of signalling pathways involved in cell polarity. These new competences will allow me to develop ambitious projects in my future academic career as an independent scientist.

Recent research has demonstrated the plasticity of the auditory system. This work has focused on changes in auditory processing mechanisms, and has not considered how these may relate to cognitive factors such as attention. In the proposed project I will characterise the neural bases of auditory perceptual improvements at a number of different levels, from those that relate to ‘low-level’ sensory processing, to those that relate to ‘higher-level’ cognitive mechanisms, such as attention. To achieve this I will acquire expertise in the neurophysiological techniques of EEG and MEG, which record the electric and magnetic fields at the human scalp during sound presentation. Using complex analysis tools developed at the host laboratory, I will be able to isolate components from these recordings that relate to different levels of sound processing, and will also be able to assess the effects of attention on sound processing. This work will provide me new skills in the field of human neurophysiology, which will complement my previous experience in auditory psychophysics. This will enable me to conduct comprehensive studies assessing auditory processing using multi-disciplinary research techniques, combining both neurophysiological and psychophysical approaches. In addition, it will form the basis of my research endeavour to study auditory perceptual processes in relation to cognitive functioning, a topic that is currently much neglected.

Mitochondria are the primary site of cellular energy generation, and as such are essential for viability. Their morphologically varied structures, which depend on a balance between opposing fission and fusion events to maintain their form, reflect differential cellular requirements and environmental conditions. The goal of the proposed project is to elucidate the molecular mechanisms involved in facilitating and regulating mammalian mitochondrial fusion. The proposal focuses on the mitofusin proteins, GTPase-like fusion components located in the mitochondrial outer membrane that have been implicated in cell development and disease. Under the proposed research plan, the functional roles of various mitofusin domains in mitochondrial fusion will be investigated by both in vivo confocal microscopy analyses of cells expressing defined mitofusin mutants, and complementary in vitro nucleotide hydrolysis assays and mitochondrial fusion assays to analyze regulatory mechanisms of fusion events. As well, the project will explore the hypothesis that mitofusins assemble into protein complexes and seeks to identify novel components of the fusion machinery. The analysis of mitochondrial morphology provides a significant opportunity to learn techniques in mammalian cell biology and confocal microscopy. In turn, the project will be complemented by my past research experience in the area of protein chemistry.

The number of species on the earth is estimated to be tens of millions, and the evolution of genes/genomes which resulted in this diversity of organisms is one of the most interesting topics in biology. I plan to work on cichlid fishes, instead of medaka, which have evolved astonishing variation within a very short period of time, in order to find out what types of genomic/genetic changes have been evolved to achieve this diversity. Because colours and colour patterns on the body surface are among the most divergent and readily recognizable features of animals, I chose pigment-cell-associated genes as the best targets of this research proposal. The aim of this study is to identify the number and chromosomal locations of genes responsible for body-colour determination as well as mating preference in cichlids for later positional cloning and functional studies. Several pairs of fishes which 1) have distinct body colour differences, 2) reach maturity quickly, 3) lay enough eggs for fine locus/QTL mapping, and 4) are closely related to each other and whose F1s are known to be fertile (e.g., The Normal and Gold morphs of A. citrinellus) are crossed/hybridised in the laboratory, and the phenotypes (i.e., body colour and mating preference) of their backcross siblings are analysed to be grouped. I perform AFLP on their genomic DNAs and phenotype-specific bands are used for STS construction. Linkage and recombination distances between the responsible locus/QTL and STSs are evaluated. Tens of backcross siblings are enough for this rough mapping, but I will construct maps as fine as possible for future gene identification by the forward genetic approaches.

All motile organisms possess means of sensing and responding to changes of the external temperature. Given a choice, an animal moves towards a cooler area when overheating, and towards a source of warmth when faced with a cold environment. This simple behaviour demonstrates the main task of the thermosensory system: to define the thermal quality of a stimulus (hot versus cold), and to trigger appropriate behavioral and physiological responses. In addition, extreme temperatures are sensed as noxious stimuli, and as such evoke unique, very rapid reactions. Despite the essential nature of thermosensation, little is known about the central mechanism of temperature discrimination and the circuits involved in behavioral choices and output. The study of temperature coding is further complicated in mammals by the existence of multiple layers of autonomic, endocrine and skeletomotor responses that are interconnected to the sensory pathways to regulate the production of endogenous heat. I propose to employ an integrated genetic, molecular and behavioural approach to elucidate the logic of temperature perception in Drosophila, a system ideally suited for a comprehensive dissection of complex circuits and behaviors.

The mammalian cerebral cortex is characterized by a modular functional organization: distinct cortical areas with specific patterns of input and output projections are devoted to different functions. In particular, sensory information of distinct modalities (vision, audition, somatosensory perception) is relayed from the periphery via specific dorsal thalamus nuclei, which project to distinct cortical areas. Recent studies have shown that the initial targeting of thalamic projections to specific cortical areas is first regulated at the level of an intermediate target, the basal ganglia and subsequently shaped within the cerebral cortex. Whereas sensory ascending pathways, including thalamic projections, form in a stereotyped manner during embryogenesis, they can be remarkably modified in response to activity. In particular, when a modality is depleted, corresponding areas can be recruited for the processing of the intact modalities (cross-modal plasticity). Understanding how thalamocortical connectivity is established and shaped during normal and pathological development is an essential question that remains to be fully investigated at the cellular and molecular levels. To this aim, we propose to use a combination of in vivo and in vitro approaches in mice to achieve three specific objectives: i) to determine the mechanisms controlling the initial targeting of thalamocortical axons in the basal ganglia, before they enter the cortex ii) to elucidate the role of regionalized cortical cues in the guidance, refinement and maturation of thalamocortical projections iii) to establish a model of cross-modal plasticity with physiological relevance.

The subject of the proposed research is the mechanism of circadian rhythms in mice. Several factors have been implicated in the central circadian clockwork, which consists of interwoven positive and negative feedback loops. At the core of this oscillator are the proteins period (PER) 1 and 2 and cryptochrome (CRY) 1 and 2, which rhythmically inhibit the heterodimeric transcription factor CLOCK:BMAL1. Insight into the precise molecular mechanisms of this process is still limited and lagging far behind the genetic progress made in the field. Therefore, I propose to characterise the PER:CRY complex with the aim of understanding how it acts as a transcriptional repressor. I will purify native PER- and CRY-containing complexes from synchronised tissue culture cells and determine their composition and the circadian changes they undergo. I expect to identify new complex components, which will be tested for a function in the circadian clock in a novel cellular system and by genetic loss-of-function studies in the mouse. To investigate how PER:CRY-mediated regulation of transcription is achieved I will analyse in detail the events taking place at regulatory sequences of genes that are under circadian control. In particular, I will address the question on how PER and CRY proteins can generate widely different phases of cyclic gene expression. In summary, the proposed combination of biochemical, molecular biological and genetic studies will make important contributions to the fields of molecular chronobiology as well as transcription regulation in general, since the PER:CRY complex appears to represent a novel type of transcriptional repressor.

DNA microarrays allow determination of global mRNA expression profiles of cells grown under various conditions. However, it is often not known how changes in mRNA expression are correlated with protein synthesis and the extent to which additional regulation occurs at the translational level. For instance, it is commonly assumed that developmental and oncogenic signaling achieve their phenotypic effects primarily by direct regulation of the transcriptional profile of cells. However, recent evidence indicates that direct effects on transcription may be preceded by effects on the translation efficiency of specific existing mRNA species. I want to establish a method for translational profiling, which involves the rapid purification of endogenously formed ribosomes and analysis of bound mRNAs with DNA microarrays. I will analyze how changes in steady state mRNA levels are related to changes in ribosome occupancy upon changing growth conditions in the yeast Saccharomyces cerevisiae. The method will also be established in mammalian cells with focus on HeLa and endothelial cells. Cells will be treated with pharmacological compounds or mixed with other cell types to explore the impact of intra-, and inter-cellular communication on translation. These studies will help to decipher the extent and nature of translational regulation and uncover new aspects of the gene expression program.