The discovery that small RNAs regulate eukaryotic gene expression poses a new paradigm for the post-transcriptional control of gene activity. We propose to study the expression, biogenesis, and mechanism of small RNA-dependent regulation through a genetic screen in Drosophila melanogaster. To avoid the potential problem of embryonic lethality, we will use targeted mitotic recombination in the eye, which creates patches of homozygous mutant tissue in an otherwise heterozygous animal. The flies will be mutagenized by transposon mobilization. Those insertions that affect small RNA-mediated post-transcriptional control will be identified using fluorescent reporter proteins whose mRNAs contain target sites for an endogenous microRNA (miRNA). Thus, we will be able to identify both mutations in the miRNA effector pathway and mutations that affect the biogenesis or transcription of endogenous miRNAs by screening for the appearance of fluorescence in mutant ommatidia. A cross of the mutant strains with flies that transgenically expresses the miRNA employed in the screen will distinguish between reduced transcription of the endogenous miRNA and mutations that affect miRNA function. We will verify that the mutations also de-repress known endogenous miRNA targets such as hid. Furthermore, we anticipate that phenotypic analysis homozygous mutant flies will indicate the range of developmental pathways regulated by miRNAs. Finally, biochemical analysis of extracts from homozygous mutant embryos will reveal at which step in the RNA silencing the genes we identify in our screen function.

Stroke is one of the leading causes of death in the world, resulting in the death of approximately five million people per year. Accumulated data demonstrates that inflammation plays an important role in the pathophysiology of ischemic stroke. Several investigators have demonstrated control of autoimmune inflammation by mucosal induction of tolerance to the central nervous system (CNS) antigens. Upon re-stimulation with the tolerizing antigen, T-cells in animals tolerized with a low-dose regimens, secrete cytokines such as IL-4, IL-10 and TGF-b1 that lead to organ-specific immunosuppression. The outcome of this tolerance may also limit toxic effects in the CNS cause by non-autoimmune process. Prof.. Weiner's laboratory has obtained extensive knowledge in mucosal tolerance against CNS antigens to minimize brain damage. We have obtained preliminary data that mucosal tolerance induced by myelin oligodendrocyte glycoprotein MOG(35-55) decreases infarct size following occlusion of the middle cerebral artery in C57BL\6 Mice. Modulation of cerebral inflammation may improve outcome after stroke and encourage mechanisms for self-recovery. During my PhD thesis I gained knowledge in the field of antibody engineering and neurology, however I had never done research on the aspects of cellular immune mechanisms involved in inflammation. The large range of knowledge obtained in Prof. Weiner laboratory about modulation of the cellular immune response to brain antigens and the ability to practice it in animal model, enable me to achieve the task during the course of the fellowship.

We propose to develop novel methods for optically tracking single molecular motor-proteins inside living cells, with a very high spatial (sub- 100nm) and temporal (sub-second) resolution over extended periods of time. We plan to achieve this goal by dark-field or video-rate confocal microscopy of proteins labeled with metallic nanoparticles, which uniquely combine high resonant scattering cross-section, lack of photobleaching and biocompatibility. We plan to imply this technique to study individual kinesin related motor proteins (KRPs) in mitotic cells. In particular, we are interested in resolving the roles of different KRPs in the assembly and function of the mitotic spindle, and in studying the important question of KRPs regulation in vivo.

Synapses are precisely assembled and organized during brain development. Despite the central role of synapses in neuronal functions, mechanisms underlying synapse formation remain unclear. The primary purpose of this project is to define molecular mechanisms for synaptic development and the relationship between synaptic morphology and synaptic function. I will focus on the role of stargazin, which is the mutated protein in an ataxic and epileptic stargazer mutant mouse and is involved in synaptic targeting of AMPA receptors. I will identify novel stargazin-interacting proteins by taking advantage of previous my biochemical techniques. Next, I will investigate the functional roles of the stargazin-interacting proteins by taking advantage of the host laboratory’s experience in neurobiological and neurophysiological approaches, which are indispensable for achievement of the proposed project. This research with Dr. David S. Bredt at UCSF, who is one of the world leaders on the mechanisms of postsynaptic protein assembly, will provide a large step toward my scientific goal to address fundamental questions in brain science. This project will also provide me with invaluable training in the discipline I plan to pursue as an independent scientist upon completion of the fellowship.

Kinesins are essential for microtubule-dependent cargo transport. While the roles of all six budding yeast kinesins have been explored, we lack a comprehensive understanding of which kinesins transport specific cargo in a single higher eukaryote that typically contains more than 20 kinesins. Furthermore, there has been no comprehensive view of the mitotic roles of kinesins in a single cell line of higher eukaryotes. I plan to analyze these issues in Drosophila S2 cell line. At first, I will perform RNAi against all kinesins in S2 and observe defects in mitosis by microscopy.

1) Embryonic stem (ES) cells have the remarkable capability to self-renew and to differentiate into all cell types of the embryo proper, a phenomenon referred to as pluripotency. The precise level of the POU-domain transcription factor Oct-3/4 governs three distinct developmental fates of ES cells (maintenance of pluripotency, differentiation into primitive endoderm and mesoderm, dedifferentiation into trophectoderm). For this reason, Oct-3/4 is regarded as a master regulator of maintenance and differentiation of ES cells. The goal of this research project is to analyze systematically the effect of altered Oct-3/4 levels on target genes on a genome-wide scale in order to identify and functionally analyze new genes which play important roles in ES cell self-renewal as well as differentiation. 2,3) In my previous studies I mainly performed analyses of gene function using the chicken embryo system. I want to switch to stem cell biology because ES cells are a powerful tool to investigate fundamental biological processes of self-renewal and multilineage differentiation. The insights gained in this research provide important contributions to our understanding of the early phase of mammalian development and may also lead to development of novel cell therapies. The lab of Austin Smith has made outstanding contributions to this field. The possibility to join his lab and participate in this exciting research offers me excellent opportunities to learn important new techniques in stem cell manipulation and in addition to gain the necessary intellectual expertise to be able to establish my own group to continue the work in the fascinating area of developmental/stem cell biology.

(i.) Proposed Project: In this application I propose studies into aspects of two of the fundamental processes that make life possible (a.) the folding of proteins and (b.) the intracellular transport of molecules. In particular I propose to investigate how these processes occur in conditions approximating the crowded milieu of the cell cytosol. Towards this purpose I describe experimental approaches that utilize in vitro approximations of an intracellular environment. Additionally I outline complimentary theoretical approaches that will help to rationalize experimental findings and advance current theory. (ii.) Difference to Prior Work: My Ph.D. studies were conducted on biological aspects of surface adsorption. My present work has been concerned with the polymerization of proteins to form fibrous aggregates. The work that forms the subject of this proposal is concerned with how proteins are folded and transported within the cell. Aside from constituting a new field of research, the proposed work will also require that I become familiar with a new set of experimental techniques. (iii.) Professional Growth: I will benefit from the acquisition of theoretical and experimental skills in nuclear magnetic resonance spectroscopy and protein folding, areas in which C.M.D. is an acknowledged world leader.

Dr. Koentges' lab is well funded by a 5-year Wellcome Trust Program grant which will enable me to perform the expensive microarray experiments required for this project. In my previous labs I encountered the obstacle that experimental testing of computational models was often impossible because of institutional divisions. In Dr. Koentges' lab I can bridge this gap by learning to do molecular biology/embryology as well as computing myself in a small and well-focused experimental system. Dr. Koentges is a very good experimentalist who can teach me all of the experimental techniques directly. I have studied medicine but was not intellectually satisfied as I was keen on directly testing stringent hypotheses. I pursued parallel studies in mathematics/computer science in Germany and Japan for 4 years, in order to introduce the rigour of mathematics into future experimental work. This made me familiar with analytical tools for network engineering that are useful for this project. However, my molecular biology expertise which I gained during my PhD (see refs. 22,23 in core text) is very limited. The opportunity to study gene regulatory networks in the developing vertebrate brain with mathematical as well as experimental tools is very exciting to me. It would make my long-hedged dream of joining computer science and developmental brain science come closer to reality.I would be most grateful if HFSPO were willing to support my career change from informatics/mathematics to molecular biology/functional genomics and help me understand complex systems in a more profound way. Many thanks for your consideration, Annika Hansen