Metastasis is the process whereby malignant cancer cells escape from a primary tumor, invade the surrounding tissue, and migrate through the circulatory system to establish secondary tumors at distant sites. Secondary metastases at vital organs are often the cause of failure in cancer therapy and subsequent mortality. Identifying the triggers of metastasis or understanding the individual steps of metastasis has been difficult due to its complex and multifaceted nature. We plan to use an interdisciplinary approach that will combine molecular and cell biology techniques with the recent advances in optics to image the behavior of individual cancer cells in their natural environment. Thus, the goal of this project is to utilize intravital imaging to specifically study cancer metastasis. Further, we plan to use this non-invasive imaging technique to study in real time metastatic growth and suicide gene therapy response. I have experience in analyzing the cell motility processes using biochemical and classical microscopic approaches. However, I do not have any experience in the research witch includes animals. Thus, in Dr. Louvard’s laboratory, I will be able to complete my training in different aspects, including tumor biology, in vivo imaging and the use of animal models to study human diseases. By obtaining training in different fields I believe that I will be able to create my unique research path.

Neural stem cells hold great promise for new treatment strategies in the central nervous system. However, little is known about the factors that determine their developmental potential. Cloning by transferring a nucleus into an oocyte provides a powerful tool to begin to identify those factors. The goal of this proposal is to begin to dissect the epigenetic factors that determine neural precursor cell fate versus the extremes of the fully differentiated neuron and the pluripotent embryonic stem cell. I will test the hypothesis whether the cellular differentiation state influences the efficiency of somatic cloning. Presumably, the more differentiated the genome, the more difficult it will be to reprogram. Finally, I will delve into the molecular basis of neural stem cell reprogramming by identifying candidate potency genes. Although the ultimate goal of this research proposal is similar to my previous research, this fellowship will focus on an completely independent research area and differ significantly in methodology. In order to develop novel transplantation-based treatment strategies with pluripotent cells from an autologous source it will be necessary to tackle two main problems: first, the development of differentiation protocols for the generation of transplantable cell types in high purity and second, the generation of pluripotent cells from adult tissue. In the last years I was involved in the differentiation of embryonic stem cells. With the help of this fellowship I now would like to extend my expertise to the area of nuclear reprogramming. This scientific maturation would be of greatest value for my future career.

Neurons communicate each other through synapses, which are formed between axons and dendrites. The dendritic branching pattern varies to a great extent with the neuronal type, and is an important determinant of the synaptic input received by a neuron. Thus, studying how dendrites take their shapes during development will give us better understanding of the wiring and function of the nervous system. I propose to study the contribution of ion channels/transmitter receptors to dendritic development by taking advantage of the powerful genetics of Drosophila system. Ion channels/transmitter receptors play important roles in dendritic signaling and there are some indications that they may also be involved in dendritic development. This project will be novel and significant because it will reveal new mechanisms of dendritic development and the developmental roles of ion channels/transmitter receptors. My past experiments focused on the axonal development using an in vitro preparation. However, in order to gain better understanding of the wiring of the brain, an in vivo study will be necessary. Moreover, the technical advances have allowed us to take the initial steps toward a comprehensive understanding of dendritic development. This fellowship will provide me the unique opportunity to study dendritic development in vivo. This fellowship experience will undoubtedly provide me with new ideas, broaden my expertise, and help me to develop a new theme independently. Furthermore, it will provide me the chance to join one of the leading labs studying dendritic development in the world, which I believe will surely help me to achieve my scientific goals.

The liver X receptors (LXR) alfa and beta, members of the superfamily of ligand activated nuclear receptors, are activated by oxysterols. The ability of LXRs to regulate genes involved in sterol and lipid metabolism has led to their identification as in vivo inhibitors of atherosclerosis. The recent finding that LXRs repress the expression of a battery of inflammatory genes in cultured cells and in vivo provides an additional synergistic mechanism to explain their anti-atherosclerotic action. Remarkably, LXRs inhibit the inflammatory response elicited by LPS, IL-1b and TNFa stimuli. The repression appears to involve antagonism of the NF-kB pathway. However, the exact mechanism is unknown. The studies proposed in this application are aimed at elucidating the molecular mechanism by which LXRs inhibit the NF-kB pathway as well as investigating the role of LXRs in inflammation. This knowledge could be exploited for future treatment of atherosclerosis and inflammatory conditions. My PhD thesis focused on multidrug resistance proteins 2-4 (MRP2-4) with the aim of evaluating their contribution to clinical multidrug resistance and elucidating their physiological roles. This fellowship will extend my training in molecular biology and biochemistry to include gene transcription and regulation, fields that I have no experience with. I expect this experience to make a major contribution to my development as an independent scientist and to my ultimate goal of better understanding complex biological mechanism and improving the treatment of human disease. Specifically, the proposed experiments will allow me to master new methodologies and to tackle an exciting biological problem.

1.) Higher brain functions are controlled by local circuits of neurons forming synaptic connections. How do synapses form during development? Much of our knowledge about synapse formation is based on studies of the vertebrate and invertebrate neuromuscular junctions. It requires inductive signaling at sites where future synapses will form. Little is known about synapse formation between neurons (e.g. in the CNS). C. elegans is an ideal model system to study these processes in vivo: It has a simple nervous system with a reproducible small number of synapses and provides powerful genetic techniques. I will create tools to visualize a selected set of synapses in C. elegans. I will characterize these synapses in molecular detail. I will study how they form during development and how their pattern is controlled by neuronal activity. I will address what cellular interactions are required to induce these synapses. Based on the obtained knowledge, I will design a screen to hunt for new genes involved in synapse formation. Identified genes will be characterized on the functional level. 2.) The proposed studies will deepen my knowledge in molecular biology and genetics, using an invertebrate system. Since my previous research experience was mainly based on cell biological and biochemical techniques this project will greatly enhance my intellectual and technical skills. 3.) I will be able to address questions in neurobiology in vivo using C. elegans genetics. In my future career C. elegans will serve as a model organism to understand higher brain functions in vertebrates. The proposed project will be extremely beneficial for this goal.

Proteolysis of key regulators of the cell cycle is controlled by ubiquitin ligases called SCFs because each is formed by three major subunits: Skp1, Cul1 and one of many F-box proteins (Fbps). The substrate specificity of SCFs is dictated by different Fbp subunits that act as substrate recognition factors. The aim of the proposed project is to investigate the function of a particular Fpb, namely Fbl4, identified by Pagano’s group group (Cenciarelli et al., Current Biology. 1999). Since joining Pagano’s lab, I have shown that Fbl4 is localized on the centrosomes. I will use cell biology and biochemical methods to understand the reason for the centrosomal localization of Fbl4. In particular, I will explore the possibility that Fbl4 is required for centriole separation and/or subsequent steps of the centrosome cycle. The inhibition of the expression and function of Fbl4 in living cells by RNA interference or by neutralizing antibodies will allow me to determine whether a lack of function induces a defect in centrosome duplication. As future directions I intend to identify and characterize specific interactors of Fbl4.

Methylation of arginines is emerging as a novel type of protein modification important for the regulation of cell growth and gene expression at many levels. The product of the HMT1 and PRMT1 genes are the major arginine methyltransferases of the yeast Saccharomyces cerevisiae and of humans, respectively. Recent evidence suggests that Hmt1/PRMT1 affect several steps associated with pre-mRNA metabolism including transcription, capping, polyadenylation, and mRNA export. The proposed research is designed to test the hypothesis that Hmt1 associates with the RNA polymerase II transcription machinery where it methylates histone tails and specific RNA-binding proteins as they are recruited to nascent pre-mRNA transcripts. We also intend to identify novel targets and cellular pathways controlled by arginine methylation. Such findings will further our understanding of the function of this poorly characterized type of post-translational modification. My experience as an Honours and M.Sc student provided me with a strong background in virology, whereas my PhD in the field of telomeres and telomerase introduced me to the biology of cancer and aging. The development of the present proposal on a poorly characterized post-translational modification, namely arginine methylation, in Dr Silver's laboratory will complement my background in molecular biology and in mammalian cell systems via experience in proteomics as well as with genetic and cell biological techniques using yeast. At the end of the Fellowship, I will able to study different basic cell biological processes in both a genetically tractable system (yeast) and in mammalian cells.