It is becoming increasingly clear that the information encoded in the linear DNA sequence is insufficient to fully account for the complex regulation of genes and genomes. Higher-order chromatin structure and epigenetic regulation via chromatin modifications are now recognized to be important mechanisms of gene regulation. An additional emerging mechanism of genome regulation is the control by higher-order organization of chromatin and chromosomes. The aim of this project is to reveal how higher-order structure of chromatin and chromosomes changes during neural cell differentiation and how nuclear architecture affects expression of gene programs. I will focus on uncovering how extra-cellular cues affect gene expression programs and differentiation in the nervous system. Specifically, I will investigate the spatial positioning of chromosomes and cell-type specifically expressed genes, and analyze kinetics of proteins and gene loci in neural stem cells and during their differentiation into neurons and astrocytes. This project takes advantage of my expertise in neural differentiation and will enable me to acquire training in new methods and a novel field of research. At the end of the fellowship, I will have technical skills in advanced imaging and expert knowledge in the area of spatial genome organization, and this will allow me to explore further the complex mechanisms of cellular differentiation in the nervous system. I believe that postdoctoral training in the field of nuclear architecture in Dr. Misteli’s laboratory will ideally complement my expertise in molecular biology and will prepare me for a career as an independent scientist.

Small non-coding RNAs play an unexpectedly large role in regulating gene expression. small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate both specific gene expression and more globally regulate chromatin structure and genomic stability. Both siRNAs and miRNAs act via the RNA interference (RNAi) pathway. At the heart of RNAi is a protein-small RNA complex, the RNA-induced silencing complex (RISC) that catalyzes target RNA destruction. Several proteins in the RISC have been identified, and many of these are required for metazoan development. My research seeks to dissect the pathway of RISC assembly and the mechanism of RISC action. I am especially keen on understanding what differentiates the RISC function in target cleavage (RNAi) from its role in translational repression via miRNAs. My previous experience dissecting the mechanism of tRNA-maturation will serve me well in this pursuit, but my study of the RNAi pathway will also give me new experience with Drosophila developmental biology and genetics. Thus, a HFSP fellowship would help me to grow beyond my biochemical expertise and will promote my professional growth through interdisciplinary research.

In this proposal, I will describe a multi-faceted approach towards establishing how functional connections in the brain are dynamically altered to link informative sensory signals with preferred behavioral outcomes. Specifically, how are the same sensory signals transformed into different types of movement signals, depending on what a subject is doing? I will ask this question in the context of a task-switching paradigm. Traditionally, studies on task switching has focused on the prefrontal cortex. This is due to the hypothesis that the prefrontal cortex operates as an “executive control” center, that modulates the “switch” between sensory information and motor commands. It is not clear, however, where and how the “switch” occurs. Here, I will train monkeys to “switch” between discriminating the direction or the depth of a single patch of dots (both discriminations are known to involve area MT) on a trial-to-trial basis, by cueing the monkey on each trial, and ask if the functional connection between MT neurons and eye movement neurons dynamically change depending on which attribute the monkey is attending to. If MT neurons switch their functional connections depending on which attribute the monkey is attending to, I expect trial-to-trial fluctuations in MT responses to correlate with different choices, depending on which attribute the monkey is attending. Moreover, I propose to see if I can evoke different eye movements using electrical microstimulation, depending on which attribute the monkey is attending to. I hope to find neural circuits that dynamically change their functional connectivity depending on what the monkey is doing.

The packaging of eukaryotic genomes into higher order chomatin hampers the accessibility of the genome for enzymes involved in processes such as DNA replication, DNA repair and transcription. Chromatin remodelling complexes help these enzymes to overcome this barrier. One class of such complexes consists of ATP-dependent machines, which contain an ATPase subunit from the SNF2/SWI2 superfamily. Recently, a new member of this class, the Ino80 ATPase, has been identified in budding yeast. It is part of the INO80 complex, which consists of 12 polypeptides. The purified complex remodels chromatin and displays 3’-5’ helicase activity. Three of the subunits, Arp4, Arp5 and Arp8, are actin related proteins and associate with histones. Mutants of Ino80, Arp4, Arp5 and Arp8 are sensitive towards DNA damaging agents and show defects in transcription, which suggests that chromatin remodelling driven by the INO80 complex may be linked to both transcription and DNA damage repair. I propose to clarify the function of the INO80 complex in both DNA repair and the response to DNA damage. Chromatin-immunoprecipitation, high resolution immunofluoresence and real-time live-imaging microscopy will be primarily used to see if INO80 is recruited to double-strand breaks (DSB) and affects histone H2A phosphorylation and/or degradation of the DNA ends at a DSB. A whole genome transcriptional microarray analysis will be performed to investigate if INO80 affects DNA damage repair by inducing expression of DNA repair genes. The proposed research allows me to change my topic of research and to acquire new skills in microscopy and cellular biology for the study of DNA damage repair in yeast.

Cell motility is crucial for many physiological and pathological processes. The objective of this project is to gain a better understanding of cell motility. An important role is played by actin. The polymerization of actin into filaments at the plasma membrane generates a force that induces cell movement. Another protein involved in cell motility is myosin. Myosins are important for controlling the actin network. Their precise role and the cooperation with actin in producing cell movement are still poorly understood, however. The approach that we suggest here is to realize a biomimetic system that reproduces the dynamical shape changes of suspended cells where microtubules have been depolymerized. These shape changes are regulated by the same mechanisms as cell movement. We will replace the cell membrane with a well-defined lipid bilayer. A liposome in suspension will be filled with cell extracts, and actin polymerization will be catalyzed at the liposome membrane. Deformations of the liposomes can then be followed and the effect of specific proteins can be investigated. The work will be compared to a biological system studied in the lab, consisting of detached motile cells that show shape oscillations, and to theory developed in parallel. My previous research experience is in the field of physical chemistry and polymer physics. In my future professional career, I hope to use my physical background to work on biological problems. This fellowship will help me to receive the training in biological sciences needed for this.

The aim of this project is to address a serious unknown in limb patterning, concerning the lineages and motions of cells that form the limb. The project draws on Fraser lab's imaging tools and in ovo techniques to assess directly some key events in limb formation in the chick. My training in the IGBMC, Strasbourg, France, was mainly focused on molecular biology, cell biology, and the study of murine embryology. All these expertises will help to pursue this project but are very far from what I'll do in Scott Fraser's lab, where I'll learn novel techniques of in vivo imaging and DNA micromanipulation. This research project has a high training content and will be very beneficial for the successful continuation of my career. The experience I am going to obtain in in vivo imaging techniques would be a valuable asset for pursuing my scientific interests in the future, after returning to France. I have chosen Caltech (Pasadena, United States) for my post-doctoral training because it is one of the best research centers in the US and because the combination of molecular biology, biochemistry, biophysics and in vivo imaging expertises available are crucial for the successful pursuit of the studies proposed here. Furthermore, the laboratory of Pr Fraser is one of the best in the field of imaging and this, in conjunction with the interdisciplinary character of the project and the innovative and interactive environment both of the laboratory and the institute as a whole, will, with no doubt, broaden my scientific horizons and help me in developing a more mature and critical attitude towards scientific research.

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.