Normal aging is associated with structural and physiological changes in prefrontal systems. However the significance of these effects to behavior is not well understood. To study this question, I will focus on orbitofrontal cortex (OFC) and basolateral amygdala (ABL) as a model system and examine the effects of aging on this system using a Pavlovian reinforcer devaluation task. In the proposed experiments, I will compare task performance in young and aged rats in order to characterize the effects of aging on the orbitofrontal-dependent functions. I expect that a subset of aged rats will be impaired on this task. To identify the neural substrate of this deficit, I will then record neural activity in OFC and ABL during training in the same Pavlovian reinforcer devaluation task. I expect to find abnormal encoding in this circuit that accounts for the age-related behavioral deficit. I believe that these experiments will further our understanding of the normal operation of this circuit and also of how this operation is disrupted in normal aging. My host laboratory has substantial experiences applying methods of chronic neural recording in awake behaving rats powerful methods to examine the cognitive brain functions. Thus I will be able to learn these neurophysiological techniques and learn to analyze animal behavior and cognitive brain functions. The fellowship will enable me to advance my career from my previous research of sensory system to that of cognitive brain functions.

Permanent memory is believed to be stored in the neocortical areas in a distributed manner. Initially the hippocampus connects these neocortical areas, and repeated reactivation of acquired activity patterns in the hippocampus eventually establishes direct cortico-cortical connections which support memory independently of a functional hippocampus. This hypothesis is supported by the reports that the spiking patterns of the hippocampal and neocortical neurons during a task are reactivated in a coordinated fashion during subsequent rest period (memory trace reactivation) however, it has never been revealed whether cortico-cortical connections are actually established as a consequence of memory trace reactivation. To study this, I will combine neuronal ensemble recording, which is a novel technique for me, with pharmacological treatments, in which I have already developed expertise. I will record neural activity of the rat neocortical areas during sleep after retrieval of recently or remotely acquired spatial memory and investigate the effect on coordinated reactivation of these areas of acute hippocampal inactivation and/or cotico-cortical disconnections. And I will investigate chronic hippocampal inactivation or chronic blockade of neocortical NMDA receptors during consolidation on coordinated reactivation for remotely acquired memory. I expect that the outcome will provide important missing evidence in support of the trace-reactivation theory of memory consolidation. Besides, by using a task schedule in accord with previous behavioral studies, this project can bridge the gap between mnemonic processes revealed by behavioral study and the neuronal activity data.

Cell surface trans-membrane (TM) proteins link the interior of the cell with the extra-cellular environment and allow the cell to transport nutrients and to respond sensory information. To preserve the function of the plasma membrane, quality control mechanisms recognize and eliminate defects in integral plasma membrane proteins, to prevent damage that might severely compromise cellular life. Quality control mechanisms that function at the Endoplasmatic reticulum (ER) and the Golgi apparatus are well understood. How cells recognize and specifically remove TM-proteins that suffer damage after insertion into the plasma membrane is not known. Therefore I propose to study cellular mechanisms that underlie the quality control of TM-plasma membrane proteins. I will employ genome wide screens in yeast to uncover how defects in damaged cell surface TM-proteins are detected and how regulatory signals link up with the components of the endocytic machinery to selectively remove the damaged protein from the cell surface. In my previous work I used mainly cell biology and reverse genetics in mouse to investigate how ERK signaling specificity is generated. In the proposed project I will learn how to employ forward genetic screens in yeast to elucidate general biological mechanisms that are essential to monitor the function integrity of the plasma membrane. The mechanisms identified in the proposed genome wide screens might have fundamental significance in understanding the pathogenesis of conformational diseases and in the maintenance of cellular homeostasis. With the results of the proposed study I should be able to launch my own independent research projects.

Calorie restriction (CR) in mammals has long been known to prevent a range of age-associated illnesses including cancer, diabetes and a variety of neurological disorders in rodents. Consistent with this, a variety of cell types from CR rodents are relatively resistant to cellular insults such as toxins and oxidative stress. Multiple lines of evidence indicate that SIRT1, a member of the sirtuin family, is a key regulator of cellular survival during stress and may also underlie the lifespan extension and health benefits of CR. In addition to its role in glucose and fat regulation, several data suggest SIRT1 involvement in neurons survival and resistance to stress. The general aim of this project is to gain insights in the mechanism by which sirtuins provide neuroprotection. We will use brain-specific SIRT1 transgenic mice crossed with a mouse model of Huntington disease. SIRT1-induced neuroprotection will be evaluated and the identification of SIRT1 specific targets in this model will be addressed. The conclusion of this project will be extended to other mouse models of neurodegenerative disorders and will provide important insights in the outbreak mechanism of these diseases. My previous work focused on the biochemical and molecular characterization of protein complexes. This fellowship is an important departure from my previous experience: neurobiology is a novel field to me and the project involves many untouched techniques. I am keen on extending my expertise to in vivo work and different approaches in order to be able to address biological questions in the future in a more extensive and comprehensive way as an independent scientist.

Many cellular processes are regulated through protein modification by conjugation of small Ubiquitin-like proteins. Recently, a new Ubiquitin-related modifier called Urm1 was identified in yeast and shown to modify proteins in vivo, a process now called urmylation. The function and mechanism of urmylation are unknown. A genetic screen conducted in the Barral lab to find regulators of spindle positioning identified a group of genes that are clearly involved in or linked to urmylation. More specifically, the genetic screen was designed to find components of one of two pathways regulating spindle positioning, the Kar9 dependent pathway. This implies that the Kar9 pathway for spindle positioning is regulated through urmylation. In my research, I will identify the urmylation target within the spindle positioning pathway through biochemical and genetic analysis. The Kar9 protein itself has been reported to be modified which makes it a strong candidate for urmylation target. Once a target is identified, I will characterize the molecular function of urmylation in spindle positioning based on unmodifyable mutants. For those experiments, I will use biochemical methods and advanced microscopic techniques. Further, the knowledge of several urmylation related genes that were identified in the screen allows me to characterize their effect on the urmylation process and will unravel the mechanism of urmylation. The laboratory of Prof. Dr. Yves Barral provides a unique environment where to conduct this research, as spindle positioning is his area of expertise. I will also closely collaborate with Prof. Dr. M. Peter, in the same building, an expert in protein conjugation.

The ubiquitin-proteasome system plays an important role in controlling diverse biological processes, ranging from signal transduction to cell cycle control. In this system the protein ubiquitin is transferred and attached to substrate proteins by the sequential action of E1, E2 and E3 enzymes. Reiteration of this cascade leads to the formation of a polyubiquitin chain, targeting the substrate to the proteasome for degradation. E3 ubiquitin ligases determine the specificity of the protein substrate, and thus ultimate control of the ubiquitination reaction. The purpose of this research is to characterize the mammalian cullin-RING ubiquitin ligase formed by Skp1-CUL7-FBXW8. To address this, I will isolate Skp1-CUL7-FBXW8 complex-associated cellular proteins using double affinity purification followed by a multidimensional capillary chromatography in line to mass spectrometry (MudPIT). I will analyze the functional role of this complex-substrate protein association established through specific FBXW8 interactions, and determine its participation in the transforming activity of SV40 large T antigen. In a broader sense, the goal of this project is to enhance our understanding about the specific cellular pathways that are being regulated by cullin-RING complexes in mammalian. Previously trained in structural biology, in particular DNA-plant homeodomain interaction, I will now move to the functional analysis of a mammalian cullin-RING ubiquitin ligase. The HFSP will give me the opportunity to vastly broaden my scientific experience both conceptually and technically in an excellent intellectual environment and to be prepared to solve the critical scientific questions in the future.

In microbiological systems as diverse as bacteria and colonial algae nature has engineered systems composed of multiple flagella, each driven by dedicated molecular motors, which exhibit collective behavior whose quantitative physical and biological explanation has remained elusive. Conceptually similar collective synchronized dynamics occur with motor proteins such as kinesins, dyneins and myosin. How does this coordination occur? This proposal aims to answer these questions through a combination of theoretical modeling and experimental work. The first of two goals of the project is the development of a quantitative mathematical model of hydrodynamically-driven motor synchronization for the two biologically relevant systems: helical prokaryotic flagella driven by rotary motors and undulating eukaryotic flagella driven by linear motors. Second, we will investigate this coupling experimentally through micromanipulation and high-speed imaging of Volvox flagella under varying light conditions to see how the phototactic response develops in terms of synchronization of nearby flagella. Together, these two approaches will shed light on the key physical features which underlie synchronization of driven, stochastic systems at low Reynolds number. My prior research experiences on the fluid dynamics have given me invaluable insight into hydrodynamic flows created by small bodies. Nevertheless, I have the strong conviction that to achieve real progress in this kind of science I must muster equally well the theoretical and the experimental practice. My main motivation to apply with this research plan is to get training in the experimental techniques in use in biology.

An intimate link exits between cell attachment to the extracellular matrix (ECM), migration, cytoskeleton organization and intracellular biochemical signaling pathways. My research goals are to study systematically the influence of physical parameters such as receptor/ligand distribution, binding energy and mechanical characteristics of the cell and the matrix on cell-ECM interaction and hence on the integrated process of cell motility. At the same time, I intend to use the insight regarding the physicochemical characteristics of the system, in order to design an artificial extracellular matrix (aECM) that will enable controlled directed cell migration. During my PhD studies, I developed statistical-mechanical models for self-assembly processes and developed and analyzed Monte-Carlo simulations. This experience provided me with the necessary tools to model the connection between cell-ECM adhesion, cytoskeleton organization and cell migration. The research I plan to perform during my postdoc training is interdisciplinary and involves both the synthesis and manipulation of artificial ECM and the modeling of the behavior encountered on the cellular level. The research is done in close collaboration with a cell biology lab which use fluoerescence techniques to monitor integrin clusters at focal adhesions and cell migration on a single cell basis. Through a continous dialogue between experiments and theory, we can gain a deeper understanding of the investigated physical phenomena.

This proposal lists two sets of experiments whose aim is to investigate the role of the Rb family of proteins in cell cycle control and tumor initiation. While Rb proteins have been identified as tumor suppressors nearly 20 years ago, the molecular basis of this activity remains partially defined. Growing evidence suggests that, within a tumor, a population of “cancer stem cells” possesses the unique ability to proliferate indefinitely and re-create a tumor. We hypothesize that cancer stem cells may arise either from poorly differentiated progenitor cells or from fully differentiated cells, depending on the type of cancer. We will investigate this hypothesis by studying two models that recapitulate both possibilities. In the first set of experiments, we will study the consequences of Rb loss in the onset of retinoblastoma. In this model, we hypothesize that cancer initiates from cycling progenitors. This project will provide us with a better comprehension of the mechanisms that lead to retinoblastoma following the loss of Rb function in retinal progenitor cells. In the second set of experiments, we will test the idea that loss of Rb function in mature hepatocytes results in cell cycle re-entry and cancer initiation. Loss of Rb family function is commonly found in hepatocarcinomas and this model may give us critical insights into the provocative idea that cancer may initiate from differentiated cells. These projects will provide me with extensive training in interdisciplinary fields such as mouse genetics and stem cell biology. Combined to my personal background, these experiments will enable me to acquire the skills necessary for a comprehensive approach on tumorigenesis.

Innate behaviors offer an excellent opportunity to explore how complex behaviors are organized in the nervous system and how they are programmed during development, because they are likely to result from the activation of developmentally specified neural circuits. Here, I propose a strategy to identify and functionally characterize the specific neuronal circuits involved in innate behaviors in fruit fly, drosophila. Especially, I will focus on the role of octopaminergic neurons, which are implicated in the modulation of various innate behaviors, by addressing the following questions: (1) What is the effect of acutely manipulating octopamine levels on aggressive and mating behaviors, and on general arousal? (2) Which octopamine receptors are involved in mediating the effects of octopamine on these behaviors? (3) Which octopamine-synthesizing neurons are important in these behaviors? (4) In which neurons are the relevant receptors required? To address these questions, I will use genetic tools to conditionally enhance or inhibit octopamine release from various subsets of octopaminergic neurons in living drosophila, and perform various behavioral assays. Thus, I will functionally dissect the role of octopaminergic neurons to understand how specific neural circuits control specific behaviors. In contrast to my previous research experience in neural development, the proposed research will focus on understanding of the function of neural circuits, which mediate innate behaviors. Therefore, this will be a good opportunity to extend my training from developmental, to functional and behavioral neuroscience.

1) The aims of this project is to develop proteomics methods to identify the substrates of protein phosphatase 1 (PP1) in neurons. The approaches will combine novel phospho-protein enrichment methods and state-of-the-art mass spectrometry. Using these methods, I will use brain tissue from wild-type and mutant mice lacking each of the three PP1 isoforms to identify substrates of each PP1 isoform. These proposed studies will establish general methods that can be used study protein phosphorylation, as well as reveal the functional differences between PP1 isoforms in neurons. I will then use the proteomics methods I have developed to study the roles of each PP1 isoforms in mice following behavioral tasks related learning and memory. 2) My previous research has focused on Cyclin-dependent kinase 5 (Cdk5). Using biochemical and molecular techniques, I have found phosphorylation-dependent proteolysis pathway in regulation of Cdk5 activity in neurons, and also revealed the functional role of Cdk5 in insulin secretion in pancreatic beta-cells. In the proposed project, I will change my focus from protein kinase to phosphatase, particularly PP1. In addition, I will develop and use various proteomics methods to identify new substrates for PP1 isoforms. 3) Together with my previous experience of kinases, the experience of studying phosphatases in this fellowship will complement my knowledge of protein phosphorylation mechanisms. I believe that the future studies will contribute to my broad and thorough understanding of phosphorylation-dependent signaling pathway. Moreover, the state-of-the-art proteomics methods will serve as a technical basis for my future research.

The actin cytoskeleton plays an important role in intracellular membrane trafficking. N-WASP, a regulator of actin assembly, is implicated in endocytosis and mediates actin-dependent rocketing of intracellular vesicles, but its precise cellular function is not well established. A recent study found that Abi1 binds to and regulates N-WASP activity in actin-based vesicular trafficking, as well as endocytosis of EGF receptor and transferrin receptor. Based on this and unpublished data that the SNARE domain of Abi1 is involved in its subcellular localization and that casein kinase 2, which is shown by others to phosphorylate N-WASP, was found to be an interactor of N-WASP by mass spectrometry studies, the following specific aims are proposed: 1) Test the hypothesis that the SH3 domain and other region(s) of Abi1 are required for full activity of Abi1 to stimulate N-WASP-dependent cellular processes. 2) Identification of the molecular determinants dictating Abi1 localization. 3) Test whether CK2 is involved in N-WASP-dependent cellular processes. This project will be a change of my research direction from membrane fusion in the early secretory pathway to the actin cytoskeleton and its regulation in intracellular trafficking. I will study a new aspect of membrane trafficking and learn and apply a wide range of biochemical, cell biological and molecular genetic approaches, which will significantly expand my knowledge and skills as a researcher in cell biology.

During development the basic wiring of the nervous system is established by connecting millions of neurons to their targets. In order to establish these connections, neurons send axons that navigate through the embryonic environment until they reach their targets and form synapses. The guidance of axons is directed by specific cues in the extracellular environment, and the past decade has seen the discovery of several families of guidance molecules and their receptors. Accurate guidance of an axonal tract requires the collaboration of multiple guidance cues. Thus, a central unanswered question in developmental neurobiology is how do multiple guidance cues spatially and temporally coordinate? With the help of the Career Development Award I would like to establish a rigorous research program that will address this question, using the guidance of sensory axons as a model system. Previous studies have demonstrated that Semaphorin-3A is an important guidance cue for sensory axons, and my work has shown that Plexin-A3 and -A4 serve as the primary signaling receptors for Semaphorin 3A. However, it is apparent that many additional cues collaborate with Semaphorin-3A to guide these neurons in an appropriate trajectory. Through the use of mouse genetics combined with in vivo and in vitro analyses I plan to identify cues that cooperate with Semaphorin-3A to guide sensory axons. Not only will these experiments increase our understanding of sensory system biology, more importantly these studies will uncover fundamental principles underlying the logic of axon guidance.

Cellular identities are defined by distinct gene expression programs that are set early during development and maintained via epigenetic mechanisms based on e.g. histone and DNA methylation as well as chromatin associated Trithorax and Polycomb group proteins. In mammalian pre-implantation embryos and primordial germ cells (PGCs), extensive changes in DNA methylation and histone modifications take place during the time of reacquisition of pluripotency. Importantly, members of the Polycomb Repressive Complex 1 (PRC1), implicated in homeotic gene silencing, X inactivation and stem cell renewal, are dynamically expressed during these developmental stages. Although epigenetic reprogramming events are thought to play a key role in establishing pluripotency, the precise underlying molecular mechanisms remain elusive. I propose to address the role of PRC1 in epigenetic reprogramming. First, I will use mice conditionally deficient for PRC1 components to examine PRC1’s regulatory role for higher order chromatin formation and directing cell fate in PGCs and pre-implantation embryos. Second, I will identify PRC1 target genes by genome transcription profiling using mutant mice. Third, I will address the significance of the PRC1 associated histone H2A lysine 119 mono-ubiquitination activity for epigenetic gene silencing in mammals. Through these projects, I will greatly extend my scientific horizon from my previous work on the regulation of chromosome segregation in fission yeast to the molecular mechanisms of epigenetic reprogramming in multi-cellular organisms. The proposed research will give me a thorough training in many aspects of epigenetic research and mouse molecular genetics.