Genomic stability and DNA repair have been suggested to affect mammalian aging. In yeast, Sir2 maintains this genomic stability and longevity by its enzymatic function of NAD dependent histone deacetylase. This protein has seven homologs in mammals and is conserved throughout evolution. However, the functions of these proteins are largely unknown and by deciphering their function, network of protein in which they are involved and the mechanisms in which they are involved will lead to better understanding of genomic stability, cellular senescence and aging in mammals. By using in-vitro biochemical assays and in-vivo mice models I would like to determine the functions of these SIRT proteins and dissect the molecular mechanisms in which these SIRT proteins are involved. I Would like to concentrate on analyzing SIRT7 knock out mice to elucidate the function of SIRT7 and its involvement in genomic stability, cellular senescence and mammalian aging. These studies have great implications towards understanding malignancies related to aging and tumorigenesis.

Epigenetic modifications impart additional layers of heritable information upon the DNA code, regulating the underlying genetic information. Cytosine DNA methylation, a major epigenetic modification in higher eukaryotes, is involved in the regulation of gene expression, but the full extent and mechanisms of control are unknown. I propose to use Arabidopsis as a model system to identify the genome-wide extent to which DNA methylation regulates gene expression, and identify genes/genetic elements that control this regulation. Whole-genome tiling microarrays will be used to detect genome-wide sites of DNA methylation and transcriptional activity between wild-type and methylation-deficient mutant plants, identifying the genes transcriptionally regulated by DNA methylation. Furthermore, genes differentially regulated by DNA methylation between 19 resequenced Arabidopsis strains (ecotypes) will be identified. By forward-genetic mapping, genetic elements responsible for the differential methylation will be identified, thus elucidating molecular mechanisms controlling the regulation of gene expression by DNA methylation. My previous research used biochemical/molecular biological techniques to investigate plant mitochondrial biogenesis. Through a significant change in my research direction, this project will use forward genetic techniques, large-scale genomic analyses, functional genomics, and bioinformatics to investigate mechanisms of epigenetic regulation. This fellowship will give me the opportunity to broaden my research and training, and will provide me with experience crucial for achieving my goal of becoming an independent researcher in the field of epigenetic regulation.

Hematopoietic stem cells (HSC) have been extensively studied and manipulated, however very little is known about their interaction with their microenvironment in the bone marrow. Direct observation of the HSC niche has revealed a more complex picture than previously believed, with HSC residing in close proximity not only of the bone endosteum but also the stromal capillaries. I intend to combine advanced imaging techniques to visualize all marrow compartments in vivo at the same time and investigate the relationship between endosteal and vascular niches. Moreover, as Notch and Wnt pathways have been linked to HSC self-renewal, I will use a cell-specific gene targeting approach to delete Jagged1 and beta-catenin in the osteoblasts and analyze their role within the niche. These studies will bring together two areas of investigation developed in Dr. Scadden’s laboratory, namely models of stem cell/niche communication and high resolution in vivo microscopy, to gain a unique perspective on the dynamic interactions between HSC and their specialized microenvironment in physiological and altered conditions. After investigating lineage commitment of epidermal stem cell progeny during my PhD, this fellowship will give me the opportunity to broaden my experimental skills and address a different question in stem cell biology by focusing directly on HSC regulation. My long-term goal is to understand how similar signaling pathways cooperate in the maintenance of different self-renewing tissues and I expect that the expertise acquired during my PhD and with Dr. Scadden will put me in a particularly favorable position for the establishment of my own research group in the future.

Embryonic stem cells (ESCs) can differentiate into any tissue of the adult organism, including blood. The goals of the proposed project are (i) to determine whether morphogens that induce blood cell fate from embryonic mesoderm will direct ESCs to differentiate into hematopoietic stem cells (HSCs), (ii) to define the genetic pathways induced by morphogen signaling in ESCs, and (iii) to test if morphogens act as true instructive signals during hematopoietic differentiation. In order to activate morphogen signaling in differentiating ESCs, I will either directly supplement growth media with Indian Hedgehog (IHH), Bone morphogenetic protein (BMP4), and Wnt3a proteins, or express these factors from an inducible transgene. I will quantify the extent of HSC differentiation using known parameters of hematopoiesis. By RT-PCR and Chromatin Immunoprecipitation, I will test whether blood cell commitment induced by morphogen signaling occurs via the direct activation of the cdx and hox master regulatory genes of hematopoiesis. Finally, I will isolate cells not fated to form blood and determine the potential of morphogens to re-direct these cells toward the hematopoietic lineage. Previously, I used Drosophila as a model to study the epigenetic inheritance of gene expression states, which is required for the maintenance of cell identities. With this fellowship, I will investigate the role of morphogens in cell fate decisions in ESCs. Thus, I will be able to study a fundamental developmental process in one of the most dynamic fields at the interface between basic biology and applied research, which will be an excellent preparation for my long-term goal to establish my own laboratory.

All cancers are caused by alterations in DNA that affect the biochemical function or expression of certain genes providing expansion capabilities to the cell with the mutations. Generally this is a multi-step process, requiring mutations in several genes that ultimately result in the uncontrolled growth of a clone derived from the cells with the mutations. A main aim in cancer research is to identify the causative genes and mutations leading to carcinogenesis. This knowledge can then be translated into new targets for diagnosis and treatment. We propose to study several aspects of the tumourigenic process using computational techniques in order to better understand the processes leading to cancer and to be able to identify candidate genes causative of cancer when mutated. We will study the evolutionary history of cancer, the variation of cancer genes in human populations, their function and role in the transcriptional regulatory network, expression profiles, genomic copy number etc. Focusing on these different aspects will help us to obtain a better understanding of the tumourigenic process and its causes. Moreover, the knowledge obtained with these studies will be integrated into the development of machine-learning methods that will be used for the identification of candidate genes with high likelihood of being involved in tumourigenesis, and for the identification of candidates specific for a tumour type. We will have close collaboration with experimental groups in order to perform mutation analysis in tumour samples for the best candidates derived from our methods and to study the functional implication of the genes and mutations in tumour development.

The most severe form of malaria is caused by the protozoan parasite Plasmodium falciparum. This parasite uses antigenic variation to avoid immune clearance and increase duration of infection in the human host. Adhesion of infected erythrocytes to host substrates is mediated by members of the PfEMP1 family of variant antigens. While each genome contains 60-70 members of this family, expression is limited to a single gene at any time. Although the molecular mechanism that determines activation and silencing of members of this gene family has been confusing, recent investigation of the nuclear biology of P falciparum has revealed several epigenetic factors that determine or are connected with the regulation of var gene activity. The main rationale for my project is the understanding of the epigenetic regulation of antigenic variation and in particular processes that determine the establishment of mutually exclusive expression of var genes such as genetic imprinting of a single member of the var gene family. The proposed project involves a novel field for me, meaning several new technologies and concepts. I will move from bacterial gene regulation of single loci to a complicated epigenetic system in the human pathogen P. falciparum, a eukaryote system. This switch involves training in techniques for Plasmodium manipulation and culture and in several new molecular and cellular skills and state-of-the-art technologies in epigenetics, which would prepare me in reaching a position of professional maturity and independence. The host lab is a world-renowned one in this field that will present a very rich and dynamic environment and several opportunities to further my interests.

Animals and plants have evolved independently as multicellular organisms. In animals, concentration gradients of signals termed morphogens are used for tissue patterning. However, existence of morphogens in higher plants remains to be demonstrated. Polar auxin transport and the subsequent accumulation of auxin in organ primordia have however been shown to be major factors in organ formation. Expression of the PLETHORA (PLT) family of root identity selector genes coincides with auxin accumulation. I propose to study whether PLTs act as morphogens that promote different pattern elements with different PLT concentrations in the Arabidopsis thaliana root. During my doctoral studies on vascular development I have obtained good skills in developmental genetics. I appreciate that the complexity of genetic interactions, such as feedback loops, can be too challenging for the human brain to derive testable predictions from experimental data sets. At the Scheres lab, together with Prof. Hogeweg’s theoretical biology group located at the same department, I employ their recently set up in silico system to model auxin transport, and develop it further by including PLT as a new component to the model. Hypotheses suggested by the model on auxin-PLT interaction on root patterning can be tested by variety of molecular and cell biological tools developed in the Scheres lab. In turn, the results from the molecular studies can be used to develop the model, and to test whether PLTs act as morphogens in plants. With the new skills on computer modeling and cell biology, I will have the capability to study the complex developmental processes in my future work as an independent researcher.

Protein-protein interactions play a critical role in signaling pathways that control a variety cellular processes. However, the crucial parameters for recognition are not fully understood. This project aims at developing a set of rules for understanding and predicting synthetic molecules that will mimic and modulate protein-protein interactions. By designing molecules that target the exterior binding surfaces of EGF and FGF growth factors, I will be able, not only to evaluate the crucial parameters for growth factors- receptor recognition, but also to turn off their function and probe their roles in cell biology. I will use a variety of techniques to determine precisely where on the protein surface the synthetic agents bind, and which residues are critical. The rules I learn in this project will pave the road towards a more challenging goal, opening up protein contacts to chemical genetics, namely, switching off specific pathways to investigate their function inside the cell. My PhD thesis focuses on the development of molecules that mimic the fundamental logic operations of electronic computers. This fellowship will give me the opportunity to get familiar with an exciting and far more complex biological world. By studying protein- protein interactions I will gain inexpressible knowledge and be exposed to a variety of ideas and techniques. I will then be able to design molecules that interrupt particular protein recognition events. I expect this new experience to contribute to my development as an independent scientist and extend my previous background to be able to explore biological problems that require a deep understanding in both chemistry and biology.

Regulation of T-helper cell polarization by the endogenous alarm signal HMGB1. HMGB1 is a highly conserved nuclear protein that is expressed in all eukaryotic cells. Recently it was discovered that HMGB1 is also active as an extracellular protein, where it displays cytokine-like activities, triggers inflammation and controls the adaptive immune response. HMGB1 is released actively by immune cells, or passively by necrotic cells and damaged tissues. HMGB1 is therefore a very interesting “cytokine”, designed to regulate both the “infectious non self” and the “injured self” response. This project is aimed at understanding the role of this endogenous alarm signal in regulating the APC/T-helper (TH) cell interaction. In this study the following areas will be addressed: Firstly the mechanisms regulating the secretion of HMGB1 by APCs will be investigated. Secondly, the downstream signal transduction cascades triggered by the HMGB1 receptor RAGE will be characterized. Finally, I will investigate the TH1/TH2 polarizing capabilities of HMGB1 and the mechanisms underlining its role in directly initiating or regulating an immune response. The comprehension of these mechanisms should give considerable insight into the mechanisms underlying autoimmune diseases and other pathophysiological immune responses. This fellowship will complement my skills in biochemistry, signal transduction and innate immunity by providing me with a background in immunology, adaptive immunity, and in-vivo studies.

Cryptochrome (CRY) is a blue/UV-A light receptor found in bacteria, plants, and animals including human. CRY phosphorylation is the only known light-dependent biochemical reaction of CRY. Although CRY contains no apparent sequence similarity to any protein kinase or ATP-binding structure, it has been demonstrated that CRY can autophosphorylate in vitro, which may represent a novel mechanism of protein phosphorylation in general. However, the underlying molecular mechanism and other important questions, such as whether CRY is phosphorylated by other kinases in vivo and whether CRY phosphorylates other proteins, remain to be elucidated. Here I propose to use a number of chemical-genetics approaches to address these questions, fully utilizing the information obtained from the recently solved crystal structure of the CRY N-terminal domain. As a graduate student, I studied structure-function relationship of plant red/far-red light receptor phytochrome using molecular genetics methods. Then I strongly felt the limitation of my previous approaches and how important the structural information of a protein to one’s understanding of its action mechanism. I proposed a chemical genetics study, not only because it is a new technique in itself, but also because I am lack of any experience in the structure-based study of protein function. The proposed research plan will allow me to gain expertise in this critical area of biological research and help me to reach my personal goal of becoming an independent investigator in photobiology.

Most physiological and behavioral events are subjected to well-controlled daily oscillations. The circadian oscillator in the suprachiasmatic nucleus (SCN) of the hypothalamus, which is regarded as the central clock in mammals, orchestrates multiple circadian biological rhythms in the organism and is entrained (synchronized) to environmental light/dark cues conveyed from the eye. The SCN consists of thousands of autonomous oscillator neurons, which are synchronized with each other by intercellular communications so that the SCN as a whole contains a self-sustained circadian oscillator. Neuronal and humoral signals from the SCN in turn entrain the peripheral circadian oscillators throughout the body, suggesting that the central clock orchestrates multiple peripheral clocks that play important roles locally in the physiology of each tissue. In the proposed study, we will aim to understand the neural mechanism of circadian rhythm as a multioscillatory system using mice with genetic modifications of specified subsets of neurons. Specifically, we will (1) characterize the network of multiple oscillator neurons that confers a self-sustained and light-entrainable circadian oscillator on the SCN and (2) reveal physiological roles of the extra-SCN circadian oscillators in the brain and interactions between the SCN and extra-SCN oscillators. The proposed plan is an expansion of my previous research during the tenure of the Long-Term Fellowship, and will be the core of my research. The CDA would provide me an essential financial support and also opportunities to discuss and collaborate with excellent HFSP awardees.

It has been said that ‘mirror neurons promise to do for neuroscience what DNA did for biology’. These cells - first discovered in monkey inferior frontal cortex using single-unit recordings - fire during execution and observation of motor actions and are thought to be important in a variety of social domains. Several imaging studies on the mirror neuron system in humans have been published over the last few years. There is still a strong empirical and theoretical gap, however, between the single-unit observations in monkeys and the imaging studies in humans. The studies proposed here are aimed at investigating the human mirror neuron system from the level of the activity of single neurons, and all the way up to the system level, by combining both imaging and electrophysiological techniques in the very same subjects. During my Ph.D. training I combined single cell recordings and functional magnetic resonance imaging (fMRI) to investigate various aspects of coding in human auditory cortex. In my post-doctoral training I intend to make use of these techniques to investigate the mirror neuron system in humans. Single unit activity and fMRI signals will be recorded from frontal cortical regions of epileptic patients undergoing a clinical procedure while observing, executing, or imitating various motor actions presented on a computer. This scheme will enable me to investigate human mirror neurons at the levels of single cells, ensemble of cells (local field potentials) and the entire network (fMRI). By probing the activity of this system at the different levels, hopefully, we will gain further understanding as to its underlying mechanism.

Tight control of gene expression in response to the environmental signal is one of the most important characteristics of life. When cells are exposed to growth factors, the signal is transduced from cell surface receptors to the cascade of signal transduction, and leads activation of specific genes. Serum Response Factor (SRF) was identified as a DNA-binding transcription factor that is required for serum-induced gene expression. Many mammalian genes that are important for regulation of cell proliferation and differentiation, including SRF-target gene c-fos, are regulated at transcriptional elongation. In the absence of serum stimuli, RNA polymerase II (Pol II) is recruited to the c-fos gene promoter, but release from the promoter and elongation of transcription can be induced only after serum treatment, allowing rapid induction of serum-induced gene expression. However, the mechanisms that specifically connect growth factor signaling and stimulation of Pol II release and elongation have not been understood. Here I propose a research plan to analyze the relationship between signal-regulated transcription factors and post-Pol II recruitment steps of transcription, particularly, by focusing on post-translational modifications of Pol II that controls elongation and other transcription-associated events. This research experience is clearly different from my thesis research, in which I studied proteolysis in yeast, as I change the model to mammalian cells, and as I characterize the steps leading to protein synthesis. I would expect to obtain important insights for my future study, by learning about multiple aspects of gene expression as an interconnected biological system.

Mutations in regulators and effectors of the Rho GTPases have been found to underlie various forms of mental retardation (MR), including syndromic and nonsyndromic X-linked forms of MR, as well as autosomal syndromic MR. Regulators of the Rho GTPases involved in MR include guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). oligophrenin-1 (OPHN1), which encodes a Rho-GAP, is expressed in neurons in major regions of the brain, including the hippocampus, cortex and cerebellum, and is present in axons, dendrites and dendritic spines. OPHN1 is required in dendritic spine morphogenesis of hippocampal neurons. At the present, the function of OPHN1 in cerebellar development remains unknown. It will be of particular interest to test the effect(s) of loss of OPHN1 on Purkinje cell development. In this proposal, we will investigate whether altering OPHN1 levels and/or its activity affect synaptic function. We will also investigate the effects of loss of OPHN1 function on the number and size of dendrites and spines in cerebellar Purkinje cells. For this purpose OPHN1 KO mice will be generated. Compared to my previous research this project will give me the possibility to work in physiological more relevant systems such as in hippocampal neurons and brain slices. In vivo studies will be performed to characterize the OPHN1 KO mouse. New techniques such as electrophysiology and two-photon imaging will be used for this project. I will gain insights and experience into in a new discipline such as neuroscience. Finally I’m convinced that this project will help to extent my view on scientific research, which can only be beneficial to me, in the future.

The research plan I propose is to determine the X-ray crystal structure of RET, a receptor tyrosine kinase that is a member of a family of tightly regulated proteins involved in tranducing extracellular signals. Through structural analysis, the regulation of related receptor tyrosine kinases such as c-Kit, FLT3 and EphB2 are now well understood and involves inactivation of the kinase domain through intramolecular interactions with the juxtamembrane region. However, structural information on RET has remained elusive and how it is regulated in the context of the entire cytoplasmic region remains a mystery that my research aims to clarify. The activity of RET is deregulated in several thyroid cancers, and it is hoped that a structural understanding of the regulatory mechanism of RET will assist in the development of improved cancer therapy. Because my expertise is limited to structural and biochemical characterization of RET, I have initiated an international and interdiscplinary collaboration with a research lab in Italy which has the ability to test hypotheses drawn from crystal structures in cellular assays and has developed methods for screening potential RET inhibitors. Most granting agencies require extensive prelimary results before funding a research program and in this regard the CDA offerred by the HFSP will substantially progress my professional growth by allowing me to establish an independent research program at my institution. The project I have chosen is in an area where I have proven to be successful in from my post-doctoral work funded by a long-term HFSP fellowship.

Over the past several years, genes have been identified that alter the lifespan of model organisms. Sir2 was originally found as the gene involved in gene silencing in yeast. Sir2 and its homologues are prominent among these. Sir2 encodes an enzyme that functions as NAD+-dependent deacetylase. In yeast, calorie restriction extends the lifespan of mother cells, and it is dependent on Sir2 activity for longevity. In rodents, calorie restriction also extends lifespan. However the molecular mechanism in rodents is not understood. In mammals, there exist seven Sir2 homologues, called sirtuins. SirT1 is the closest to yeast Sir2, and it also has NAD+-dependent deacetylase activity. SirT1 deacetylates not only histones but also other proteins such as p53 and FOXO. Though yeast Sir 2 only localizes in nuclei, mammal sirtuins localize in various compartments in cell. Surprisingly, SirT3, SirT4 and SirT5 are found in mitochondria. It is known that mitochondria play the most important role in energy production and metabolism in cells. Moreover the connection between aging and mitochondria has been strongly suggested for long time. Therefore I believe that Sirtuins in mitochondria also have an important role in metabolism regulation and lifespan extension during calorie restriction. Among the mitochondrial sirtuins, I will especially focus on SirT5 because the functional role of SirT5 is totally unknown. SirT5 possesses the NAD+-dependent protein deacetylace activity in vitro, but the functional targets are not identified yet. In this project, I will try to identify the functional targets of SirT5 and the SirT5 containing complexes, and elucidate the function of SirT5 in mitochondria.