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.

The occurrence and advancement of metabolic syndromes resulting from obesity is closely associated with adipocyte differentiation and the extent of subsequent fat accumulation. To elucidate the mechanisms underlying adipocyte differentiation as well as the commitment of mesenchymal cells into adipocytes is important to understand the symptoms of obesity-related metabolic syndromes. My research interest is to investigate the mechanisms of mesenchymal cell commitment in detail and the relationships between the molecules that govern the commitment and obesity-related metabolic diseases. To examine them, first I will identify key genes that influence the commitment of adipocytes. Then I will examine their functions in adult using murine models. To identify the key genes, I will make use of two methods-namely gene silencing by RNA interference (RNAi) in mesenchymal cells and gene array analysis. To apply a large-scale library of RNAi-inducing shRNA expression retroviral vectors, I will newly develop a cell system to screen for the genes that are involved in the commitment and/or differentiation of mesenchymal stem cells into adipocytes. I will also make full use of the method of gene array analysis to identify the key genes. Following the identification stage, by generating the candidate gene-deficient mice, I will further investigate the effects of the genes on metabolic disorders from the standpoint of maintenance of homeostasis of energy through the regulation of mesenchymal cell commitment and/or differentiation. The study proposed in this application will greatly broaden my technical abilities and allow me to pursue scientific interests.

How cells regulate their size is a major unsolved problem in basic cell biology. Comparing related organisms, cell size scales with nuclear parameters (size, DNA-content). This led to the hypothesis that nuclear size may control cell size, but this idea has not been tested by direct experiments, and possible molecular mechanisms are unclear. In this study will ask how the nucleus is involved in cell size regulation. For this, we will evaluate the relative roles of protein synthesis versus degradation in response to variation of nuclear and cytoplasmic parameters. Initial descriptive work using optical sensors for protein synthesis and degradation combined with experimental manipulation of the nucleo-/cytoplasmic (N/C) ratio will be followed by molecular perturbations to test specific hypotheses. This project implies a considerable change of scientific focus for me from cytoskeletal to cell size research. I will get theoretically and methodologically acquainted with the field of protein degradation, -synthesis, ribosomal and proteasomal regulation, cell cycle timing, early embryonic development and mathematical biology. For most of these aspects, expertise is directly accessible in the Systems Biology Department and at HMS and close interactions will be established. I will contribute my knowledge in advanced fluorescence microscopy that I gained during my PhD research. The experience and professional contacts that I can gather during my postdoctoral time at HMS, I anticipate to be highly beneficial for the development of a scientific long-term perspective/career. I hope that the HSFP will support this.

In addition to neurons, the brain contains three major types of glial cells. Among these, the role of astrocytes, including Bergmann glia (BG) in the cerebellum, has remained enigmatic. In recent years, astrocytes have been shown to have some surprising functions, including control of synapse formation and function. Furthermore, both synaptic and structural plasticity processes thought to underlie learning and memory in the brain involve astrocytes. In addition, gap junctions (GJs) known to be important in neural synchronization and information processing in multiple brain regions, are highly expressed on astrocytes, allowing them to form an extensive reticulate network and presumably to mediate long-range signalling. To study the potential role of BG cells in cerebellum-dependent motor learning I propose to monitor neuronal and BG network function in live mammalian subjects using two-photon fluorescence imaging of cellular calcium dynamics. The form of motor learning I will study is classical eyeblink conditioning. By using transgenic mice with targeted deletion of BG connexin proteins that form GJs, I will examine how this selective interference perturbs the acquisition and expression of learned behaviour. If we can understand how BG cells as part of the highly conserved cerebellar architecture communicate with the neural circuit, the impact on many cerebellum-dependent behaviours may be considerable. Moreover, the proposed project provides an outstanding training opportunity in behavioural and molecular neurobiology, complementing the set of capabilities I already have. This will allow me to independently study the role of glia in cognitive function at various levels.

Exploring molecular mechanism for asymmetric cell division is important to understand not only the development of an organism but also the differentiation and proliferation of multi-potent stem cells. Imbalance of such cell division results in tumorigenesis caused by overproliferation. I previously had focused on the intracellular signaling events for establishing neuronal polarity in vitro by using biochemical and cell biological approaches. In this project, I will investigate how neural stem cells precisely and continuously generate differentiating and self-renewing daughter cells by asymmetric cell divisions in Drosophila nervous system during development. Recently, the host laboratory has found that a protein Brain tumor (Brat) localized asymmetrically in neuroblasts and inhibited cell growth in the daughter cells. Homozygous brat mutants result in the production of a tumor-like neoplasm in the larval brain, suggesting that asymmetric segregation of Brat in neuroblasts controls self-renewal versus differentiation. Specific aims of my research proposal are 1) to examine how Brat inhibits neuroblast self-renewal by systematically isolating Brat binding proteins, and 2) to search mutants causing overproliferation or underproliferation in the larval brain by genome-wide RNAi screening for systematic surveys of gene function in vivo. I would like to combine my biochemical and cell biological talents with developmental biology and Drosophila genetics in the host laboratory for my future career, which could allow rapid progress especially in a field analyzing complex mechanisms of living organisms.

DNA gyrase, an enzyme essential for cell viability and unique in its ability to introduce negative DNA supercoils, is responsible for maintaining (-) DNA supercoiling in vivo, involved in DNA replication, transcription, repair and recombination, and the prime target of important antibacterial and anticancer drugs. Understanding the mechanism of gyrase may help in producing new or improving existing drugs by elucidating how such drugs interrupt cellular functions in vivo, and contribute in general to the study of protein-DNA interactions. The objective of the work proposed here is to understand the full mechanochemical cycle of DNA gyrase, which will contribute to a better understanding of its overall physiological function. Using single molecule and in bulk biochemical techniques I plan to: (1) characterize the DNA tension dependence of DNA wrapping, the role of ATP, the effect of drugs, and the nature of the rate-limiting step in the enzymatic cycle of gyrase (2) investigate the origin of the rate dependence of strand-passage on DNA topology, the dynamics of DNA wrapping in the presence and absence of ATP and drugs, and the role of the C-terminal domains of gyrase A in the wrapping mechanism and (3) investigate the (-) sc DNA relaxation activity of gyrase and various gyrase mutants. My previous research involved the study of structural aspects of site-specific DNA recombination using small angle scattering, hydrodynamic, fluorescence and computational techniques. The work proposed in this application comprises the study of biological topics and the use of techniques (single molecule manipulation) that represent a clear departure from my previous research.

Despite enormous progress made in elucidating enzyme structure and functions and the extensive database of atomic-level structural and functional information currently available, the versatility, specificity, and efficiency of natural enzymes are yet unmatched by any man-made catalyst. This is mainly because evolution through natural selection has made biological systems extremely complex and redundant, which often obscures the essential functional elements. Nonetheless, our recent survey of the extensive structural and mechanistic information from light harvesting and redox enzymes affiliated with photosynthesis and respiration shows that the basic physics of energy and charge transfer processes can tolerate a broad range of structural and environmental variation. This research project will test this tolerance by constructing modular and functional protein-cofactor assemblies according to the simple engineering guidelines that were derived from investigating the architectures of natural light energy and charge transfer units in photosynthetic and respiratory enzyme complexes. The building blocks for these constructions will be synthetic, de novo designed protein scaffolds, known as maquettes, together with versatile and tunable cofactors such as heme, chlorophylls and bacteriochlorophylls, and their synthetic derivatives. The structure and function of new designs will be examined by a variety of physical methods exploiting the unique possibility to initiate of truly single-turnover catalytic cycles with short light flashes, and the distinct fluorescence and absorbance spectroscopic markers of the cofactors for probing catalytic intermediates and structural variations.

Type I diabetes (also called juvenile diabetes) is an autoimmune disease in which insulin-producing beta cells in the pancreatic islets are destroyed by autoreactive T cells. We have shown that pancreatic islet cells in pre-diabetic NOD mice inappropriately express retinoic acid early inducible-1 (RAE-1) proteins and that autoreactive CD8+ T cells expressing the costimulatory NKG2D receptor infiltrate into the pancreas of pre-diabetic NOD mice. However, why the pancreas in NOD mice inappropriately expresses the RAE-1 genes and whether NKG2D on NK cells, in addition to T cells, contributes to disease progression is unknown. Thus, my proposal consists of two aims: 1) To determine whether aberrant expression of the RAE-1 genes is unique to the genetic background of the NOD mouse strain, and 2) to evaluate the role of NK cells in progression to diabetes. NKG2D and genes very similar to the mouse RAE-1 genes are present in humans. Therefore, by understanding better how this process works, I may provide useful information for the diagnosis or treatment of human juvenile diabetes. The goal of my research is to apply the knowledge obtained through basic research to the clinic.

Notch receptors mediate an evolutionarily conserved intercellular signaling that regulates a wide range of cell fate decision processes. For precise control of Notch activity, various modulators act at the different level of the signaling pathway. One of them is O-fucose on EGF repeat found in the extracellular domain of Notch receptors. O-fucose modification is a rare type of post-translational modifications, which is catalyzed by O-fucosyltransferase1 (OFUT1). Previous research supported by HFSP Long-Term Fellowship, revealed that OFUT1 is absolutely required for Notch signaling. I also found out that Ofut1 can act as a Notch-specific molecular chaperone and this activity is required for cell surface expression of Notch. The fact that OFUT1 can act as both an enzyme and a chaperone raised the question for roles of O-fucose for Notch functions. The re-evaluation of roles of O-fucose constitutes first part of my proposal for CDA. My well-designed approaches will segregate enzyme activity of OFUT1 from chaperone activity, and clearly demonstrate the importance of O-fucose modification for Notch receptors. The CDA will not only serve for pursuing my previous lines of research direction, but also serve for originating new filed of research. The most significant part of my proposal is to elucidate the molecular mechanisms for OFUT1-dependent folding of Notch EGF repeat and elaborate the mechanisms by isolating the novel components involved in the process. The proposed research for CDA will pave a way for understanding the high-order structure of EGF repeat of Notch, which is totally unknown due to limitation of methodology.

Evolution of phenotypic complexity is believed to emerge largely through the generation of novel connectivities in regulatory signaling and genetic circuits. Signaling networks orchestrate diverse cellular activities, yet they are made of a limited number of modular protein components assembled in a combinatorial manner. New pathways likely emerge by duplication and divergence of promiscuous components, but little is known about how new specificities arise. Our goal is to use directed evolution to probe mechanisms by which distinct pathways can be generated from pre-existing parts. Using yeast as a model organism, we will start with a mutant high-osmolarity responsive MAPK pathway that, in addition to its wild-type response, yields a low level of mating pathway activation. We will determine the genotypic changes that most effectively convert this promiscuous pathway into a novel pathway in which osmo-input leads efficiently and specifically to mating output. We will focus on the effects of altering catalytic specificity of the kinases, protein-protein interactions between kinases and systems-level feedback loops. The design principles that emerge should advance our understanding of pathway mechanisms and evolution, as well as our ability to engineer pathways with potential therapeutic and biotechnological benefits. This fellowship, by allowing me to explore Eukaryotic signaling networks evolution, will complement my previous work in protein fold evolution and my Ph.D. studies on protein-membrane interactions. This combined knowledge shall enable me to develop an independent career, deciphering the principles of biological evolution at a molecular level.