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.

Chromosomes are non-randomly positioned in distinct territories within the interphase nucleus. This higher order nuclear organization is linked to gene expression and genome stability. However, the functional significance and mechanisms of how chromosomes are positioned are poorly understood, and in particular, chromosome organization has not yet been systematically studied in intact animals. I propose to investigate chromosome organization in the nematode Caenorhabditis elegans as a simple multicellular model organism. Chromosome positions will be visualized by whole chromosome staining using fluorescence in-situ hybridization, as well as live fluorescence imaging of tagged genomic loci whose expression is regulated during development. The Gasser laboratory has extensive expertise in live cell imaging techniques. This project should reveal if chromosome positions are constant or change during animal development, if chromosome positions vary in different tissues, and if positions of specific loci are modulated by gene activity. In addition, I will use RNAi to investigate how nuclear structural proteins contribute to the regulation of chromosome positioning during development. During my Ph.D. studies of the DNA damage response, I have developed extensive research skills in yeast molecular biology and genetics. This fellowship will allow me to expand my scientific expertise into the use of C. elegans as a model organism and in advanced live cell imaging technologies. This training and experience in a new field of research would greatly benefit the development of my independent research career.

We perceive and understand the world on the basis of how our brain encodes and decodes information. The neural "code" takes the form of electrical pulses called action potentials or spikes. We want to decipher the neural code in order to understand what information is being conveyed by different neurons. Information Theory, which was originally developed for communication systems, has been used to investigate neural coding with notable success. By correlating the response with the stimulus, relationships can be determined that allow one to estimate the stimulus given the spike sequence. This is called reverse reconstruction, and comparing the estimated stimulus with the original stimulus tells you what information about the original stimulus was encoded in the spike sequence. Using this technique, we will investigate the biophysical basis of neuron encoding properties focusing on the role of intrinsic voltage- and calcium-sensitive currents. We will also explore the counter-intuitive possibility that background synaptic noise improves rather than degrades encoding through novel forms of stochastic resonance. My past research focused on cellular mechanisms important for pain. It involved electrophysiology and some simulations, but did not include the more complex analysis tools derived from Information Theory. The current project will therefore involve application of new tools for the investigation of fundamental questions on how neurons encode dynamic stimuli under noisy conditions. This understanding is crucial because if neurons do not encode or transmit information correctly, neurologic function can be disrupted leading to conditions like psychosis and dementia.

The neuronal mechanisms that convert visual information reaching our eyes into signals associated with perception are poorly understood. Cortical visual area MT contains neurons specialised for motion analysis and its activity underlies motion perception. I will study the activity of single neurons in MT of awake monkeys as they view complex visual stimuli and perform behavioural tasks. These tasks will require them to indicate specific parameters of the stimulus, such as what direction it moves in or when it changes direction. Thus the neuronal responses to a visual stimulus can be correlated with the animal’s attentional state and perception during the behavioural tasks, giving insights into the neuronal mechanisms underlying perception. The proposed work differs dramatically from my previous research, which focused on low-level motion processing mechanisms in anaesthetised or fixating animals and was motivated by the desire to understand the neuronal mechanisms underlying motion sensitivity. The proposed work will involve behavioural tasks and probes higher-level processing associated with attentional and perceptual mechanisms. It will examine how the responses of a neuron are used to generate perceptual awareness of a stimulus and how they influence behaviour. The experience with training and testing animals in such behavioural tasks will equip me with the skills to run my own research laboratory investigating the neuronal basis of visual perception.

Microtubule (MT) is one of the major elements of the eukaryotic cytoskeleton, exhibiting complex dynamic behavior. The activity of MT is regulated by MT associated proteins, such as tau, whose malfunction is involved in a number of neurodegenerative diseases, including Alzheimer’s disease and Pick’s disease. While the physiological role of tau is unclear, it is believed to stabilize and bundle MTs and mediates the interaction between MTs and neurofilaments. Certain neurodegenerative diseases affect the amount of tau bound to microtubules and the relative ratio of specific tau isoforms and their aggregation properties. We propose to combine synchrotron x-ray scattering experiments in solutions with high-resolution transmission electron microscopy to elucidate the precise manner by which tau and MT self-assemble and interact. We will study the interactions between tau isoforms and between tau and MTs and the manner in which tau alters MT-MT interactions and influence MT assembly and dynamics. We also plan to study how mutations and phosphorylation, both of which may occur in vivo and cause diseases, alter those structures and interactions. The Career Development Award grant will be a very important source of funding for this project. It will support graduate students devoting full time to this project and will help me in setting up a unique state-of-the-art ultra small angle to wide angle x-ray lab. This lab will be the first of its kind in Israel. It will be essential for extending my research into dynamical aspects of self-assembled systems and will allow flexibility to explore novel ideas.