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.

The appropriate connections of retinal ganglion cells (RGC) to their thalamic target, the lateral geniculate nucleus (LGN), are essential for visual function and involve precise topographic mapping and arbor reorganization. My project will link molecular specification of both retina and LGN to the innervation of retinal fibers, still poorly understood in the mouse. The transcription factor Zic2, the guidance receptor EphB1, and the adhesion molecule NrCAM have been shown by Dr. Mason’s lab to specify the RGC axon growth in the optic chiasm. After delineating the spatiotemporal aspects of RGC innervation, I will assess the role of these molecules one step further in the pathway, in the targeting and patterning of retinal afferents in different topographic and eye-specific subdivisions of the LGN. My preliminary data implicates EphB1 in the topographic mapping of RGC fibers. Furthermore, EphB1 and Zic2 are expressed differentially in subregions of the LGN and might therefore also participate in target specification. My previous training in developmental neuroanatomy will be applied to a more defined and malleable system. I will learn additional approaches in Dr Mason’s lab as I determine a role for these genes by perturbing gene expression in transgenic models and by blocking or ectopically expressing genes in the system in vitro and in vivo. This work is essential to our understanding of the interplay between activity-dependent and molecular directives for retinogeniculate development. I will also benefit from the rich environment of Columbia University and aim to initiate collaborations important for my future career as researcher in France.

Tumors that have reached a critical size almost invariably encounter an intracellular environment characterized by insufficient nutrient supply and oxygen shortage (hypoxia) due to a poorly developed vasculature. These conditions are known to induce hypoxic and endoplasmic reticulum (ER) stress triggering adaptive gene expression programs that provide the tumor cells with the capability to survive in adverse environments. To identify genes important for growth and viability under conditions that are met by tumors in an intact organism, I will perform systematic genome-wide loss of function screens in mammalian tissue culture in a high throughput format based on lentivirus-delivered shRNA (small hairpin RNA) under distinct stress conditions including hypoxia and ER stress. Genes identified in these screens and their subsequent biochemical characterization may eventually have an impact on our understanding of pathological responses and in developing cures for specific diseases such as diabetes, cancer and neurodegenerative diseases. My graduate research expertise is based on genetic work with an invertebrate model system, the fruitfly Drosophila. For my work as a postdoctoral fellow I will turn my research focus to mammalian cell culture systems implementing high-throughput screening and biochemical methods to study gene functions related to cancer development. This goes along with the use of robots for virus production, harvesting and target cell infection, high-throughput microscopy, and biochemical follow-ups on candidate genes identified in the proposed screens. My project will allow me to discover novel biology and genes that will lay the foundation for my own lab.

Any living organism occupying a particular habitat is the result of an evolutionary success story having provided it with both abilities to survive and sufficient advantages over competing organisms. Accordingly, the characteristics of an organism are a reflection of the environment. A complete understanding of the functions and diversity of molecular mechanisms therefore requires integrating an ecological dimension, and viewing their roles on the evolutionary scale. My project aims at theoretically investigating the relations of the properties of the lower, molecular, level, with those of the higher, environmental, level. I will build on the recent step in this direction undertaken by E. Kussell and S. Leibler who put forward a model for comparing different phenotype switching mechanisms as alternative strategies for adaptation to variable environments. With their collaboration, and in contact with experimental groups, I plan to apply and extend their approach to the problem of switching between different ploidy states, as it occurs in yeast, depending on the environmental conditions. I expect the study of this particular system to contribute to the highly debated issue of the conditions under which diploidy confers or not a competitive adaptive advantage over haploidy. Biology is for me an entirely new subject and this fellowship experience will offer me a unique opportunity to learn about its current progress and questions. My general goal is to be able to identify relevant problems in this area which can beneficit from an approach using the concepts and methods that I have acquired during my PhD studies and already succesfully applied to problems from computer science.

Cellular asymmetries are important for many developmental processes, including axon guidance, epithelial function, and asymmetric cell division. The asymmetric first cell division of C. elegans is a powerful model for studying how cellular asymmetries are established and tranduced. In C. elegans and most or all animal cells, conserved PAR proteins play a central role in cell polarity, but it is still not well understood how they function. I propose to identify positive and negative regulators of PAR signalling pathways through genome-wide RNAi screens, in which nearly every gene in the genome can be rapidly tested. I will also biochemically screen for proteins that interact with PAR proteins. I will study the new proteins identified to fit them into a pathway of PAR function. I have chosen C. elegans as a model for this project because it will enable me to do large scale genomic screenings that, together with proteomic approaches, should rapidly yield a lot of information. Conservation of PAR protein function across animals means that the results I obtain will be useful for understanding other developmental processes. My PhD work concerned axon guidance in a non-genetic system. I decided to change disciplines and use the power of genetics to study the basic process of how cells establish and use asymmetries. The Ahringer lab is the perfect place to do this work as they have pioneered genome-wide RNAi screening and will provide a great environment to learn the C. elegans model and genetic approaches. This will give me a broader perspective in science, putting me in a good position to do independent research in the future.

If neuronal plasticity depends on synaptic activity, the mechanisms responsible for synapse efficacy are not yet well understood. Recent data suggest an active participation of astrocytes in central functions by dynamic interactions with neurons through their calcium signaling. However the contribution of astrocytes may not only result from individual cells, but also from cellular networks, as they express numerous gap junctions. Thus the goal of this project is to determine whether the connectivity and calcium signaling of astrocytic networks participate to neuronal transmission and plasticity in the hippocampus. The specific aims will determine: 1)How astrocytic networks are organized in the hippocampus and does a plasticity of these networks, dependent on neuronal activity, exist? 2)If astrocytes network organization contributes to neuronal activity and plasticity? 3)What are the astrocytic mechanisms of neuronal modulation? As a major technical limit to the study of neuroglial interactions is to act selectively on astrocytes, targeted pharmacological and molecular tools will be used, such as photolysis of encaged molecules, RNAi and KO mice. Moreover, an interdisciplinary approach combining molecular biology, physiology and mathematical modeling will be adopted in a collaborative effort. The proposed research should provide a better understanding of neuronal transmission and plasticity by identifying new mechanisms of neuroglial interactions. This should reinforce the emerging concept of an active contribution of astrocytes to information processing. This award will be essential for the acquisition of an imaging system and the development of a young research group.