The mechanisms by which CD4 T cell help facilitates the generation of functional memory CD8 T cells are unknown. This proposal aims to determine whether Th1 and Th2 CD4 T cells are equally able to provide appropriate help to generate effective CD8 T cell memory. CD4 T cells could also facilitate memory generation by promoting complete differentiation to the memory T cell lineage. To test this hypothesis, the differentiation state of the CD8 T cells generated in the absence of CD4 T cell help will be analyzed. Finally, gene regulation is tightly regulated during lineage commitment steps. Little is known about the molecular changes that occur during CD8 lineage commitment. Memory T cells which develop in the presence and absence to CD4 T cell help will be compared to identify a molecular profile of functional versus non-functional memory CD8 T cells. My previous research was focused on signal transduction in T cells, which largely utilized mouse genetics and biochemical and cell biology analysis as experimental approaches. The described proposal is focused on questions surrounding host immunity and experimentally involves infectious models in vivo. This proposal will significantly strengthen my knowledge and expertise in cellular immunology and in vivo model systems. Furthermore, I will maintain a presence in the signal transduction/molecular immunology fields by performing novel molecular analyses on memory T cells generated in vivo. This training environment will provide groundwork for a multifaceted approach to the study in vivo immune responses during infection.

TCRalphabeta (TCRab) precursors are selected in the thymus based on TCR/MHC/self-peptide interactions and this process results in survival (positive selection) or deletion (negative selection) of the precursors. This selection pathway may not be the only one. Indeed some specialized T cells are selected upon agonist signals that otherwise delete conventional thymocytes. The aims of this project are to characterize precursors and selecting cells that participate in this novel selection process and to investigate the functional specialization T cells acquired during this selection process. This project covers novel aspects of T cell selection as well as functional aspects of immune regulation. Thymic selection is a new area for me and my future study will also examine the functional specialization acquired during agonist selection using animal disease models. This new line of research will widen significantly my knowledge and understanding of how the immune system provides protection while preventing self-destruction. I am convinced that this unique experience will prepare me for a successful and productive research career, and that my previous expertise during which I have characterized IEL precursors and differentiation pathways of these precursors in the gut will undoubtedly form a strong basis.

(1) Adenoviral vectors are powerful vectors for gene transfer into the brain. However, systemic anti-vector immune responses abolish vector-encoded transgene expression, and potentially inhibit endogenous brain gene expression. I will test the hypothesis that activated CD8+ T-lymphocytes down-regulate vector and brain transgene expression through IFN-gamma dependent non-cytolytic transcriptional inhibitory mechanisms. To test this hypothesis I will use molecular approaches and knockout animals in a novel paradigm of immune-mediated down-regulation of vector-encoded gene expression developed in Dr. Lowenstein's lab. (2 and 3) The proposed research complements my background in molecular biology and neuroscience to include the area of brain immunology and gene therapy. The research proposed here has far-reaching consequences for gene therapy, and our understanding of brain immune responses in health and disease. This project will broaden my capacity to address complex neurobiological issues in whole animals. Neuroimmunology, gene therapy, and whole animal approaches are completely novel research areas for me. Pursuing my training in a different country will provide me with experience in a different research organization, research field, and scientific approaches. Furthermore, because Dr. Lowenstein is a world leader in neuro-gene therapy and the neuroimmunology of viral vectors, this postdoctoral training will give me the opportunity to start collaborations with researchers all over the world. Also, the HFSP postdoctoral fellowship will provide me with necessary knowledge and autonomy, to allow me, in the future, to establish myself as an independent investigator in Canada.

Neurogenesis is a highly ordered multistep process in which multipotent neuronal stem cells (NSCs) first commit to a specific lineage and, following a proliferative phase, differentiate into a remarkable variety of neuronal subtypes of different morphologies and functions. Recently the Crabtree laboratory has purified a neuron-specific family of dedicated SWI/SNF-like chromatin remodeling complexes. Intriguingly, these complexes appear to undergo subunit exchange as neurons develop from proliferating paraventricular stem/progenitors to post-mitotic mature units. During my post doctoral fellowship, I would like to investigate the functions of these complexes as they evolve from neural stem cells to functionally mature neurons. Specifically, I will use proteomic methods to define and clone each of the subunits of the neural-specific complexes in the paraventricular and post-mitotic regions of the spinal cord. I will then use existing strains of mice in the laboratory and Cre-lox recombination to define the roles of the ATPase subunit Brg1 in proliferating paraventricular neural stem cells and post-mitotic differentiated neurons. Finally, I will define the role of the neural-specific repression of the BAF53a subunit gene in the post-mitotic neuronal compartment. These studies should provide critical insights into the developmental roles of ATP-dependent chromatin remodeling complexes in neurons as well as generating essential reagents for my transition to an independent researcher in this field. This work will be a significant departure from my graduate studies on hemopoiesis and introduce me to proteomic approaches to complex biological problems.

Apoptosis is central to the processes of organogenesis, tissue remodeling, cell homeostasis, and defence processes. Apoptosis is executed by members of the caspase protease family. Although many caspases have been identified, little is known about their physiological role, how they are activated and how their activation is regulated. Thus, using Drosophila melanogaster as a model system, I propose to conduct two complementary strategies to investigate the physiological function of caspases: First, I will employ the RNA interference technique to generate potent and specific gene-silencing of the seven currently known caspases to reveal their role in Drosophila development. Second, I will uncover genes essential for caspase activation and inhibition using a novel EMS mitotic recombination screen. This genetic analysis is thought to reveal new members of the machinery that execute and control apoptosis in Drosophila. The combination of genetic with molecular and biochemical techniques will increase our understanding of how apoptosis is used as a constructive tool in development and how it may function as a defence strategy against the emergence of cancer. During my previous experiences, I mainly have been trained in Drosophila Genetics and Immunology. In the receiving lab, however, I intend to extend my scientific and technical knowledge in Development, Cell Biology and Biochemistry. Apoptosis is a fundamental process regulating immune responses. Since my long-term goal is to establish myself as an independent research scientist in the field of immunology, I expect to gain important insights from working in a laboratory focusing on apoptosis.

The neural identity and connectivity are generated from multiple, combinatorial fusion events during the production of mature RNA transcripts from genomic template in a process termed alternative splicing. Malfunction of this process in the mammalian central nervous system (CNS) causes neurological diseases (e.g. Alzheimer’s), yet little is known about the mechanism. Given that CNS is well known for its cell diversity, it is important to understand where these alternative splicing events occur and being regulated. To address this, I will develop a multiplex fluoresent in situ hybridisation (FISH)-based assay for dissecting the alternative splicing pattern of transcripts at single-cell level, starting from known neuronal examples. Proteins responsible for the CNS-specific alternative splicing will be identified by gene silencing techniques while using the assay to monitor visually and quantitatively the level of alternative splicing. In parallel, I will work closely with an expert on RNA bioinformatics, Dr. Chris Burge at MIT, and use a proven method RESCUE (Relative Enhancer and Silencer Classification by Unanimous Enrichment) developed by his group to identify sequence elements within these transcripts that are responsible for CNS-specific alternative splicing and eventually correlate the regulation of these elements to the cellular context using the FISH assay. Studying RNA-related phenomenon has been my long-term interest and this fellowship allows me to transfer my existing skills in quantitative imaging and bioinformatics, which are now mainly on protein, to the RNA field, whilst learning new techniques in RNA biochemistry and gene silencing in the host laboratory.

1)Light is an important environmental factor for plant growth and development. Previous analysis has revealed that the expression of about one-third of the Arabidopsis genome are up- or down-regulated by light. However, the transcription network and cascade to achieve such a vast change in gene expression is not known. In this proposed project, I will employ both the dynamic genome expression profiling at different time points after light treatment and mathematical modeling to reveal insights about the transcription network and cascade involved. Further, I will also investigate whether chromatin domain or gene-specific DNA methylation and histone modification are involved in the light-regulated genome expression. 2)My Ph.D work is about the morphological and molecular analysis of rice tiller development through examining a single gene. However, in this proposed project, the approaches to addressing biological problem will be changed to the whole genome level and holistic appreciation. I will use a combination of genomic and mathematical modeling approaches to study the light control of Arabidopsis development. I will obtain integrated expertise in diverse disciplines such as biology, genomics, bioinformatics, and mathematics. 3)This fellowship will allow me a postdoctoral training according to my optimal design and not subjected to any specific limitation that may be otherwise linked to a specific support from a host laboratory research grant. My selected post-doctoral research will not only bring about fundamental changes in the approaches, but also introduce me to a new biological problem. These new experiences will be invaluable to my future independent research career.

My long-term interest is to study the molecular events leading to neurodegenerative diseases. Alzheimer's disease and related age dependent dementia are major diseases affecting the aging population globally. Recent studies from our laboratory uncover an enzyme, prolyl isomerase Pin1 that plays a pivotal role in guarding against age-dependent neurodegeneration of Alzheimer's disease and suggest a new concept for therapy. However, how Pin1 protects the brain from neurodegeneration is not clear. The goal of my proposal is to elucidate how Pin1 interacts with the known pathogenic factors and how their interactions affect the development of Alzheimer's disease. The results should help understand how Alzheimer's disease and related diseases develop and might eventually lead to new therapies. This postdoctoral training, if funded, would allow me to apply my knowledge and experience that I learnt from delineating the signaling mechanisms during my PhD training to neuroscience, a completely different and exciting field. Furthermore, it would allow me to learn how to study neurodegeneration using various experimental models, including mice and cultured cells. Finally, my host lab is the leading laboratory in studying Pin1 and Alzheimer's disease, in addition to Harvard Medical School, which is rich in scientific tradition and vibrant in international collaborations. These outstanding training environments would enhance my ability to approach scientific questions with original ideas and methods. After the training, I hope to set up my own research program on neurodegeneration in Singapore, which is becoming the scientific research center in South East Asia region.

Ras homologue enriched in striatum (Rhes), a new member of the Ras GTPase superfamily, has been implicated in several cellular responses, one of which is insulin secretion from pancreatic beta-cells after exposure to insulin secratogues. The molecular mechanism culminating in increased insulin secretion and the role of Rhes in this process is currently unknown. The proposed research effort seeks to define Rhes function in beta-cells by identifying Rhes effector proteins and signaling pathways as well as by mapping the subcellular localization of this signaling molecule. My research background is heavily rooted in mechanistic enzymology, so the proposed research effort differs in several aspects from my Ph.D. research. I will enter an entirely new field where I will be exposed to many new techniques and, more importantly, perspectives unique to studying complex processes within the context of the whole cell. The primary research focus of Prof. Hans Bos’ laboratory is characterization of small GTPase function therefore, I anticipate mastery of relevant techniques in a timely manner. This fellowship will promote my development as a researcher due to the scientific excellence of the host laboratory and my enthusiasm for the proposed research.

The ability to analyze global gene expression patterns using DNA microarrays has revolutionized basic research in the life sciences, however, its usefulness has been precluded by the difficulty in gene transcripts from specific tissues or cellular populations. The goal of this project is to develop a system by which to isolate mRNAs whose expression is specifically enriched in the C. elegans nervous system or neuronal sub-type populations with the intent of developing a comprehensive understanding of the unique patterns of neuronal gene expression and how these patterns are affected by thermal stimulation. The nature of this project demands the coordinated contribution of multiple research disciplines including genomics, molecular biology, physiology, and behavior. Although my previous research training in cellular neurobiology will be an asset, the current project represents a significant departure from that field, both in the nature of the questions being asked and the tools being used to address them. This fellowship will be my first formal exposure to DNA microarray technology and to molecular genetic experimental techniques. I believe this broad experience is essential for the increasingly multi-disciplinary field of neurobiology. The training opportunity available to me as a member of the laboratory of Dr. Goodman and of the greater scientific community of Stanford University is first-class, and will provide a solid foundation as I continue my scientific career in neuroscience at an academic or government institution.

1) Bidirectional signaling by Ephs and ephrins regulates a number of processes during nervous system development. In addition, ephrinB2 signaling has an important role in vascular development. In spite of the recent progress in understanding the importance and basic activation mechanisms of ephrinB signaling the critical determinants and downstream effectors mediating ephrinB2 transduced cellular responses remain to be elucidated. The objectives of this project are to understand these molecular signals and to identify mechanisms by which ephrinB2 signaling is mediated in endothelial cells and in neurons. The studies will be carried out using mutant mice which have specific mutations in ephrinB2 and display more subtle phenotypes when compared to mice completely deficient of ephrinB2 signaling. Phenotypic analyses and cellular and biochemical studies using cells isolated from these mice can facilitate a detailed study of the critical ephrinB2 signaling components involved in vascular and neuronal development. 2) In my PhD work I studied the molecular mechanisms of lymphangiogenesis – in the present work I will focus on the regulation of nervous system development by ephrins and Ephs. Part of my project involves analysis of lymphatic phenotype in an ephrinB2 mutant, which enables me to use my previous expertise. However, my main goal is to enter the field of neurobiology. 3) Several molecules regulating vascular development appear to be involved also in the regulation of nervous system development and vice versa. Therefore knowledge of both research fields may give me an ability to produce novel ideas and to carry out my future studies using both systems in a flexible way.

The aim of the present project is to study the process of spontaneous polarization in budding yeast. In order to do that we will perform quantitative experiments and build mathematical models of the dynamics of an activated form of Cdc42 in the yeast Saccharomyces cerevisiae. In our project we will first establish a mathematical model that describes the processes of diffusion, transport, endocytosis of Cdc42 and nucleation of new actin cables. We will then experimentally determine the model parameters with in house analysis tools. The experiments that we will have to make include the analysis of the Fluorescence recovery after photobleaching (FRAP) of Cdc42 tagged with GFP and of photoactivated fluorescence (PAF) of variants of Cdc42-GFP. We will finally predict with our model the behavior of the system under different conditions and will make experimental tests. My previous research experience was in modeling different processes in nuclear and particle physics. In this project I will be able to apply my analytical and computational skills to a completely different problem: the dynamics of Cdc42 and actin in the process of spontaneous polarization. Unlike in my previous projects, I will have now to take part in experiments and analyze the experimental data in order to extract the model parameters. This will require learning techniques in yeast genetics and microscopy. The HFSP long term fellowship will allow me to deepen my knowledge of biology and especially of experimental techniques in biology. This will be very important in my future career and will help me to formulate new questions and looking for new problems.

Epithelial cells form sheets that line internal organs and spaces. Most fatal malignancies in adults arise from epithelial organs, such as colon, mammary, and lung. Epithelial cells are highly polarized, with an apical surface facing the lumen of the organ, and a basolateral surface facing other cells and the underlying basement membrane. Epithelial polarization depends on a common molecular mechanism that is conserved throughout metazoa. A complex of Par3/Par6/aPKC and Cdc42 is a master regulator of epithelial polarization. Prof. Mostov's lab has shown that the orientation of the polarity of epithelial cells can be separated from the polarization itself. In three dimensional culture, epithelial cells form hollow cysts lined by a monolayer of cells. The cells are all polarized in a uniform orientation, with the apical surface facing the central lumen. I will examine how Par3/Par6/aPKC orient polarity. First, I will localize each of the components, and then, I will examine the functional roles of Par3, Par6 and aPKC in orientation of polarity. My previous research focused on the biochemical analysis of a raft-associated proteins involved in protein trafficking. Now, I would like to focus on a more physiological field, such as the study of differentiation and cell polarity development, and its relationships to cancer progression. In this project I will use novel techniques such as 3D cultures that resembles organs and time-lapse confocal microscopy. I will address the important scientific question of how polarization is controlled in multicellular structures. This work will also give me the conceptual framework and experience that I need to become an independent researcher.

The Efficient Coding Hypothesis (ECH) states that the brain utilises the extensive redundancy of natural sensory signals to represent information about the environment in an efficient way. Despite its success in explaining linear response properties of visual neurons, it is unclear whether the ECH is a general principle of brain function. To address this issue, I propose to test whether the ECH can account for neuronal response properties in somatosensory cortex. I will measure the statistics of natural tactile signals and electrophysiologically record the cortical response to these signals. By deriving optimal filters for processing the tactile data, I will test whether the cortical neurons are efficient encoders. Finally, I will apply computational modelling to investigate how the cortical output might be efficiently decoded by downstream neural structures. This is a highly interdisciplinary project that combines neurophysiological experiment with sophisticated mathematical and computational analysis. I will apply the skills and frame of mind that I developed in theoretical physics to the challenge of understanding brain function. Drs. Panzeri and Petersen are leading researchers in the field of neural coding with an active and productive collaboration. The combination of Dr. Panzeri's computational expertise and Dr Petersen's neurophysiological expertise, offering a near unique level of theory-experiment interaction, makes UMIST an exceptional training environment. An HFSP award will be an excellent foundation for my long-term objective of establishing myself as an independent computational neuroscientist working at the interface of theory and experiment.

A general concept about pattern formation in early plant embryogenesis is still lacking. The analysis of mutants disrupting embryonic development of the model plant Arabidopsis suggests a role for plant hormones and sugar signals in providing positional information during embryogenesis. However, due to redundancy many mutants defective in potential signaling components show weak phenotypes only. In order to develop a model of how positive and negative regulators of the different signaling pathways cooperate to organize embryogenesis, I plan to manipulate the temporal and spatial patterns of key signaling regulators and see how the phenotype and expression of marker genes are altered. Microarray analysis of wild type embryonic development at a cellular resolution will reveal the active set of potential signaling components and provide new molecular marker genes that will be further analyzed in embryos by whole-mount in situ hybridization or in situ RT-PCR. This data will be used for a functional analysis that includes mutational analysis as well as targeted overexpression of transcribed signaling components. The experiments can be carried out in transgenic embryos as well as in a somatic embryos system that I propose to establish. The high level of plasticity, the genomic and functional redundancy of plants differs from Drosophila and represents an attractive challenge. Like Drosophila, Arabidopsis allows the use of sophisticated genetic and genomic tools. Many basic questions of early pattern formation in plants need to be addressed - I am looking forward to entering the plant field and joining the laboratory of Jen Sheen.

1) For decades it was thought that one of the major differences between bacteria and eukaryotic cells was that bacteria lack an actin cytoskeleton. However, recent studies have identified a homolog of actin in bacteria that forms dynamic helical structures adjacent to the cell membrane and is essential for cell wall synthesis, suggesting that bacteria contain an actin-like cytoskeleton involved in cell morphogenesis similar to that in eukaryotes. Therefore, understanding the mechanisms governing cell wall biosynthesis in simple model organisms such as bacteria will provide a framework to better understand how complex eukaryotes control their cell wall growth, division, and shape. 2) I will begin working on a different bacterial model organism, Bacillus subtilis, where I will be trained in various aspects of its biology and genetics. This is a Gram-positive bacterium, only distantly related to the Gram-negative E. coli with which I am presently most familiar. I will also learn about the microscopical imaging systems used to study sub-cellular organization of living organisms. The Errington lab is extremely well set up to carry out such a research program, with state-of-the-art microscopic imaging facilities equipped with digital CCD cameras and image processing software. 3) My career goal is to obtain an academic position in the United States. Towards this end I will be provided with a range of training opportunities. I will be assigned at least one undergraduate student project to supervise, thereby developing skills including leadership, general mentoring, report writing, etc. I will also have the opportunity to lecture in several undergraduate classes.