1) The goal of my project is to understand the biological function of adult neurogenesis. To this end, I will perform targeted in vivo manipulations and functional analyses of newly generated neurons in the adult mouse brain. Two types of experiments will be conducted. First, in order to map the fate of new neurons, I will create transgenic mice in which a marker gene can be turned on only upon drug induction and only in newborn neurons. Second, I will produce transgenic lines where newly generated neurons can be specifically ablated upon drug induction to achieve targeted inhibition of neurogenesis. Collectively, these experiments should help elucidate the functional significance of adult neurogenesis in such processes as learning and memory, emotional response, and repair of brain injury. 2) The topic of my Ph.D. research was circadian rhythm. The project proposed here entails different biological questions and experimental tools. I therefore see little overlap between my prior work and the current proposal. 3) My career goal is to establish an independent research program to study the basic biology of neural stem cells, and to explore their clinical applications. The proposed project will allow me to understand the behavior of neural stem cells, such as how they proliferate, differentiate and migrate in response to physiological signals, and how newly born neurons integrate and function in the neural network. Additionally, it should give me an appreciation of the clinical potential of stem cells, and familiarize me with the latest technologies in mouse transgenics and stem cell biology. As such, this work should prepare me well for my career objectives.

Every aspect of mature brain function is dependent on a precisely sculptured neuronal network. This complex wiring of the nervous system is controlled by an ordered series of guidance decisions during neural development. The semaphorins compromise a large and conserved family of neuronal guidance cues, many of which can mediate repulsive axon guidance. Binding of semaphorins to receptor complexes on the surface of axonal growth cones activates intracellular signal transduction pathways that ultimately infringe upon the actin cytoskeleton. Although we have learned a great deal about the biology of semaphorins and their receptors, our understanding of the cytosolic signaling cascades that link repulsive semaphorins to the actin cytoskeleton is still limited. The work proposed here involves a detailed characterization of a novel family of intracellular signaling cues, MICALs, recently implicated in semaphorin signaling, as well as the identification of novel signaling proteins critical for transducing semaphorin signals. Together these studies are aimed at better defining the molecular basis of intracellular semaphorin signaling during vertebrate axon guidance. This project will give me the opportunity to study familiar aspects of neurobiology while initiating new research through the anticipated identification of novel signaling proteins. In addition to neural development, my future research will focus on a function for axon guidance cues during adult structural plasticity. Therefore, a better understanding of the molecular mechanisms of axon guidance cue signaling will be critical for achieving several of my research goals.

Dosage compensation is required to equalise the expression of X-linked genes in males and females. In Drosophila melanogaster, this is achieved by a 2-fold transcriptional upregulation of the single male X chromosome in comparison to the two female X chromosomes. The components involved have been identified in genetic screens, these male specific lethal (MSL) proteins, together with 2 non-coding RNAs, roX1 and roX2, form the dosage compensation complex (DCC). However, the mechanism underlying the dynamic regulation of the DCC is far from clear. The aim of my project is to study the molecular dynamics of the DCC on chromatin in live cells. To achieve this goal I will take an interdisciplinary approach, combining cell biology, chemistry, biophysical and biochemical techniques. We plan to fluorescently label all the DCC members using novel chemical techniques, using complex confocal microscopy we will study their dynamics in real time in vivo. These studies will be combined with a well-established RNA interference strategy to study the integrity & stability of the complex in the absence of specific components. During this fellowship experience I will completely change fields, and will now gain experience in the Drosophila system as a model organism. I will learn new skills such as fly genetics, sophisticated confocal microscopy & live cell imaging, RNA interference strategies and novel techniques to label & track RNA molecules in vivo. These new skills will serve to broaden my scientific knowledge but additionally, as I plan to continue studying the dynamic role of RNA components in chromatin templated processes, I believe this is the perfect project to launch such a career.

The molecular circadian oscillator in hepatocytes must control a variety of physiologically relevant liver functions around the clock. At least in part, the circadian liver clock accomplishes this task by controlling the cyclic expression of transcriptional regulatory proteins at different phase angles. The circadian activity of such transcription factors may be regulated at multiple levels. Transcription factors whose expression is regulated at the level of cyclic mRNA accumulation can in principle be identified by functional genomics strategies, such as microarray hybridization or the like. However, all regulatory mechanisms that occur on the protein level, e.g. covalent modification, multimerization, subcellular localization etc., necessitate the analysis of the proteome. Here I propose to develop a novel technique, Differential Display of DNA-binding Proteins (DDDP), that may allow the identification of a large fraction of circadian transcription factors present in the nuclear proteome of rat liver hepatocytes. The mode of regulation and the functional relevance of some of these transcription factors will be studied, in order to shed light on how hepatocytes coordinate circadian metabolism, detoxification and DNA repair. The proposed research differs from my previous work in the experimental methodology as well as the model organism used. However, it will draw on my experience on the field of transcriptional regulation and specifically on the initial steps of promoter activation. At the same time it will introduce me to the field of circadian regulation, whith its outstanding opportunities to study complex behavioral processes on a molecular level.

I intend to characterize the mechanisms of prefrontal control in cognitive behavior and to investigate how the mechanisms contribute to our intelligence. The research plan consists of three steps. First I will establish the method of estimating the causal relationship of activity between the prefrontal cortex and posterior association areas second I will clarify the mechanisms of prefrontal control in complex cognitive behaviors and third I will test the hypothesis that the efficiency of prefrontal control is associated with psychometric intelligence. I will conduct experiments using multiple modalities of functional imaging on normal human subjects. Subjects are to perform visual attention, task switching and working memory paradigms and I will estimate the efficiency of prefrontal control by computing its strength, selectivity, and flexibility. I will also conduct correlation analysis between these biological measures and psychometric intelligence across subjects. This project will form the basis of a long-term interdisciplinary project. Thus the ultimate goal of the project is to set up a common framework within which one can investigate biological mechanisms of cognitive development, test the effects of genetic variation on intelligence, and build up realistic artificial intelligence models. Through such large-scale interdisciplinary approach, I will address the issues on whether there exists single intelligence or multiple intelligences, how genes and environment affect intelligence, what is the intelligence that is unique to humans. The Career Development Award will promote the development of such novel direction of interdisciplinary, international research.

Vascular smooth muscle cell (vaSMC) development, a process termed vascular myogenesis, is an important event during cardiovascular system formation and maturation. VaSMC development is regulated by a complex array of local environmental cues including growth factors, cell-cell and cell-matrix interactions, and inflammatory stimuli. However, the molecular mechanisms by which these diverse signals regulate vaSMC development are largely unknown. The goal of the proposed project is to study the mechanisms governing vaSMC origin, differentiation and plasticity combining cell biology, molecular biology and genetics. To this end I will use the zebrafish as an embryological and genetic model system. First, I will characterise vaSMC patterning during zebrafish development using several vaSMC marker genes. Then, I will generate transgenic zebrafish with fluorescently (GFP) labelled vaSMC to monitor in vivo vaSMC development in wild-type and (cardio)vascular mutants. Using this GFP-vaSMC fish line I will conduct a forward genetic screen in order to identify genes involved in vaSMC development. Moreover, to elucidate the interplay between vascular smooth muscle and endothelial cells, vaSMC mutants will be examined for vascular impairment both at the level of circulating blood cells and endothelial cells. This experience will be very different from my previous studies and will be essential for my professional growth focused on developmental biology and zebrafish. This period will help me to establish my own developmental biology laboratory working with zebrafish when I come back to Italy.

The proposed project will explore function/form relationships during cardiac development and will more generally illuminate the contribution of epigenetic factors such as flow-induced shear stress to gene regulation and tissue remodelling in organogenesis. The proposed research significantly differs from my previous studies and, importantly, will extend my scientific experience to medically relevant issues. Having worked mainly on transcriptional regulation via chromatin modification in the simple organism, yeast, the proposed research will represent a broadening change of field and experimental model. The aims will be achieved using state-of-the-art methodologies: I will gain practical experience in mouse embryological methods, gene targeting, microarray studies and novel imaging techniques by working with experts in each of the aforementioned fields. However, the project will also mesh synergistically with my expertise and perspective in transcriptional regulation. The chosen host laboratory has an international track record in cardiac developmental biology as well as international and interdisciplinary collaborations. The Victor Chang Cardiac Research Institute and its adjacent St.Vincent’s Hospital also have a strong international profile. I am convinced that the host laboratory and its immediate precinct will provide both a stimulating medical research environment and scope for my professional growth. It will also create for me new career options in the future, not only in molecular and developmental biology, but also in an area of research with clear clinical relevance.

Mitochondria integrate several cellular processes, from Ca2+ signalling to apoptosis. The structural organization of mitochondria influences their participation in such aspects of cellular physiology. For example, during apoptosis mitochondria undergo a profound remodelling of their internal structure and they often fragment in order to release cytochrome c into the cytosol, where it grants competent activation of caspases. Mitochondrial shape during life and death of the cell is the result of the equilibrium between fusion and fission processes, controlled by a growing family of proteins including mitochondrial dynamin-related proteins such as OPA1, affected in the human disease dominant OPtic Atrophy. Little is known about the molecular details of these processes and about the physiological consequences of changes in the fusion-fission equilibrium. The objective of this project is to understand how dynamin related proteins regulate mitochondrial fusion and fission and if they can influence cellular and mitochondrial physiology. We will study (i) if and how upstream second messengers influence mitochondrial fusion-fission (ii) how fusion of the four membranes of two fusing mitochondria is accomplished (iii) how mitochondrial physiology and integrated cellular signalling (such as Ca2+ signalling and apoptosis) are affected when the fusion-fission equilibrium is altered. This project will contribute to establish myself as an independent investigator in the novel, unexplored field of the control and consequences of mitochondrial fusion-fission, allowing me to transition from the field of apoptosis, the focus of my postdoctoral experience.

Whereas Structural Biology is typically confined to an in-vitro experimental setup and samples are studied in an artificial and isolated environment, my emphasis is to develop a structural method to study biological processes in-vivo and inside a cell. I propose to establish in-cell NMR spectroscopy in the Xenopus laevis oocyte. The general feasibility of in-vivo spectral studies in this eukaryotic cell and in its different intracellular environments will be determined. Conformational differences between cytoplasmic and nuclear localization of a model protein domain will be analyzed and the suitability of this system for the study of larger protein assemblies evaluated. This project differs from my previous research experience as it aims to apply NMR to study structural biology inside a cellular, in-vivo system. It describes a novel implementation of NMR and, for the first time, introduces this physical technique to the eukaryotic, living cell. This approach will provide a new tool for studying biological processes inside a cell and, if successful, will be of substantial interest to the general scientific community. The novelty of this application requires expert advice and sophisticated infrastructure both in the fields of Xenopus oocyte biology and NMR technology. This project will be carried out in close collaboration with Harvard research groups of Profs. Reed, Ruderman and Kirschner. The excellent NMR facilities and profound expertise of Prof. Wagner will ideally complement this effort. Together these groups provide a unique scientific environment to successfully pursue the outlined project and the HSFP fellowship will greatly support my effort to achieve this goal.

Abstract of research plan TGF-b and activin regulate tissue homeostasis by activating the Smad2/3 intracellular signaling pathway leading to potent inhibition of proliferation of multiple cell types including epithelial cells and disruption of this signaling pathway is associated with oncogenesis and tumorigenesis. Cripto is a developmental oncoprotein that is highly expressed in human tumors but not their normal tissue counterparts. Overexpression of Cripto transforms mammary epithelial cells in vitro. It was previously shown that Cripto can antagonize activin signaling and it is important to detect if Cripto can similarly antagonize TGF-b signaling. I therefore propose that Cripto may be generally capable of blocking antiproliferateve Smad2/3 signals providing a novel mechanism of oncogenic action with multiple therapeutic implications. This proposed project focuses on characterizing the molecular mechanisms by which Cripto antagonizes the activin and possibly TGF-b signaling pathways in in-vitro models of breast cancer. The proposed project is clearly different from my previous background dealing with a family of protein kinases that are taking part in apoptosis. The hole field of TGF-beta signaling and its involvement in growth inhibition is interlay new to me but is well established and should expose me to various techniques of hormonal regulation. Oncogenesis induced by Cripto and its potential mediation via inhibition of TGF-beta signaling is a totally new concept. It may provide a handle to study the initial steps of Tumorigenesis, and in this way extend my knowledge form the apoptotic contribution to cancer to the more causal aspects of this phenomena.

It is becoming increasingly clear that many kinetochore proteins localize to nuclear pore complexes (NPCs) before mitosis. Vice versa, after nuclear envelope breakdown, several nucleoporins (Nups) localize at kinetochores from late prophase to anaphase. However the function of this connection remains unclear. The most trivial explanation is that NPCs and kinetochores respectively behave as docking sites for checkpoint proteins before mitosis, and Nups before nuclear envelope reformation. However, a more likely hypothesis is that higher eukaryotes have evolved a functional link between cytoplasmic microtubules, the NPC and nuclear kinetochores. The goal of my research project will be to clarify this link. To achieve this I will (I) carry out an RNAi screen of the various Nups to identify mitotic phenotypes by automated microscopy (II) search for the mitotic binding partners of the positive scoring Nups by iTAP purification approaches (III) characterize selected Nups and their kinetochore interactors during the G2/M transition by 4D live cell imaging (IV) measure in vivo interactions between these Nups and their kinetochore partners by photobleaching-photoactivation techniques and fluorescence resonance energy transfer (FRET). This project, therefore, will give me the chance to continue working in mitosis but from a completely different point of view, learning new techniques and novel aspects of cell biology that I am nor familiar with. I believe that Dr Ellenberg’s laboratory provides the best environment for the success of the project and for my personal growth as it offers all the necessary equipment but also a group of qualified expertise.

We propose to develop trophoblast stem cells (TSC) as an efficient in vitro model allowing to decipher the role of genomic imprinting in trophoblast biology. We plan to use this model (1) to better characterize imprinting in trophoblast (2) to decipher the biological function of all the known imprinted genes in trophoblast (3) to discover new gene products implicated in trophoblast genomic imprinting regulation (4) to better characterize the biology of TSC. In order to realize this: (a) we will have to adapt to TSC many different techniques of molecular and cell biology which are established in the Reik lab: e.g.we will have to set up RNAi technology in TSC, which has not been described to date (b) we will have to molecularly manipulate expression of all imprinted genes in TSC (c) we will set up a new screening technique for the imprinting status. This project is important for my training because (1) I will learn and practice many techniques with which I am not familiar: RNAi, transgenesis, FISH, cell biology, fluorescent proteins, blastocyst manipulation,... (2) I will discover mouse development, genetics and genomics fields. At long-term, I want to become a full-time researcher: I hope to divide my research activity in three themes: (1) molecular biology of the placenta and early embryo development (2) trophoblast and early embryo epigenetics (3) finding gene mutations in patients to explain obstetrical and gynecological pathologies. My feeling and hope is that to go and work in the laboratory of W. Reik at the Babraham Institute of Cambridge UK, will allow me to progress in the most productive way towards these aims.

Bacterial pathogens have evolved elaborate mechanisms to subvert host defense systems. The induction of apoptosis in host cells is a pathogenic strategy employed by several different bacteria, including Salmonella and Shigella. Both Salmonella and Shigella induce macrophage apoptosis through the activation of Caspase 1 (Casp-1). Caspases are a family of cysteine proteases involved in both apoptosis and the generation of a pro-inflammatory cytokine response. Casp-1 was originally identified as interleukin (IL)-1beta converting enzyme (ICE), and not thought to participate in apoptosis. The activation of Casp-1 by Salmonella and Shigella therefore induces both apoptosis and a pro-inflammatory response. Unlike macrophages, epithelial cells are resistant to S. flexneri-induced death. S. flexneri invades epithelial cells and replicates to high numbers. Despite the presence of Casp-1, however, this cell type is refractory to Shigella-induced apoptosis. It is not known why Casp-1 activation in macrophages culminates in cell death, or why epithelial cells are resistant to S. flexneri-induced death. Here, I propose to use two independent genetic approaches to identify the host cell factors necessary for Casp-1-mediated apoptosis. The majority of my research to date has been directed at understanding the molecular basis of virulence from the bacterial standpoint, focusing on individual virulence factors and their regulation. My postdoctoral training will be geared at understanding the host response to infection.

Protein ubiquitination plays an important role in various cellular processes. Protein modification by the attachment of ubiquitin regulates these processes by selectively targeting proteins for degradation by the proteasome. Ubiquitination also implicated in protein activation, endocytosis, and histone regulation. The ubiquitination pathway involves three key enzymes which attaches the small ubiquitin molecule to proteins. Of interest is the E3 ubiquitin ligase, which facilitates the transfer of ubiquitin to the substrate. The large number and different types of E3 lends to the specificity of this pathway. The purpose of this research is to characterize and analyze a sub-set Arabidopsis RING type E3 ligases so as to gain insight into how this modification is regulated and how it influence cellular responses. Very few Arabidopsis RING proteins have been studied therefore analysis to demonstrate activity and function of these proteins is of importance. My previous research focused on receptor kinase signaling by studying self-incompatibility in Brassica. In a broader sense, this work contributed to the understanding of flowering plant reproduction. The proposed research has broader implications in that it involves the functional characterization of a family of Arabidopsis proteins. This new area of research will serve to introduce me to the tools commonly used by Arabidopsis researchers but are not available to researchers utilizing other species such as Brassica. The RING E3s are predicted to be involved in many aspects of plant development, therefore this research will allow me to work on other areas of plant biology. The possibilities are endless.