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.

It is becoming increasingly clear that the information encoded in the linear DNA sequence is insufficient to fully account for the complex regulation of genes and genomes. Higher-order chromatin structure and epigenetic regulation via chromatin modifications are now recognized to be important mechanisms of gene regulation. An additional emerging mechanism of genome regulation is the control by higher-order organization of chromatin and chromosomes. The aim of this project is to reveal how higher-order structure of chromatin and chromosomes changes during neural cell differentiation and how nuclear architecture affects expression of gene programs. I will focus on uncovering how extra-cellular cues affect gene expression programs and differentiation in the nervous system. Specifically, I will investigate the spatial positioning of chromosomes and cell-type specifically expressed genes, and analyze kinetics of proteins and gene loci in neural stem cells and during their differentiation into neurons and astrocytes. This project takes advantage of my expertise in neural differentiation and will enable me to acquire training in new methods and a novel field of research. At the end of the fellowship, I will have technical skills in advanced imaging and expert knowledge in the area of spatial genome organization, and this will allow me to explore further the complex mechanisms of cellular differentiation in the nervous system. I believe that postdoctoral training in the field of nuclear architecture in Dr. Misteli’s laboratory will ideally complement my expertise in molecular biology and will prepare me for a career as an independent scientist.

Small non-coding RNAs play an unexpectedly large role in regulating gene expression. small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate both specific gene expression and more globally regulate chromatin structure and genomic stability. Both siRNAs and miRNAs act via the RNA interference (RNAi) pathway. At the heart of RNAi is a protein-small RNA complex, the RNA-induced silencing complex (RISC) that catalyzes target RNA destruction. Several proteins in the RISC have been identified, and many of these are required for metazoan development. My research seeks to dissect the pathway of RISC assembly and the mechanism of RISC action. I am especially keen on understanding what differentiates the RISC function in target cleavage (RNAi) from its role in translational repression via miRNAs. My previous experience dissecting the mechanism of tRNA-maturation will serve me well in this pursuit, but my study of the RNAi pathway will also give me new experience with Drosophila developmental biology and genetics. Thus, a HFSP fellowship would help me to grow beyond my biochemical expertise and will promote my professional growth through interdisciplinary research.

In this proposal, I will describe a multi-faceted approach towards establishing how functional connections in the brain are dynamically altered to link informative sensory signals with preferred behavioral outcomes. Specifically, how are the same sensory signals transformed into different types of movement signals, depending on what a subject is doing? I will ask this question in the context of a task-switching paradigm. Traditionally, studies on task switching has focused on the prefrontal cortex. This is due to the hypothesis that the prefrontal cortex operates as an “executive control” center, that modulates the “switch” between sensory information and motor commands. It is not clear, however, where and how the “switch” occurs. Here, I will train monkeys to “switch” between discriminating the direction or the depth of a single patch of dots (both discriminations are known to involve area MT) on a trial-to-trial basis, by cueing the monkey on each trial, and ask if the functional connection between MT neurons and eye movement neurons dynamically change depending on which attribute the monkey is attending to. If MT neurons switch their functional connections depending on which attribute the monkey is attending to, I expect trial-to-trial fluctuations in MT responses to correlate with different choices, depending on which attribute the monkey is attending. Moreover, I propose to see if I can evoke different eye movements using electrical microstimulation, depending on which attribute the monkey is attending to. I hope to find neural circuits that dynamically change their functional connectivity depending on what the monkey is doing.

The packaging of eukaryotic genomes into higher order chomatin hampers the accessibility of the genome for enzymes involved in processes such as DNA replication, DNA repair and transcription. Chromatin remodelling complexes help these enzymes to overcome this barrier. One class of such complexes consists of ATP-dependent machines, which contain an ATPase subunit from the SNF2/SWI2 superfamily. Recently, a new member of this class, the Ino80 ATPase, has been identified in budding yeast. It is part of the INO80 complex, which consists of 12 polypeptides. The purified complex remodels chromatin and displays 3’-5’ helicase activity. Three of the subunits, Arp4, Arp5 and Arp8, are actin related proteins and associate with histones. Mutants of Ino80, Arp4, Arp5 and Arp8 are sensitive towards DNA damaging agents and show defects in transcription, which suggests that chromatin remodelling driven by the INO80 complex may be linked to both transcription and DNA damage repair. I propose to clarify the function of the INO80 complex in both DNA repair and the response to DNA damage. Chromatin-immunoprecipitation, high resolution immunofluoresence and real-time live-imaging microscopy will be primarily used to see if INO80 is recruited to double-strand breaks (DSB) and affects histone H2A phosphorylation and/or degradation of the DNA ends at a DSB. A whole genome transcriptional microarray analysis will be performed to investigate if INO80 affects DNA damage repair by inducing expression of DNA repair genes. The proposed research allows me to change my topic of research and to acquire new skills in microscopy and cellular biology for the study of DNA damage repair in yeast.

Cell motility is crucial for many physiological and pathological processes. The objective of this project is to gain a better understanding of cell motility. An important role is played by actin. The polymerization of actin into filaments at the plasma membrane generates a force that induces cell movement. Another protein involved in cell motility is myosin. Myosins are important for controlling the actin network. Their precise role and the cooperation with actin in producing cell movement are still poorly understood, however. The approach that we suggest here is to realize a biomimetic system that reproduces the dynamical shape changes of suspended cells where microtubules have been depolymerized. These shape changes are regulated by the same mechanisms as cell movement. We will replace the cell membrane with a well-defined lipid bilayer. A liposome in suspension will be filled with cell extracts, and actin polymerization will be catalyzed at the liposome membrane. Deformations of the liposomes can then be followed and the effect of specific proteins can be investigated. The work will be compared to a biological system studied in the lab, consisting of detached motile cells that show shape oscillations, and to theory developed in parallel. My previous research experience is in the field of physical chemistry and polymer physics. In my future professional career, I hope to use my physical background to work on biological problems. This fellowship will help me to receive the training in biological sciences needed for this.

The aim of this project is to address a serious unknown in limb patterning, concerning the lineages and motions of cells that form the limb. The project draws on Fraser lab's imaging tools and in ovo techniques to assess directly some key events in limb formation in the chick. My training in the IGBMC, Strasbourg, France, was mainly focused on molecular biology, cell biology, and the study of murine embryology. All these expertises will help to pursue this project but are very far from what I'll do in Scott Fraser's lab, where I'll learn novel techniques of in vivo imaging and DNA micromanipulation. This research project has a high training content and will be very beneficial for the successful continuation of my career. The experience I am going to obtain in in vivo imaging techniques would be a valuable asset for pursuing my scientific interests in the future, after returning to France. I have chosen Caltech (Pasadena, United States) for my post-doctoral training because it is one of the best research centers in the US and because the combination of molecular biology, biochemistry, biophysics and in vivo imaging expertises available are crucial for the successful pursuit of the studies proposed here. Furthermore, the laboratory of Pr Fraser is one of the best in the field of imaging and this, in conjunction with the interdisciplinary character of the project and the innovative and interactive environment both of the laboratory and the institute as a whole, will, with no doubt, broaden my scientific horizons and help me in developing a more mature and critical attitude towards scientific research.