1) The proposed project will elucidate the biochemical nature of meiotic DSB formation. I will use isolated meiotic nuclei to develop a novel in vitro system competent for DSB formation. I will biochemically fractionate the nuclei and use the DSB assay to identify protein activities essential for the reaction. I will use small molecule inhibitors and purified recombinant protein to investigate the role of chromatin structure on DSB formation. 2) My previous research experience was centred firmly on the molecular characterisation of DSB repair in mutant yeast strains. The project was successful, but I soon became aware that I lacked sufficient biochemical knowledge and training to ask more complex questions about the processes under study. The proposed fellowship project will provide me with essential training in protein biochemistry that I feel is indispensable for my ability to achieve my long-term research goal: to unify protein function with the molecular structure of DNA intermediates that form during repair and recombination. 3) As a research fellow I will have the freedom to think independently about the research project, whilst retaining advisory contact and supervision as required. I will present my research observations to peers within the institute, and at international conferences, and use such events as opportunities to broaden my scientific knowledge and to forge new links with other motivated researchers. I will have adequate opportunity to write research papers, gain experience of grant writing, and to provide supervision to graduate students. These factors will provide me with attributes essential to my professional development as an academic researcher.

Reverse gyrase is a unique, ATP-dependent, Type I topoisomerase that positively supercoils DNA. Supercoiling is achieved through the concerted activity of a topoisomerase domain and a helicase domain on a single polypeptide, though the mechanism remains unknown. An association between a helicase and a topoisomerase has been documented in eukaryotic systems where it is thought to play a role in genomic stability. Reverse gyrase represents a model system in which to study the function and regulation of a combined helicase and topoisomerase. The proposed experiments examine the mechanism of supercoiling by direct, real-time measurements of topological changes induced in a single DNA molecule by reverse gyrase. Initial experiments will focus on the mechanism of ATP-driven unidirectional winding of DNA. Subsequent experiments will examine the coupling between the two domains through the study of mutants. Further experiments will test whether reverse gyrase can resolve non-native DNA structures, which has been postulated to be among its in vivo functions. This proposal affords me the opportunity to gain expertise in single-molecule magnetic manipulation, particularly as applied to DNA topology, while being able to concentrate on the biologically relevant questions that can be addressed with this technique, rather than the technique itself. Furthermore, I will expand my molecular biology skills and knowledge through a close collaboration with Michel Duguet, a recognized expert in topoisomerase molecular biology and enzymology. Expanding my background in biology is an important step in my development as an independent investigator working at the interface of biology and physics.

Language-based numerical competence does not emerge de novo in evolution but arises from biological predispositions. This hypothesis is supported by studies on pre-linguistic representations of numerosity in human infants and non-verbal numerical competence in animals. The aim of the project is to investigate the neural formation of numerical categories and concepts in behaving macaque monkeys in combined psychophysical/neurophysiological studies. Non-verbal representation of numerosity in monkeys is of special interest because of the relative similarity between the human and monkey brain. The target brain areas will constitute the prefrontal (PFC) and the posterior parietal cortices (PPC), which play dominant roles in coding of numerical information. The results of this study should be suited to develop a more detailed understanding of the neural mechanisms of counting. Results obtained in non-human primates might help to understand neurological disorders in human patients that involve problems with numerical processing. On the long run, it may help to elucidate the neural basis of other cognitive functions closely related to numerical competence, like mathematics, logic or language.

Legionella pneumophila is an intracellular bacterium that can infect and replicate within macrophages of a human host. To establish this infection, it uses a specialized secretion system, belonging to the type IV family of transporters, to inject protein substrates directly into the host cell cytoplasm. These proteins are believed to be modulating vesicle traffic in the host cell to help generate a vacuole that supports bacterial replication. Despite their importance, only three of the proteins secreted by the L. pneumophila type IV transporter have been recognized. The goal of this work is to identify novel substrates of this transporter, and characterize their role in L. pneumophila pathogenesis. Two independent approaches are proposed. The first one is an informatics-based screen for bacterial effectors, based on homology to eukaryotic domains, and the second approach describes a biochemical in-vitro screen utilizing host plasma-membranes to simulate host cell interaction. Together these approaches will enhance our understanding of the processes that govern L. pneumophila pathogenesis and the fundamental cell functions it subverts. The research proposed here differs from my previous research experience in the field of research, the research methodology and the level of complexity of the system investigated. I am shifting from the field of membrane transport physiology, to cell-biology of eukaryotes, and from in-vitro single protein analysis to the study of whole cell, multi-protein interactions. In the future I intend to combine the analytic viewpoints of cell-biology and membrane biochemistry in order to develop new ways of attacking unsolved biological questions.

Sister chromatid cohesion is the process whereby the two chromatids produced by DNA replication are held together until the mitotic cell division event. This process is mediated by the multi-subunit protein complex called cohesin. Recent biochemical analyses suggest that cohesin's subunits form a ring structure. The ring hypothesis proposes that sister DNA molecules are embraced within the cohesin ring. According to the hypothesis, the cohesin ring must open to allow chromosomal DNA to enter the ring. After cohesion has been established during S-phase, the ring needs to be 'locked' until anaphase to hold sister chromatids together. The 'locked' ring can only be opened by proteolytic cleavage of cohesin's Scc1 subunit. In comparison to cohesion disruption, little is known about how the cohesin subunits assemble during S-phase when cohesion is established. Furthermore, there have been no studies addressing the loading of cohesin onto DNA or chromosomes in vitro. My research activity will be to characterize the cohesion establishment process in vivo and in vitro. First, assembly or the exchange of the cohesin subunits will be studied in vivo. In parallel, purified cohesin subunits will be prepared to determine the molecular basis of assembly coupled with crystal structure determination. Consequently, in vitro loading of cohesins onto DNA and establishment will be examined. These studies are different from the previous ones that I am aiming to study cohesion at molecular and cellular level. Nonetheless, my training of structural biology will help me greatly to carry out this project. Thus, HFSP award should provide me the firm foundations for my professional goal.

My aim is to analyze the robustness of developmental precision and self/non-self recognition in biological systems. I will use Dictyostelium as a model system owing to its genetic tractability, its multi-cellular development and the role of self/non-self recognition in several aspects of its existence in nature. Dictyostelium cells will be individually manipulated in a controlled environment in microfluidics devices and observed by video-microscopy as they develop into a multi-cellular organism or perform self/non-self recognition. Through modeling and theoretical approaches, I will attempt to characterize the stability of and the evolution towards developmental precision and self/non-self recognition in the presence of external and internal perturbations. After studying molecular mechanisms of intracellular transport, setting up antibody phage display and live cell fluorescence microscopy techniques during my PhD, I have decided to approach general problems in biology using the powerful tools of physics and engineering sciences. The problems I intend to address are therefore clearly distinct from my PhD dealing with the intracellular dynamics of Golgi membranes. The model system (Dictyostelium) and some techniques such as microfluidics I have chosen are also entirely new for me. This fellowship experience will enable me to combine the tools that I acquired during my undergraduate education in physics and engineering sciences with the experience of my PhD in cell biology. Indeed, I had decided to learn cell biology concepts and techniques during my PhD to be able to reach my initial goal: to study relevant problems that are central in biology with a physicist’s perspective.

P. falciparum malaria severely affects pregnant women and children in Africa. Despite immunity through lifelong exposure to malaria, pregnant women become susceptible to infections causing anemia, abortions and low birth weight. The placental vessels become filled with infected erythrocytes (IEs). Adhesion of IEs to endothelial cells is mediated by members of a large diverse protein family called P. falciparum erythrocyte membrane protein1 (PfEMP1). Pregnancy malaria is associated with the emergence of a distinct subset of parasite variants that bind the host-receptor chondroitin sulfate A (CSA). Resistance to pregnancy malaria is associated with the acquisition of antibodies that block parasite binding to CSA. I seek to understand why women who are otherwise clinically immune develop symptomatic infections during their first pregnancy. The lack of antibodies to the surface of placental isolates without pregnancy suggests that these parasites become virulent only in the presence of the placenta. The aim of my project is to investigate the mechanisms that induce or modulate the expression of placental PfEMP1 molecules. The proposed project will completely shift my field of research from stem cell biology to molecular and cellular biology of P. falciparum. The host lab has a longstanding interest in cytoadhesion and antigenic variation. I am confident this will provide a very rich and dynamic environment where I will be able to work on a significant human pathogen, learning state-of–the-art techniques. This will enable me to analyze complex cellular host-parasite interactions with the aim of developing preventive therapies against malaria, which is my long-term career goal.

Fat storage disorders are main risk factors in many serious illnesses such as diabetes, heart disease and cancer. In mammals and Caenorhabditis elegans a direct link between feeding rate and body weight has been observed. A previous study from our laboratory unveiled a plethora of genes that regulate fat storage in C.elegans. Some of these genes and some undetected genes must be feeding regulators. To distinguish basic fat metabolism from intake regulator genes and to find new feeding regulator genes I will perfom two genetic screens based on measuring feeding rates. I will measure feeding rates of the previously reported mutants with fat storage defects and in parallel I will perform a genome-wide screening in a C.elegans mutant background hypersensitive to gene inactivation. I will measure feeding rates by quantifying the rate of consumption of fluorescent food. The characterization of the genes affecting intake behavior will include: analysis of their sites of action with GFP fusions, complementation of the deficiencies by feeding neurotransmitters or hormons, and the analysis of their genetic interactions with already known neuroendocrine pathway components. The goal is to identify and characterize conserved neuroendocrine signals and receptors that regulate food sensation and consequently body weight. The HFSP will help me to train in a complete new field, neurobiology. My graduate work was focused in the study of a bacterial pathogen. Even in an animal as simple as C.elegans, more complex genetic and physiological interactions will pose difficulties that I expect to overcome while acquiring the necessary skills to become an independent researcher.

1) My proposed project focuses on one of the GTPases of the Ras superfamily that are involved in membrane traffic. In particular on the human Arf-like GTPase, Arl8, a member of the Arf family whose other members recruit vesicle coats and other effectors to the endoplasmic reticulum and Golgi apparatus. However, Arl8 is unexpectedly located to lysosomes and late endosomes. The existence of such an Arf family GTPase in the endocytic pathway was not anticipated, but its importance is suggested by it being conserved from plants to humans. This project is intended to examine the function of Arl8, and to identify the effectors that it recruits to membranes. 2) My previous work was in the elucidation of molecular mechanisms of morphogenesis in plants. For my postdoctoral training I will change my research field to yeast and mammalian cells. Before, I focused on transcription factors and identifying their target genes, I will change my focus onto the membrane traffic system using biochemical and cell biological approaches. 3) I will move my research field from the multicellular plant to the unicellular yeast and cultured human cells. This shift will give me a new and different point of view to see eukaryotic organisms. The cell biological techniques and the methods of analyzing protein-protein interactions used in the proposed institute are now indispensable for understanding biological phenomena, and the scientific activity in the host laboratory is very high. Acquiring these techniques and working in such a favorable circumstance will develop my ability for my professional career and give me a great opportunity to discuss with people at a high level.

Long-lasting forms of synaptic plasticity, which underlie essential aspects of cognitive functions in the brain, require gene transcription and translation. However, little is known about how the gene activation in the nucleus contributes to the activity-dependent, synapse-specific modifications at dendrites. An immediate-early gene, Arc, is rapidly induced in response to synaptic stimulation, and its mRNA is sorted into the activated dendritic regions. Local translation of Arc mRNA has also been reported. As Arc protein is present in the postsynaptic density and can bind several synaptic proteins, Arc may link nuclear events with the activated synapses. In order to test this, I will examine the dynamics of activity-dependent Arc expression and translocation from the nucleus to dendrites. In slice culture preparation, I will first establish a method to manipulate nuclear gene induction by local synaptic activation. I will then develop strategies to specifically tag Arc translation in either the cell body or dendrites. Finally, I will examine whether Arc induced by synaptic activation is actually targeted back to the activated synapses. The long-term goal of my research is to understand the molecular mechanisms conferring synapse specificity to protein synthesis-dependent synaptic plasticity, and to understand how such plasticity contributes to reorganization of local neuronal circuits. With support of the CDA, I will direct my research to optical molecular biology/physiology. The techniques developed in the proposed projects will be essential tools in my future studies, in which I hope to investigate the role of activity-regulated genes in cortical reorganization in vivo.

Neuroimaging techniques have revealed several regions in the human and macaque brain that are selectively activated by object shape. The major aim of this research plan is to focus on visual experience as a possibly very important determinant of functional organization in these shape-selective regions. First, we will investigate some general characteristics of the activation pattern that is associated with a range of novel and parametrically controlled object classes prior to any training. Afterwards, we will train humans and macaque monkeys for a relatively long period of time in the discrimination of exemplars of a specific object class. We will compare the activation pattern after training with the data obtained before training for trained and non-trained object classes. The results will allow us to target brain sub-regions with specific training effects by techniques such as single-cell recordings and micro-stimulation. My previous behavioral and single-cell work has confronted me with the limitations of techniques such as single-cell recordings in case one is interested in understanding the determinants of the organization of the human brain and the way in which it affects behavior. However, fMRI is not an ideal tool either. Thus, I believe that a combination of behavioral work, human fMRI, monkey fMRI, and single-cell recordings would be more than just ‘the sum of its parts’. I want to be able to perform each of these techniques and their combination. I expect to apply this powerful combination in my future research to other manifestations of human flexibility, such as perceptual learning, rule learning and the first steps towards intelligent behavior.

Myc is an essential transcription factor that is profoundly involved in the genesis of cancers. In order to understand how Myc functions we need to define: (i) developmental signals that regulate Myc protein levels (ii) the direct target genes whose expression Myc modulates, and (iii) the biological consequences of target gene expression. The recent identification of the Myc ortholog (dMyc) in Drosophila provides a powerful genetic system for analyzing Myc biology. My initial characterization of dMyc mutants suggests that they are female sterile and these phenotypes are due to a failure in stem cell proliferation. I am interested in elucidating the role of dMyc in the early Drosophila development in particular within the stem cell niche. I will investigate a role for Myc as an integrator of distinct and critical signaling pathways regulating early stem cell proliferation and fate. Earlier I used the Drosophila system to identify global dMyc binding sites. A subset of novel dMyc targets and cis-acting factors are potentially involved in regulating the stem cell niche will be studied in detail. Finally, my recent discovery that Myc directly regulates the expression of hundreds of genes motivated me to search for conserved negative regulators of Myc. To date I have identified the Archipalago (Ago) gene product and its mammalian ortholoug FW7 as such candidates. I will systematically identify other negative regulators utilizing a genetic screen. Thus, I propose to derive a more encompassing view of Myc function by utilizing an integrated approach combining Drosophila molecular genetics, functional genomics, and Drosophila/mammlian tissue culture systems.

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.