Chromosome condensation is an essential process that ensures proper segregation of the genome during mitosis and meiosis. In vertebrate cells, two different condensin complexes, known as condensin I and condensin II, play central roles in this process. It remains unknown exactly how the two complexes are regulated and how their functions are coordinated during the cell cycle. In this proposal, I will take biochemical and cell biological approaches to this problem by using Xenopus egg cell-free extracts and human tissue culture cells. First, I will determine the distribution and dynamics of condensins I and II from prophase through metaphase. Their relative distributions on the metaphase chromatid axis will be assessed at high resolution by deconvolution and electron microscopy. Second, I will investigate the mechanism by which the recruitment and activity of condensins I and II are regulated by mitotic kinases. The hypothesis will be tested that different cyclin-Cdk complexes sequentially phosphorylate the two condensin complexes and thereby orchestrate their actions. Third and finally, I will test the idea that condensins may have a critical role in the functional organization of the interphase nucleus, with a major focus on its potential involvement in checkpoint activation and DNA repair. During my thesis work, my primary approach was genetics in fission yeast. The current application proposes to take biochemical and cell biological approaches in vertebrate cells. I believe that these new experiences will be important not only to deepen my understanding of chromosome function but also to broaden my scientific and technical scopes.

Mitosis requires profound cellular rearrangements and movements, leading to the physical separation of two identical sets of chromosomes into two newborn cells. Mitotic activities are orchestrated by crucial kinases conserved in all eukaryotes. These include kinases of the cyclin-dependent, Polo-like and Aurora groups. Each of these enzymes follows a specific pattern of localization during mitosis, suggestive of different targets and substrates at multiple locations. Understanding the specific functions of such protein kinases in mitosis requires the knowledge of their binding partners and substrates. The focus of my PhD was to identify and elucidate the functions of interactions involving cyclin-Cdks in yeast starting with a proteomic approach. It is a goal of my career to apply similar methods to model metazoans with more complex proteomes, so we might begin to understand how protein kinases coordinate the events cell division in organisms more similar to humans than yeasts. Here, I propose a strategy for elucidating the molecular functions of key mitotic kinases in the fly Drosophila melanogaster, with an initial focus on Polo. The structural domains of Polo, as well as biochemical, functional and localization data suggest that it interacts with multiple proteins to direct mitosis. I will use cultured Drosophila cells and embryos, from which to purify protein complexes using affinity-tagged kinases. I will identify co-purifying proteins using mass spectrometry. The functions of novel interactions will be investigated using an array of in vitro assays, microscopic imaging and genetic manipulations that include RNAi in cultured cells and in the whole organism.

Genetic robustness is the capacity of phenotypes to minimize the effects of mutations. Robustness at the level of the nucleotide sequence has not been studied. We suggest that the very sequence of a gene may evolve to produce robustness at the phenotypic level. Moreover we conjecture that in certain cases a hypersensitivity to mutations (anti-robustness) will evolve. We will develop a method to measure genetic robustness from DNA sequences and we will study the causes of genetic robustness, to understand under which conditions and for which genes it evolves. We will also study the effects of robustness on development and on cell populations undergoing somatic mutations to unravel its importance for phenotypic variation and for genetic diseases. We will use a combination of mathematical methods and computer simulations and integrate them with empirical studies of genomic data and of genes involved in development, in particular homeobox genes. My background is in theoretical evolutionary ecology: I have studied insect-plant coevolution and the evolution of life cycles. More recently I became interested in the evolution of the genetic code and codon usage. In the future I would like to devote myself more to the molecular aspects of genetics and this project will help me to pursue this new research field. I view training in this project as essential to my goal of integrating evolutionary theory with the study of molecular and development genetics. The Grafen and Holland labs in Oxford have an excellent expertise in all aspects of the proposed project, and thus offer an ideal environment for this research.

Independent research on DNA repair in mammalian cells and on photomorphogenesis in plants led to the discovery of common factors, the DNA Damage Binding complex, which is likely to act in a similar way in these two different processes by modulating access to chromatin-packed DNA. The DDB complex is not an integral component of the nucleotide excision repair (NER) machinery but becomes tightly bound to chromatin upon UV irradiation and associates with chromatin remodelling and ubiquitination activities. Thus, it could rather participate in early steps, helping the DNA repair machinery to access the DNA lesions. Considering its binding to nonacetylated histone H2B tails, a similar biochemical function of the plant DDB1/DET1 complex has been proposed by the host group, in order to regulate transcription of light-responsive genes at a global level during photomorphogenesis. We propose to purify the Arabidopsis DDB1/DET1 complex and to characterize its role in chromatin remodelling at loci of UV-damaged DNA and developmental genes in the broad context of plant photomorphogenesis. The complex will be immobilized on sepharose and binding assays will be performed with mononucleosomes purifed from dark/light-grown and UV-irradiated A. thaliana seedlings. Its function will also be assessed using a recently established plant in vitro DNA repair system, and its genomic targets will be investigated by chromatin immunoprecipitation. This project contributes to the current strong interest of plant scientists in studying epigenetic control of gene expression, and would allow me to investigate, on a longer term, a challenging and fascinating new field of gene regulatory mechanisms.

Many surveillance mechanisms operate in eukaryotic cells to ensure the stability of the genome. Genomic instability occurs in normal cells at a low rate, but is enhanced in cancer cells. Colorectal cancer is the fourth most common cancer in men. 85% of colorectal cancers are associated with chromosomal instability (CIN), a condition in which cells gain or lose whole chromosomes. The molecular basis for CIN has not been defined, and still remained mysterious. The basic mechanisms and pathways involved in faithful chromosome transmission are conserved throughout eukaryotic evolution. The molecular basis of CIN is likely to be determined by studying the mechanisms of chromosome transmission in model organism such as the baker yeast. The technical accessibility of yeast strongly recommends this organism for the study of fundamental biological problems. The specific aims of this proposal are to comprehensively define the entire set of genes that are mutable to a CIN phenotype. The data obtained from yeast will be used to test directly whether the loss of function of the new CIN candidate genes causes cancer in human cells. This will result in the establishment of genetic interaction maps for CIN genes mutated in human cancer. In my PhD thesis I investigated the mechanisms that keep genome stability in yeast. My studies provided me with a strong basis in yeast genetic and molecular biology, which will contribute to the genome wide screens of the proposed project. By studying CIN mechanisms in higher eukaryotic systems I will have the opportunity to apply my skills to different organisms and to get acquainted with new approaches necessary to proceed with my academic career.

Prof. Davidson’s laboratory is investigating gene networks that control development. The laboratory scope of research is exceptionally wide Starting from the basic building blocks that are the sea urchin genomics, through transcription factor-DNA interactions that control spatial and temporal expression of specific genes in the embryo, onto the systems level analysis of large regulatory networks and their evolution. The purpose of the proposed research is to reengineer the gene regulatory network components by constructing altered cis-regulatory elements (e.g., self-repression elements which oscillate in output or reinforcing feedback loops) with altered functional behavior, reintroduce them clonally into the embryo, and demonstrate programmed alteration of the developmental process. Besides demonstrating an impressive control of the biological system in study this approach enables the researcher to investigate his assumptions on the connectivity and the functionality of the genetic network in a pure and clean way. Prof. Davidson’s lab is a most promising place for such a research due to the extensive background and experience in developmental genetic regulatory networks and the unique combination of the experimental and computational tools. For me joining Prof. Davidson’s lab and working on this project is a unique opportunity to gain a deep understanding of a complicated biological system, to study a wide range of biological methods and tools and to combine my experimental and modeling skills.

Satellite cells are adult stem cells found in vertebrate skeletal muscle. They possess the general features of stem cells like the ability for self-renewal and the production of large numbers of differentiated daughter cells. Quiescent satellite cells become activated during normal muscle growth, muscle injury and in degenerative diseases such as muscular dystrophy. Despite their importance, the molecular mechanisms that regulate satellite cell activity are poorly understood and most of the current data is based on in vitro and ex vivo experiments performed in mammalian systems. So far no animal model has been used to systematically investigate the genetics of satellite cells in vivo. For this fellowship, I suggest to introduce zebrafish (Danio rerio) as a vertebrate model system to identify new genes that control satellite cell activity. To this end, I will perform a mutagenesis screen in order to identify mutants with satellite cell phenotypes. Furthermore, existing mutants with defects in muscle development and growth will be analysed for satellite cell phenotypes using satellite cell specific molecular markers. New insights into the genetics of satellite cell biology will contribute to the development of new therapeutical approaches, including stem cell therapy, for degenerative muscular diseases. My research experience is based on invertebrate developmental genetics. For this fellowship, I’ll change my research focus from invertebrate genetics to the investigation of genes that regulate adult stem cell biology and adult muscle tissue homeostasis in vertebrates. This will allow me to gain insights into new biological concepts and to acquire new technologies.

The centrosome has long been known to promote accurate chromosome and cell division through the regulated nucleation of microtubules. Defects in centrosome function are thought to lead to chromosomal instability, an important factor in the initiation and progression of cancer. The goal of the research proposed here is to investigate the molecular mechanism of centrosome assembly and pericentriolar material (PCM) recruitment in human cells. Studies will initially focus on the human homolog of the C. elegans centrosome component SPD-2, established in the host lab to play a key role in these processes, and will ultimately be directed towards elucidating components of these processes through RNAi-based human genomic screens. The following aims employ proteomic, cell biological, and genomic approaches, including novel techniques for the study of protein function in human cells. Aim 1: Characterize hSPD-2 involvement in human centrosome assembly and function. Aim 2: Identify hSPD-2-interacting proteins and complexes and elucidate specific functions of hSPD-2 in centrosome assembly and PCM recruitment. Aim 3: Develop genome-scale screens in human cells to study centrosome composition and PCM recruitment.

We propose to study long term thermal memory formation and resetting in the worm C. elegans. The details of this phenomenon remain largely unknown (although it was reported some 30 years ago) but novel tools have improved the ability to investigate it on several levels. The development of automated methods to quantify behavior has enabled us to detect changes in behavior induced by genetic manipulations which were too subtle to notice previously. Genetically encoded neural activity reporters can be targeted to specific neurons in intact worms. Ultra-short laser pulses can induce subcellular damage to neurons and gene chip technology makes it possible to identify genes that are primarily expressed in a given neuron. Preliminary results from a collaboration between a physics laboratory and a biology laboratory suggest that certain proteins may play specific roles in temperature memory formation or resetting. We suggest to extend this collaboration. Combining molecular biology tools with quantitative methods and cutting edge laser and imaging techniques we will focus on the molecular mechanisms of memory formation inside a neuron and on the dynamics of signals in the neural network. In the long run we would like to shed light on the issue of how the molecular and neural mechanisms conspire to determine behavior in worms. My own scientific background is physical and computational. Having developed an increasing scientific interest in biology and biophysics, training in a biology lab is of major importance for my scientific development. Moreover, I am a newcomer to neuroscience. Working on memory and learning in worms will introduce me to a new and exciting field of research.

Cell surface display of antigenic peptides by MHC I molecules allows priming of appropriate CD8 T cell responses and elimination of abnormal or infected cells. MHC I antigen processing occurs in the cytosol and continues in the ER through an N-terminal trimming activity. ERAAP, the ER aminopeptidase associated with antigen processing, plays a key role in these last steps of processing. My research proposal addresses unresolved questions about ERAAP function in endogenous and exogenous (cross-presentation) MHC I antigen presentation. It is divided into two specific aims. On the one hand I will study the molecular mechanisms responsible for ERAAP retention in the ER and the regulation of ERAAP's activity. I will carry out a structure/function analysis and seek for potential partners through experiments combining molecular biology, biochemistry and advanced fluorescent techniques. On the other hand, I will analyze the role of ERAAP in cross-presentation of exogenous antigens. To this purpose, I will use a model of infection by Toxoplasma gondii parasites secreting the OVA protein, well characterized by our collaborators (E. Robey's group). The ERAAP ko mice generated in my host laboratory will be valuable for the whole study. While the fluorescence-related techniques acquired during my PhD should be helpful in setting up confocal and FRET experiments, most concepts, models and methodologies about antigen processing mechanisms are new to me. Undoubtedly this complementary experience will widen my immunological background and my technical skills, two factors that are critical to achieve my professional objective to become an independent researcher.

My long-term research goal is to understand how gene expression is regulated in regulated during development. For this purpose, I will study the b-globin gene, which represents a highly useful system for examining developmental-specific gene expression. Each b-like globin genes is expressed at a specific development stage and only in erythroid cells. Despite about 30 years of studies, the molecular mechanism regulating the expression of the b-globin genes is still unclear. To better understand the process of b-globin activation in erythroid cells, we decided to combine a newly developed quantitative proteomics technology with protein purification techniques in order to identify changes in interactions between transcription factors controlling b-globin expression during erythroid differentiation. In this application, I propose to apply this technique to uncover the role of all the other b-globin transcription factors as well as to decipher how these factors interconnect to regulate b-globin transcription. It is expected that these studies will provide us with a complete understanding of the molecular mechanism governing b-globin activation of transcription during terminal erythroid differentiation. If successful, I will use this award in order to hire a technician with previous experience in mass spectrometry. The sophistication of the instrumentation used for these highly sensitive studies requires someone with a very good knowledge of this technique and extensive experience. Thus, a Human Frontier CDA will be crucial to start my lab as an independent investigator.

The claustrum is a puzzling structure which is widespread and reciprocally connected with the cerebral cortex. Although its function remains obscure, it has been related to sensory integration among different modalities (i.e. audio-visual, tactile-visual). Different experimental paradigms had led to some inconsistent findings on the claustrum function. In the proposed project, we take advantage of two new techniques, recently developed at the host institution, to overcome a multidisciplinary approach exploring the function of this structure. We plan to study the dynamic connectivity of the claustrum in the context of a variety of perceptual tasks in the trained monkey. This methodology combine behavioural, electrophysiological and functional magnetic resonance imaging (fMRI) experiments guided by connectivity maps, in an individual-animal basis. My background covers from phenotype differentiation, cell viability and molecular signalling in neurons to dendrite electrophysiology and neuron-glia crosstalk. I also had experience in behavioural studies. In the proposed investigation I will focus on system and cognitive neuroscience. The research plan will allow me to learn new techniques, analytical methods and experimental paradigms in the environment where they were developed. Additionally, this fellowship will facilitate multidisciplinary collaboration between our groups in the future. Overall, this fellowship will represent the ideal start-point to initiate my own research group at my home country.