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.

The innate immune system protects us from bacteria, viruses, and cancer. Natural killer (NK) cells are innate immune cells that can detect and destroy cancerous and infected cells. How NK cells do this requires understanding of the molecules they use to sense what is harmless or dangerous, and how these molecules assist in the decision to ignore or kill the target cell. We have identified a receptor-ligand pair (NKR-P1B:Ocil) on NK cells and their targets, respectively, that mediates immune recognition at the molecular level. Because the NKR-P1B receptor is inhibitory, normal cells express the "self" Ocil protein to prevent NK cell attack, while infected and cancerous cells lose Ocil protein ("non-self") and are killed by NK cells. We have already shown that the NKR-P1:Ocil interaction is important in fighting cancer, and we anticipate a novel role for this interaction in infectious disease. I propose to advance our understanding of cancer biology by studying what molecular or genetic signals cause the loss of "self" Ocil protein during formation of tumour cells. I also intend to study the role of this molecular interaction in immunity to viral and bacterial infection, and how pathogens can evade this immune recognition. The HFSP fellowship award facilitated my repatriation to my home country, but I now need further support to continue this important work. An HFSP CDA award would give me the financial and scientific independence, support, and tools that I need to make this work possible. The CDA award would also facilitate my promotion from Lecturer and Principal Investigator to Scientist, so that I may pursue this research full-time without my current teaching duties.

The eukaryotic cell division cycle, including DNA replication and mitosis, is driven by a complex regulatory network. Stochastic elementary steps involved in these processes as well as large morphological transformations of the cell (such as spindle formation, or bud formation in yeast) introduce time variability in the duration of the cycle. How does the cell control the timings and ensure the robustness of the cell cycle oscillator? Many pathways of the cycle are duplicated and over-determined is the role of such redundancy to reduce the temporal fluctuations? The molecular pathways that control the cell cycle are well known, and the emerging challenge in systems biology is to connect the molecular data to the dynamical properties of the whole system. Such dynamical understanding requires study at the single cell level, to eliminate averaging effects encountered in bulk studies. Here, I propose to investigate quantitatively the temporal variability of the division cycle using time-lapse microscopy on monolayer yeast microcolonies. First, by tracking the expression of fluorescent reporters, I will precisely time the different events occurring during the cycle, to determine the noise contribution of each step in the cycle, and check the effect of gene redundancy. Second, using an external trigger signal, I will force the cycle to beat at an imposed frequency, in order to examine the non-linear properties of this essential biological oscillator. This project pools disparate approaches including yeast molecular genetics, cell biology, biophysics, and physics of dynamical systems, and thus constitutes a promising novel cross-disciplinary approach in biology.

T cells play a key role in maintaining immunity against pathogens. T cell progenitors originate in the bone marrow. They then move to the thymus where they undergo education and development which ensure that resulting T cells are able to recognize pathogens and infected cells and tolerate normal healthy cells. These key immunological processes occur in several stages within distinct anatomical compartments of the thymus. To develop normally a T cell progenitor must travel between these compartments in a carefully orchestrated manner. Certain receptors expressed on the surface of thymocytes at different stages of development may play important roles in regulating cell migration. The aim of my postdoctoral project is identify the mechanisms which control thymocyte migration. Using multi-photon microscopy I will be able to track thymocytes in intact thymi. I will use cells, labeled with fluorescent markers, which either lack or have increased expression of particular receptors to identify the roles of these receptors in thymocyte migration. I will also look at how developmental signals influence thymocyte migration. This project will complement my PhD work, where I studied mature T cells at the molecular level looking at the patterns of gene expression in particular subsets. For my postdoctoral project I will examine the development of T cell progenitors at the cellular level using multiphoton imaging of intact thymus. My postdoctoral project will address fundamental issues in immunology and will also extend my knowledge in Developmental and Cell biology and give me an opportunity to learn new cutting-edge techniques.