Phagocytosis is the process by which eukaryotic cells engulf and digest particles like bacteria. The amoeboid Dictyostelium discoideum uses phagocytosis for feeding while in mammals, professional phagocytes preserve the organism from infection and inflammation. Phagocytosis and macropinocytosis, a similar mechanism for the ingestion of fluid phase, have been widely studied but a lot of gaps persist in our knowledge. The first objective of the project is to identify the molecular mechanisms involved in the trafficking of the plasma membrane proteins during phagocytosis and macropinocytosis. This will be achieved by the microscopic analysis of different membrane and cytoskeleton proteins used as markers. The second aim of the project is to identify and characterize, in Dictyostelium discoideum, new genes required for the phagocytic/macropinocytic processes. This will be done by generating mutant cells by directed and/or random mutagenesis and by the analysis of the consequent phenotype. To ensure their propagation, bacterial pathogens have developed strategies to disrupt the phagocytic process in amoebae and in humans. The last step of the project is to determine if the membrane protein trafficking and the function of the genes newly identified are disturbed by pathogenic action. The results of this project may bring new light to our understanding of phagocytosis, as well as host-pathogen interaction.

The hereditary breast cancer susceptibility genes BRCA1 and BRCA2 are required for the cellular response to DNA damage. The host laboratory has demonstrated a role for both of these proteins in homology-directed DNA repair (HDR), suggesting that they serve as genomic caretakers to preserve chromosome structure, as do other HDR proteins. The exact role that a defect in HDR plays in promoting tumorigenesis has proven difficult to define, because BRCA1 and BRCA2 have been implicated in multiple cellular processes. The aim of my project is to dissect the specific function of BRCA2 in HDR in mice to determine if HDR defects in the mammary gland are tumor-promoting. We will generate a reporter in the mouse to monitor deficiencies in HDR during sensitive stages of mammary development. We then plan to cause such deficiencies by specific interference of BRCA2 function in HDR. The proposed project will make an important contribution towards the understanding of the cause and mechanism of breast cancer and will allow the evaluation of new therapeutic agents in a mouse tumor model. The members of the host laboratory have a longstanding interest in genome instability, HDR and cancer. Working in this environment will allow me to broaden my knowledge of DNA repair in organisms and its analysis through mouse genetics and physiology. The proposed project will shift my field of inquiry from developing site-specific recombinases as research tools to the role of a specific DNA repair pathway in maintaining genomic integrity in a larger cellular and organismic context. This will enable me to analyse complex cellular functions with the aim of developing preventative therapies against cancer.

One of the RNA-protein interactions essential for HIV replication involves the interaction of the viral Rev protein with its RNA target, Rev response element (RRE). The objective of this proposal is to use the RRE as a model system to explore general modes of RNA-recognition, particularly focusing on designed zinc fingers and arginine-rich peptides. Combinatorial libraries will be screened in search of variants that bind with higher affinities or with altered recognition specificities, and novel binders will be studied biochemically and by NMR. My previous research experience was focused on the study of mucin gene expression in trypanosomes at the level of mRNA maturation and stability, primarily using molecular biological techniques. This led us to the identification of novel regulatory cis-elements and trans-acting factors. My post-doctoral position will expand my expertise in RNA-protein interactions in the context of a very different system, allowing me to learn new techniques including combinatorial library screening, biochemical mapping, circular dichroism, and NMR spectroscopy. I am very interested in gene regulation at the RNA level. The combination of molecular biology learned as a student and the biophysical and structural perspectives that I will gain during my post-doctoral work will provide me with a broad background to approach problems in RNA structure and biology. I am interested in obtaining new insights into the molecular recognition problem and in understanding how different RNA-protein molecules interact and are assembled in complexes. This model may also provide an attractive target for the development of non-conventional therapeutic agents against HIV.

1) The aim of this project is to study from a structural point of view the enzymatic and regulatory protein factors involved in mRNA decapping in yeast and/or human. The obtained information would allow a better understand about the role of decapping in mRNA decay and also about the specific interactions between proteins and the mRNA cap structure. 2) My previous research experience has been mainly focused on the areas of Genetics, Biochemistry and Molecular Biology of RNA-based pathogens and ribozymes. The project proposed here is involved in the field of Structural Biology and Biophysics of protein-RNA interactions. This is for me a movement to a new discipline, which I consider it is actually one of the most promising areas of research in life sciences, especially with structural genomics on the near horizon. It will allow me to get used with many novel experimental techniques like protein purification, RNA and protein crystallization or structural determination of macromolecules by X-ray diffraction analysis among others. 3) This new direction in my research, although clearly different from that of my doctoral studies, would allow me to improve my knowledge on the powerful experimental techniques available so far to solve the structure of biological macromolecules at atomic resolution and to understand their corresponding functions, interactions or mechanisms of regulation.

The aim of this project is to elucidate how cell fate determinations are translated into cell shape changes and intra-cellular remodeling during cell differentiation using Drosophila embryogenesis as a model system. In particular, I will focus on how mesodermal programming by the transcription factor snail is coupled to and required for the formation of the ventral furrow at the onset of gastrulation. Preliminary experiments indicate that in mesodermal cells components of the Notch signaling pathway are specifically localized to internal vesicles and that this distribution depends on snail. This observation establishes a link between a transcription factor regulating cell fate and a specific trafficking event. To characterize the molecular regulation underlying the snail-dependent modulation of Notch trafficking and cell shape changes accompanying ventral furrow formation, two complementary strategies will be undertaken. First, the genes required for the localization of Notch will be identified using a genetic approach. Second, I plan to undertake a systematic analysis of the transcriptional network controlled by snail. To this end, I will conduct a microarray analysis of gene expression in wild type and snail mutant embryos. Subsequently, the exact functions of the genes isolated by both methods in regulating Notch trafficking and cell shape changes in the mesoderm will be analyzed using genetic and cell biological methods. In conclusion, this project will help reveal how a transcription factor regulating a specific cell fate choice, remodels intracellular membranes and signaling events to control a correspondent change in cell shape.

My aim is to identify and study novel neurodevelopmental genes by using bristle number quantitative trait analysis in Drosophila. This strategy is geared toward identifying pleiotropic loci that have not been isolated with other screening methods in the past decades. Mutations in important neurodevelopmental genes often result in changes in bristle number, since bristles are integral structures of the Drosophila peripheral nervous system. Dr. Bellen’s laboratory has, in collaboration with Dr. Trudi Mackay (NC State) screened a collection of strains that carry single P-element insertions causing quantitative changes in bristle number. Sequencing data show that as many as 50% of the identified quantitative trait loci (QTL) correspond to known neurodevelopmental genes such as Delta, extramacrochaetae, hairy, scabrous, lola, and pointed. The present project involves the comprehensive analysis of lambik, one of the novel genes identified in this screen. lambik encodes a single-pass transmembrane Leucine-Rich-Repeat/Immunoglobulin domain protein that antagonizes the activity of the Notch receptor. Since lambik has a mammalian homolog and since many Notch pathway and neurodevelopmental genes have been implicated in highly prevalent and genetically complex diseases like cancer and Alzheimer’s disease, I am confident that insights gained from this study will enhance our understanding of epidemiologically important human diseases that have major impact on public health.

The proposed project is a fundamental, theoretical/experimental investigation of the hydrodynamics of droplets and biomolecules in microdevices, including the interactions between biomolecules and novel surface treatments. The latter understanding will be applied to the development of a rapid, inexpensive, accurate microscale system for the detection of cancerous point mutations, particularly those responsible for breast cancer. The development of this system will require solving numerous hydrodynamic problems which plague a novel "liquid belt" protocol proposed for moving samples and analytes along the chip, as well as for automated polymerase chain reaction (PCR) amplification. Initially utilizing the candidate's existing expertise in abstract topics in theoretical transport phenomena, the proposed studies will quickly differentiate themselves from his doctoral research by incorporating a great deal of experimental investigation, along with a very strong emphasis on biomedical applications. The attainment of experimental expertise is crucial to his professional goal of directing a combined theoretical/experimental academic research program in the U.S. upon completing this fellowship. Moreover, the proposed program will aid the candidate's professional development by broadening his culture to include a thorough understanding of biomedical issues, as well as furnishing an international perspective on addressing problems in the life sciences.

Considerable evidence has been accumulated over the past 30 years demonstrating the implication of sleep in learning and memory. However, it is still unknown what characteristics of sleep underlie this involvement. The purpose of this study is to investigate whether the replay of specific patterns of neuronal activity in the hippocampus is necessary during sleep, and particularly during rapid-eye movement (REM) episodes, for memory consolidation. We will use the multi-tetrode technique to record from the hippocampal CA1 region of behaving rats. The animals will be trained daily in two spatial learning tasks known to involve the hippocampus. The neuronal ensemble activity patterns recorded during the two tasks will be used, during subsequent REM sleep periods, as templates against which the activity of hippocampal cells will be compared continuously. Electrical stimulation disrupting hippocampal activity will be selectively triggered by the replay of neuronal patterns corresponding to one of the tasks. The behavioral performance of the animals will be compared in the two tasks in order to determine whether a specific deficit is induced for the task for which REM sleep disruption is performed. In parallel, we will monitor ongoing changes in the discharges of the neurons (e.g., place fields modifications). This work, which will be realized in the laboratory of Pr. Wilson at MIT, will allow me to extend the skills I acquired in electrophysiology during my PhD to the use of multiple tetrodes in an awake behaving animal. Also, in line with my interest in theoretical neuroscience, I will develop new computational methods both for implementation of the protocol and data analysis.