Driven by the appearance of antibiotic-resistant bacterial pathogens, there is an ever-pressing need for new antibiotics. Complete genome sequencing of many microbial species has highlighted, during the last decade, a large number of potential molecular targets. Among them, nicotinamide adenosine dinucleotide kinase (NAD kinase) is a ubiquitous enzyme involved in the last step of NADP biosynthesis. NAD(P) biosynthesis is a promising, albeit relatively unexplored, target pathway for the development of novel antimicrobial agents. Thanks to significant functional and structural characterization, bacterial NAD kinases can be regarded as novel and attractive for the development of selective antibacterial drugs. We have recently identified following a « target-guided synthesis » approach, a series of micromolar inhibitors of NAD kinase. Some showed promising bactericidal activities on Staphylococcus aureus. A patent was deposited protecting a first family of nucleoside derivatives. In this project, we aim to demonstrate the antibacterial activity of our compounds in vivo using a mouse model of staphylococcal infection. We will also confirmed in vitro the mechanism of action and validate NAD kinase as the actual target of these new antibacterial series. In parallel, biological activity will be improved by chemical optimization of our current hits. All together these data will strengthen our results recently patented (EP 10290679.9).

Extracellular vesicles (EVs) serve a direct role in intercellular communication by sampling the biochemical content (such as RNAs and proteins) in a donor cell and transferring the sample to an acceptor cell. Little is known of the delivery process of EVs to the recipient cells. This stands in contrast to the translational opportunities that mastering insights into delivery would promise. EVs have been implicated in multiple physiological functions., including Antigen presentation. Development of adaptive immune responses requires capture of extracellular antigens (Ag) by dendritic cells (DCs), followed by processing, and finally presentation of these antigens on DCs' major histocompatibility Complex (MHC) class I and class II molecules to, respectively, CD8+ and CD4+ T lymphocytes. We have previously demonstrated that EVs play a significant role in Ag presentation. In addition We also showed that EVs represent a very heterogeneous population, which suggests that specific EV sub-types containing specific cargo may be dedicated to specific acceptor cell type and functions. We will focus on the mechanism of EV-mediated transfer of Antigen to dendritic cells, because excellent experimental paradigms in this area have been developed, and because the possibility of manipulating the immune response is a high impact translational area. The present research proposal aims at i) identifying which (if any) EV subtype(s) is/are dedicated to specific acceptor cells and functions (i.e Ag presentation within acceptor Dendritic Cells), ii) identifying the docking and the core fusion machinery that is required for the EV-content delivery within the acceptor cells.

Most anticancer therapeutic drugs target molecules controlling cell proliferation and have deleterious side effects on rapidly renewing cell populations such as those of the hematopoietic lineage or the intestinal lining. In contrast to the antiproliferative therapeutic approach, little success was attained in developing therapeutics effective against cancer cell metastasis. However, secondary metastasis to vital organs is most often the cause for failure in cancer therapy and results in subsequent mortality. Identifying the molecular mechanisms that trigger metastasis or understanding the individual steps in metastasis has been difficult owing to its complex and multifaceted nature. The broad objective of my research is to understand how malignant cancer cells escape from a primary tumor, invade into the surrounding tissue, and migrate through the circulatory system to establish secondary tumors at distant sites. To reach their final destination, metastatic cells must recognize environmental cues, determine the direction of locomotion and degrade the extracellular matrix (ECM) to open a passage for migration. Filopodia are actin-rich protrusive structures believed to be the cell's guidance organelles that sense and regulate directional motility. Fascin is a key regulator of filopodia formation because of its ability to bundle actin filament allowing for efficient pushing of the membrane and protrusion. Invadopodia are another kind of actin-rich protrusive structure specialized in ECM degradation. However, the structure of invadopodia and the mechanisms leading to their formation are poorly understood. Fascin is expressed predominantly in neuronal tissue, and is absent from normal epithelial cells. However, we previously demonstrated that colon cancer cells at the invasive front of the tumor express high level of fascin under the regulation of the beta-catenin pathway. Moreover, we showed that forced expression of fascin in colon cancer cells increases migration and invasion in cultured cells; and cell dissemination and formation of lung metastasis in xenograft mouse models. Our hypothesis is that during progression from adenoma to carcinoma, there is a transient activation of 'invasion' genes, one of which is fascin. We further suggest that expression of fascin promotes cell invasion due to its role in invadopodia formation; and cell migration through its role in filopodia formation and uropod retraction. First, we will determine if fascin can promote tumor cell metastasis. For this purpose, transgenic and xenograft mouse models for colorectal cancer will be established. Whether fascin expression affects the survival rate of these mice will be determined. Aggressivity of the primary tumors and presence of metastases will be evaluated. In parallel, we aim to characterize invadopodia at the molecular and ultrastructural level and to determine the role of fascin in their formation. Involvement of fascin in ECM degradation and molecular composition of invadopodia will be evaluated on 2D and 3D matrices by immunofluorescence. Ultrastructural organization of actin filaments in invadopodia will be studied by electron microscopy. Presence of invadopodia in vivo will be evaluated using mouse and human tumor section followed by electron microscopy. Also, we plan to determine the mechanisms controlling fascin activity and more specifically the role of fascin phosphorylation in cancer cell migration. Using biochemical approach coupled with mass spectrometry, fascin binding partners specific for its phosphorylation state will be searched for. The effect of fascin phoshorylation status on filopodia-dependent motility will be tested on 2D and 3D matrices using live cell imaging and the mechanism of fascin activation will be addressed. Finally, we will analyze invasive migration of cancer cells and their interaction with the microenvironment in vivo. Initial steps of metastasis, namely invasion and migration of cancer cells and their interaction with stromal cells will be analysed at the single cell level using two-photon microscopy. Whether and when the fascin gene is activated during the tumorigenesis process will also be addressed in vivo. In conclusion, while much work has gone into characterizing metastasis at the late steps (extravasation from the circulatory system and establishment of the secondary tumor), the primary tumor has remained a black box at the single-cell level, mainly because of a limitation in the available imaging tools. My research strategy will employ an interdisciplinary approach that combines molecular and cell biology techniques with recent advances in optics to visualize the behavior of individual cancer cells in the living animal. This strategy is unique in its ability to fill the existing gap between properties of individual molecules, like fascin, and their role in cell motility in the natural environment.