The genetic network that underlies the first steps of sex determination and gonad differentiation has been elucitated over the 15 past years. During the last few years, we have contributed to major advances in our understanding of genes driving testicular differentiation in mammals and largely contributed to demonstrate, by biochemical and genetic approaches, that Sox9 is the master gene controlling Sertoli cell fate and testis differentiation, although detailed cellular and molecular processes downstream of Sox9 are still largely unelucidated. - Our previous experiments demonstrated the crucial function of Sox9 in the sex determination pathway, and suggested that Amh/Mis and Sox8 represent direct targets of Sox9. However, since neither Sox8 nor Amh/Mis knockout mice show XY sex reversal, it is clear that important additional targets of Sox9 must exist. Furthermore, we demonstrated that nuclear import of Sox9 was sufficient to induce testis differentiation. Besides the Sox9/Fgf9 regulatory loop, we characterized the functional relationships between Sox9 and Pgds by analysing Sox9 and Pgds knockout mice and identified the Pgds gene as a new gene of the sex determination cascade. - - In this project, we will decipher in more detail the molecular and cellular mechanisms leading to the formation of a functional testis. Sox9, the effector of the testicular differentiation, must activate a complex program of gene transcription resulting in expression of secreted signalling molecules, receptors, hormones, matrix molecules and enzymes that should drive testis differentiation. - Using our established cellular and animal models and the complementarities of both partners, we will thus identify and analyse new genes involved in early sex determination by the available ChIP on chip technique, with a particular focus on Sox9 target genes and we will define new signalling pathways activated during the sex differentiation period. - We will thus directly link Sox9 to key transcription factors and signalling molecules that could serve as amplifiers of differentiation signals and effectors of specific Sertoli functions. Signalling is crucial for key cellular and developmental decisions. A number of signalling pathways involved in the regulation of Sox9 activity have been described. By identifying new genes, we will clarify the relationships and the hierarchy of these molecular pathways; this will also bring new insights since these genes could potentially represent excellent candidates to explain the genetic causes of many human sex disorders that are still unclear. - The aims of this project are thus : to find the direct target genes for Sox9 by the high-thoughput Chip on chip technique, to validate this approach by using transcriptomic approach of established mouse models, to study the functional analysis of the Sox9 target genes in genetically modified mice and by biochemical approaches, to elucidate the functional interplay of these new genes with known pathways: Fgf9, Pgds, SOX9 and Wnt4 and finally to screen for mutation(s) in panels of human sex reversal patients. - ...

Protein misfolding diseases (PMDs) are associated with either aggregation of misfolded proteins leading to toxic gain-of-function phenotypes or with protein degradation leading to loss-of-function phenotypes. Cystic Fibrosis (CF) is an example of a loss of function resulting from genetic mutations within CFTR. F508del, by far the most frequent mutation, is associated with protein misfolding, reduced channel function and cell surface stability. Mutant channel maturation and function can be partially rescued with small molecules referred to as correctors, while channel function is efficiently enhanced by potentiators. Nonetheless, this treatment combination is still suboptimal in clinics and there are still no means to increase channel cell surface stability. Our working hypothesis is to increase channel rescue by targeting specific protein-protein interactions, which retain abnormally folded proteins. This novel strategy is supported by our previous work, which revealed that an interaction with intermediary filament, Keratin 8, retains F508del-CFTR in the endoplasmic reticulum (ER) and that disruption of this interaction restores CFTR-F508del functional expression, leading to the identification of a new class of CFTR correctors. Preliminary data indicate that the two classes of correctors, targeting either CFTR directly or protein-protein interactions, present an additive effect. Our new results identified PRAF2 as a novel key regulator of CFTR exit from the ER, suggesting that PRAF2/F508del-CFTR interaction could also be a target for pharmacotherapy. We have previously shown that PRAF2 controls the ER exit of cargo transmembrane proteins (i.e. receptors, transporters…) in a stoichiometric manner and that this regulation involves the presence of a molecular code-bar (RXR motifs) located in the intracellular domains of PRAF2-regulated proteins. The first aim of this project is therefore to understand the role of PRAF2 in the ER exit of CFTR by identifying both the specific binding motifs and the additional molecular actors implicated. This will set the basis for a molecular screen aimed at identifying chemical compounds targeting the PRAF2/CFTR interaction. In parallel, we will identify new (i) differential (WT or mutant CFTR-specific) interatomics and (ii) compartment-specific protein-protein interactions for F508del-CFTR using the newest proteomic approaches: proximity labeling with APEX2 and APEX2-complementation coupled to mass spectrometry. The importance of these “new” molecular partners will be evaluated both functionally and biochemically. The best targets will be combined with CFTR modulators available today to enhance treatment efficacy and tested on primary epithelial cells. The end point to these studies would be the identification of new drugs capable of enhancing the efficacy of current treatments. We believe, indeed, that the release of partially functional misfolded proteins from specific protein-protein interactions will restore to some extent functional activity. For cystic fibrosis, this could be further enhanced by combination with available CFTR modulators. The data obtained so far support our strategy of targeting protein-protein interactions in PMDs and provide the flowchart to study other loss of function PMDs such as alpha-1-antitrypsin deficiency, diabetes, nephritic syndrome, and also to some extend other diseases such Chronic obstructive pulmonary disease (COPD)…. Consequently, the methodology developed in this project could in fine lead to the identification of key protein-protein interactions in other diseases which could be targeted and represent potentially treatments, which could similarly be improved be improved with a new class of drugs.

Cohort studies are studies in which the same variables are measured repeatedly over time. Each sequence of measures, known as the variable-trajectory, reflects the evolution of a phenomenon. In recent years we have seen the development of new tools to analyse these trajectories. The most widely used are partitioning techniques (Proc Traj or KmL for instance). They consist in grouping individuals whose trajectories are similar, thus defining "typical trajectories" that reflect the "mean" behaviour of the individuals in a given sub-group. However these techniques have a certain number of limitations. Collaboration between methodologists and physicians in our different teams has enabled priority lines of action for the development of methods to overcome these limitations, and thus considerably enhance the use made of the cohorts that have already been subjected to the more classic analyses. Thus our project has a threefold objective: i) to create new methods for partitioning longitudinal data, ii) to demonstrate their efficacy on existing cohorts, and iii) to make the newly-developed tools available to the scientific community in the form of computing libraries. The cohorts selected to implement these methodological developments are all prominent studies, and concern important societal issues: standardised care plan and Alzheimer's disease, long-term outcome of severely premature infants, access to and compliance with retroviral treatments in Senegal, hormone profiles of women without fertility problems, social and behavioural outcomes of children in Quebec. The analysis of these programmes has identified five methodological issues to be addressed by the present research programme: - KmL3D: To date, partitioning techniques consider the temporal evolution of a single variable-trajectory. However it is possible to envisage complex interactions between trajectories. KmL3D will enable work on the concurrent evolution of several variable-trajectories by partitioning them simultaneously. - KmLCov will partition the data integrating the effects of covariables (time-dependant or other) on the trajectories. Their effects may be specific for each class of trajectory. - KmLShape: In certain circumstances, the exact moment of the appearance of a phenomenon is less important than the type of evolution that it exhibits. The ability to partition trajectories according to their shape or profile and to group individuals whose trajectories are close despite atime discrepancy is the objective of KmLShape. - KmLVar: It may also be relevant to classify individuals according to whether their variable-trajectory is stable or fluctuates. Indeed, the degree of instability of a marker may provide more information than the evolution of its value. The aim of KmLVar is to enable the modelling and the identification of groups or trajectories with the same variance. - Finally, partitioning techniques enable new approaches for imputing missing data in trajectories, based on both the known values for an individual and the mean trajectory of the group. The validation of the Copy Mean method is thus also an objective of this project. In this way our project combines methodological research and application to real data, in response to research questionings from clinicians and epidemiologists. Each team will be in a position to propose original analyses of cohorts using the new tools developed within the programme. Ultimately, these new statistical techniques will be programmed and made available to the scientific community on a dedicated website, thus ensuring wide diffusion.

Actin-based adhesion structures, especially those involved in cell adhesion to the extracellular matrix via integrins that we will call here ABS, are complex molecular assemblies with a wide range of shapes and dynamics. By participating in the remodeling of the extracellular matrix and in cell migration, ABS perform essential functions in many biological processes such as embryonic development, tissue repair, immune response, angiogenesis... The transport of mRNAs and their local translation participate in the spatial and temporal control of protein distribution, notably in the lamellipod, a well-known example of ABS. However, very little is yet known about the diversity of mRNAs enriched in ABS. Furthermore, the mRNAs whose local translation is required for the formation and maintenance of ABS are not known. Finally, although the general mechanisms of mRNA translation initiation and elongation have been characterized, the mechanisms that control local translation remain mostly unidentified. Cell adhesion to the extracellular matrix, extracellular matrix remodeling and cell migration are key processes in cell biology, which also have implications in tissue morphogenesis and organogenesis. This highlights the importance of studying the impact of local mRNA translation on ABS dynamics. In this project, we aim to understand the importance of local translation of mRNAs in the establishment and maintenance of ABS. For this, our consortium combines the expertise in the study of molecular mechanisms controlling the dynamics and functions of different types of ABS with expertise in translation initiation factors, protein synthesis machinery and genome-wide (translatomic) mRNA translation analyses. For this project, we will use 3 models of ABS based on a dynamic branched actin network dependent on the Arp2/3 complex, but with distinct molecular organization, dynamics and functions: the lamellipod of the migrating cell, the invadosomes that remodel the extracellular matrix and the actin ring of osteoclasts that supports their bone resorption apparatus. We will rely on state-of-the-art techniques gathered in our consortium: subcellular laser microdissection combined with proteomics and transcriptomics, local and global detection of protein synthesis, super-resolved microscopy and single molecule imaging, and time-resolved translatomics. We propose: (WP1) to establish and compare the local landscape of mRNAs and proteins in ABS and to characterize the local translation activity of mRNAs during ABS formation; (WP2) to establish the localization and nanoscale dynamics of key components of the translation machinery in ABS, and to assess the impact of intra- and extracellular forces on local translation at ABS; (WP3) to establish the hierarchy of translation events during the assembly of each ABS and to identify the specific translation factors that control protein synthesis in each ABS. In this way, we hope to discover mRNAs whose local translation is key to the maintenance of the different ABS, determine whether the different translation steps occur at the same location or in different subdomains of the ABS, define the mechanisms that couple local translation of mRNAs to the dynamics of the ABS, and identify translation factors and locally translated mRNAs that are critical for the establishment and function of the different ABS.