Growing evidences indicate that metabolism plays a pivotal role in regulating directly proliferation, differentiation or stemness. Mainly studied in the hematopoietic and central nervous system, these concepts remain poorly investigated in other tissues. My project aims at addressing this question in the context of the epidermis. I recently identified the multifunctional protein E4F1, an important component of the p53 pathway, as a new essential regulator of pyruvate metabolism implicated in skin homeostasis. Using several genetically engineered mouse models (GEMM) that display genetic inactivation of E4f1 in the epidermis, I previously showed that E4f1-associated metabolic functions regulate epidermal stem cell (ESC) maintenance and keratinocyte differentiation (Lacroix et al., PNAS 2010; Lacroix et al., PNAS 2016; Goguet-Rubio et al., PNAS 2016). At the molecular level, we highlighted that E4F1 controls at the transcriptional level the pyruvate dehydrogenase (PDH) complex (PDC), a mitochondrial complex that catalyzes the oxidation of pyruvate into Acetyl-CoenzymeA (AcCoA). We showed that E4F1-mediated control of PDH is important for ESC maintenance. However, our data obtained with other GEMM suggest that E4F1 functions in metabolism extend beyond the regulation of the PDC and also impacted lipid metabolism in a p53-dependent manner (Lacroix et al., in revision at Nat Comm). Based on this solid set of data, I propose to further investigate the molecular mechanisms by which E4f1-deficiency impact on keratinocyte differentiation and ESC maintenance. I will explore whether perturbation of AcCoA production impinges on histone acetylation and epigenetic regulation of genes implicated in epidermal differentiation and ESC functions. Because PDH-derived AcCoA and citrate production is also important for de novo fatty acid synthesis, I will also investigate whether the epidermal barrier defects previously observed in vivo in E4f1cKO keratinocytes result form defective lipid metabolism. This part of my project should highlight new molecular mechanisms linking pyruvate metabolism to keratinocyte differentiation and functions. The second axis of my project corresponds to a unbiased screen aiming at identifying new metabolic regulators of skin homeostasis. I will combine whole-genome gene expression and metabolic profiling of purified basal (undifferentiated), suprabasal (differentiated) keratinocytes, and follicular stem cells to generate the first genome-scale metabolic network related to normal skin homeostasis. Using computational modeling of these metabolic networks, I wish to highlight metabolic pathways that play important roles in ESC function and keratinocyte differentiation. Their role will then be functionally validated upon shRNA-mediated depletion of key components of these metabolic pathways in primary keratinocytes that I will use to perform clonogenic assays, 3D in vitro skin reconstruction and in vitro keratinocyte differentiation assays. On the long term, this ambitious project should shed light on some of the unexplained skin defects that commonly occur in some metabolic diseases, may lead to the development of new therapeutic strategies for skin diseases, and could have important perspectives in regenerative medecine.

Peptimprint relies on the complementarity and expertise of five teams specialized in the conception of bioactive compounds (Partner 1-IBMM-team amino acids), analytical development (Partner 1-IBMM-team analytical sciences), sol-gel process and hybrid materials (Partner 2-ICG-team CMOS), microfluidics devices (Partner 3-L2C-team POMM) and study of biomolecular interactions by biosensors development (Partner 4-IRCM-team criblage) Objectives The main objective of Peptimprint is to set-up a ground-breaking technology to prepare specific tridimensional and functionalized imprints of peptides and large proteins by sol-gel process, polymerizing original hybrid building blocks mimicking amino acids around the biomolecular template. Unfunctionalized blocks (e.g. tetraethoxysilane, dimethyldichlorosilane) will be used concomitantly to create the network. Once the template biomolecule removed, the hollow cavities will be able to capture these biomolecules with very high selectivity. The second objective of Peptimprint is the preparation of unprecedented imprinted devices for the detection, separation of biomolecules and their extraction and concentration in biological samples. Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM) chips as well as polydimethylsiloxane (PDMS) based microfluidics channels will be modified with hybrid biomimetic imprints. Scientific challenges Classical molecular imprinted polymers (MIPs) suffer important limitations. Firstly, the polymerization conditions (organic solvents, non-selectivity vs amino acid side-chains…) affect the structure of the template yielding a non-relevant MIPs. Secondly, the obtainment of a large cavities deprived of functional groups generate non-specific imprints that failed to mimic the diversity of weak interactions found in natural recognitions systems. Peptimprint approach is bringing down such barriers. The sol-gel process takes place at physiological pH, in aqueous conditions and at room temperature preserving the structural and functional integrity of the protein template. Moreover, the original hybrid monomers (amino acid mimics) used in Peptimprint will recapitulate all types of interactions (ionic/hydrogen bonding/hydrophobic/aromatic stacking) involved in real interactions between biomolecules. Proceeding much more slowly than photopolymerization used in classical MIPS, sol-gel approach of Peptimprint favours a self-organization of all the functionalized hybrid blocks around the template. First models and applications Several peptide and proteins templates covering a wide range of size (from 1.5 to 150 kDa) and functions will be used as models for Peptimprint. It includes vancomycin, C-peptide, human kallikrein1, antibody fragments and therapeutic antibody. Hybrid imprints of such proteins will be prepared on the surface of QCM and SPR devices and the interaction will be studied. On the other hand, models will be either adsorbed or covalently grafted on the silicon mold to cast hybrid-PDMS microchannels. The later will be used for electrophoric analyses. Expected impacts Fundamental knowledge on molecular imprinting will be gathered thanks to Peptimprint project including (i) a generic and straightforward sol-gel method and (ii) tailored reagents (hybrid amino acids) for the inorganic polymerization of functionalized imprints. From a technological point of view, if convincing analytical data are obtained using imprinted microfluidics devices or sensors, the development of relevant selective sensors for the detection and the quantification of biomarkers will be envisioned. Indeed, Peptimprint concept can be generalized to any other types of biomolecules (oligosaccharides, oligonucleotides etc.) or even objects (viruses, bacteria) enlarging the scope of biomedical applications. A strategic advisory board will be gathered (month 24) to discuss the technological transfer opportunities and to maximize the dissemination impact.

The present project focuses on substitutes and derivatives of bisphenol A (BPA) which are used in the manufacture of polycarbonate and epoxy resins, including food contact materials. Indeed, a growing attention has been paid to BPA in recent years, giving rise to an intense debate concerning its endocrine disrupting properties and in fine the associated risk for humans. If BPA is still under the spotlights, the study of other related substances used as derivatives or substitutes must be given a high priority. Indeed, a gap was observed during the “BPA crisis” between the scientifically documented risk assessment approach and a risk management position rapidly adopting the precautionary principle. This was a damageable experience from which lessons have to be learnt. The present project aims to avoid the reoccurrence of such a situation for a new generation of BPA analogues for which risk assessment has not yet been performed. Our primary targeted substances will be two main BPA substitutes/derivatives namely bisphenol S (BPS) and bisphenol A diglycidyl ether (BADGE), and our aim will be to generate appropriate data as regards (i) their biotransformation and biological impact on the human hepatic function, (ii) their biotransformation and biological impact on the human reproductive function, (iii) their modes of actions at molecular level through ligand-receptor binding / transactivation mechanisms, (iv) the human external and internal exposure assessment. A set of secondary targeted substances (BPA analogues including bisphenols B, C, E, F, M, AP and AF) will also be considered for human exposure and ligand-receptor activities. The common guiding principles considered throughout the project will concern: the considered biological models / sub-populations (humans, and more particularly pregnant women, foetus/newborn and adult males), and the consideration of realistic exposure conditions for toxicological tests (low doses, compounds alone versus mixtures representative of the real human exposure).

Most current knowledge of endocrine disrupting chemicals (EDC) action is derived from data sets that use single molecule exposure, with few studies taking into account the more realistic situation where humans and wildlife are simultaneously and chronically exposed to low doses of multiple EDCs. The TOXSYN consortium will address the question of synergistic toxic effects and analyse how low dose combinations of EDCs affect physiology and homeostasis. We focus on two nuclear receptors directly implicated in xenobiotic responses: the pregnane X receptor (PXR) and peroxisome proliferator activated receptor gamma (PPARg), that both heterodimerize with the Retinoid X receptors (RXRs). The focus on these nuclear receptors is determined by the key observation that both have sufficiently large ligand-binding domains to accommodate two ligands simultaneously. We hypothesise that each signalling pathway is a front line candidate for activation by cocktails of endocrine disrupting compounds. A number of preliminary results support our hypothesis including: • X ray crystallography on PPAR? ligand binding domain that shows simultaneous binding of two molecules of mono (2-ethylhexyl) phthalate (MEHP), • in vitro tests that show synergistic activation effects of MEHP and perfluorooctanoic acid (PFOA) on human PPARg dependent-transcriptional activation. These demonstrations of synergistic effects, lead TOXSYN to investigate the molecular basis and physiological consequences of combinatorial effects of low doses of emerging endocrine disruptors through PPAR?/RXR and on PXR/RXR signalling. Besides addressing PPAR for which we have strong preliminary data, we are extending our analysis to PXR signalling for two main reasons. First, like PPARg the ligand-binding pocket of PXR is large enough to accommodate simultaneously two molecules and second, PXR is one of the principal players in mediating the mammalian xenobiotic response. Notably, PXR plays a critical role in the regulation of phase I (CYP), phase II (conjugating), and phase III (ABC family transporters) detoxifying enzymes, co-ordinately regulating steroid, drug, and xenobiotic clearance in the liver and intestine. TOXSYN brings together a highly qualified partnership, from the academic and biotech sectors, with state of the art expertise ranging from molecular and biochemical conformation assays to alternative testing models using cells and small model organisms (SMOs). Our overall aim is to exploit our complementary leading-edge technologies to set-up a panel of structural tests and novel cell-based in vitro and in vivo, embryo-based assays for studying effects of low dose mixtures of EDCs. These mechanism-based assays will furnish extensive data sets on how low dose mixtures can interact with PPARg and PXR receptors at the molecular and biophysical levels (X-ray crystallography, binding affinity assays), cellular levels (high-throughput mammalian and non-mammalian in vitro assays) and whole organism levels (embryo-based SMOs, Zebrafish and the amphibian Xenopus leavis). TOXSYN will: • Identify EDC mixtures that exert synergistic or additive effects on PPARg-RXR activity • Provide a detailed mechanistic view of these affects through structural studies • Validate the physiological relevance of these mechanisms in vivo using reporter assays and tests of adipogenesis • Determine whether these synergistic mechanisms also apply to PXR signalling • Compare the data for each receptor and placing the results in their physiological and environmental contexts Impact: Results from TOXSYN will improve current screening tests and strategies for understanding EDCs action. They will provide data on the molecular mechanisms underlying the physiological consequences of exposure to low-dose effects of mixtures. Their dissemination will help fill current knowledge gaps for regulators concerned with public health and environmental issues and drive new hypotheses for further research on combinatorial effects.

Chlordecone (CLD) is an organochlorine insecticide that was used on the banana plantations of the French West Indies from 1973 to 1993. Because of its low dissipation in the environment, it has now caused long-term contamination of the environment, soil and water, and local agriculture and fisheries products. The West Indian population is continuously exposed to CLD, as shown by its presence in the blood of the entire population of these territories with likely consequences on health. The health issues are currently being evaluated. An epidemiological study (Multigner, 2010) has shown a link between exposure to CLD and prostate cancer for a fraction of the Caribbean population of African origin, with the condition of having resided in mainland France. The risk is increased compared to the same type of sedentary population of the West Indies. However, banana plantation workers directly exposed to massive doses and with generally higher blood levels are not impacted more than the rest of the population. It appears from this study that chlordecone potentializes carcinogens and that exposure would be diet related, with the origin in mainland France. In addition, a preliminary study (F. Lawrence, 2012) carried out on rats showed that the prostate tissue is a major target, apart from liver tissue, for the distribution of CLD in the body. The objective of this project is to complement the existing pharmacological data by focusing our studies on chronic exposure to CLD; on the male genital tract, particularly the prostate gland, not studied so far, as it represents a major target of CLD and toxic action in the West Indies; evaluate the affinity of CLD for different tissues of this tract; determine the impact of this particular tissue exposure on the functioning of the prostate tissue. Our project, in the beginning, will involve a toxicokinetic study to confirm our initial experimental results and evaluate the exposure parameters and retention of CLD in the prostate and due to the direct functional link existing in other tissues, of the male genital tract. This study will be complemented by a MALDI mass spectrometry imaging of different tissues of the genital tract in animals exposed to CLD, to locate and quantify the CLD at a sub-tissue level. This point is related to the program ANR-11-CESA-0017 / HEPATOCHLOR that develops this technique on the liver. In a second step, this project will highlight the impact of CLD on the functioning of the prostate tissue. The CLD interacts with estrogen receptor a and ER ß. The expression of these varies, depending on the specific prostate tissue but also on the function of neoplastic development in these tissues. The link between the activity of endocrine disruption and the development of prostate cancer will be evaluated in vivo in this tumor model (TRAMP transgenic mouse) by determining the aberrant expression of genes related to prostate cancer (by transcriptomics) and the overall metabolic disturbance (by NMR profiling of metabolites) in this model "mouse", treated or not with the CLD at different stages of tumor development. A link will be established with the distribution and quantification of exposure to chlordecone in prostate tissue by statistical analysis aggregating all the data. This project will highlight the determinants of retention of CLD in prostatic tissue and provide a better understanding of mechanisms of the predisposition development of prostate cancer induced by exposure to the CLD.