PROOFS is an interdisciplinary project aiming at studying the potential impact of environmental progestins on the neurodevelopment and on the reproduction in fish but also at investigating the progestative activities of environmental samples and identifying the compounds responsible these biological activities. The rationale behind PROOFS is that, during the last two decades, a large number of studies have investigated the risk posed by natural and synthetic steroidal estrogens on aquatic organism reproductive health while virtually no study have been conducted on the potential endocrine disrupting effect of natural and synthetic progestins. This results in a significant lack of data to characterize the hazard and the risk of this class of environmental contaminant on fish reproductive health. However, there exists evidence to show that there is increasing concentrations of progestagens in the European river waters and that progestins can adversely affect the reproductive function of fish. Progesterone and synthetic progestins (used in contraceptive pills) are present in effluents as well as in surface waters (and probably sediments) at concentration that often exceed those of natural and synthetic estrogens, i.e. in the ng/L range. Second, progesterone are endogenous steroid hormones are key players of important reproductive functions in both male and female, since they are directly involved in stimulation of spermiation and sperm motility in male, in the stimulation of oocyte final maturation and ovaulation in females, and the initiation of meiosis in both sexes. It is also known that progesterone and progesterone derivatives are major neurosteroids, i.e. steroids that are produced by the brain itself. Both nuclear and membrane progesterone receptors are highly expressed in the brain, including in neuronal progenitors that sustain the growth of fish brain throughout the entire lifespan. This suggest that neuronal progenitors are targeted by endogenous progestagens and thus potentially by progestagens present in the environment. The PROOFS consortium aims to (i) to establish new in vitro tools to assess the progestative activities of environmental samples and (ii) to better understand the effects of progestagens on neurodevelopment in zebrafish as well as to characterize their effect on fish reproduction by studying a set of known and relevant hormone-regulated genes. The expected output are 1) the improvement of knowledge of progestins in fish and 2) a better characterization of hazard of these compounds as regards to their endocrine disrupting potency in aquatic vertebrates which is a prerequisite for future risk assessment.

A large body of scientific evidence reports that exposure to pollutants early in life – during pregnancy and early childhood – might have an impact on development resulting in damaging effects on health across the lifespan. Pregnant women and children are exposed to a large number of environmental and occupational pollutants with the potential to act on the endocrine systems. The mother-child PELAGIE cohort conducted in Brittany from 2002, included approximately 3500 pregnant women before 19 weeks of gestation. The women and their children were followed until birth and at ages 2 and 6. The cohort provided evidence that the early exposures to toxicants, such as solvents, current-used pesticides, polychlorinated biphenyls, may affect reproductive and developmental health, including fertility, intrauterine and postnatal growth, behavior, and asthma and allergy. It thus appears relevant to pursue the assessment of the long term impact of these toxicants on later development, in particular pubertal development. The present project will investigate two research hypotheses. (1) Most of the persistent organic pollutants (POPs), such as organochlorine insecticides, polychlorinated biphenyls, brominated flame retardants, or perfluorinated compounds, have endocrine disrupting properties. Biomonitoring studies, including the PELAGIE cohort, have shown the presence of these POPs in biological samples in various European populations, and suggest that humans, including pregnant women, are regularly exposed to these compounds. In light of the various findings of the literature in which longitudinal studies with prospective exposure measurements are recommended, the present project aims at assessing the potential impact of the early exposure to POP compounds on growth, obesity and pubertal development. (2) Organic solvents, including glycol ethers, are present in numerous products of our domestic and occupational environments. They have well-known neurotoxic properties in adults. Our previous findings in the PELAGIE cohort reported higher levels of hyperactivity/impulsivity and aggression in 2-years-old children who were prenatally exposed to solvents; only three other studies have addressed the same issue and there is still insufficient evidence to draw firm conclusions. The present project will aim at pursuing the study of the effects of prenatal solvent exposure on neurobehavioral development in older children. The program of the present project is to set up the follow-up of the PELAGIE children during their pubertal development. The follow-up will be conducted in two ways, using repeated self-questionnaires (every year between 9 and 16 years old), and with clinical examinations (at 12 years old). Measurements of puberty development will include the secondary sexual characteristics (breast, body and pubic hair, testicular and menarche development), anthropometric parameters (bone age, height, weight and body fat), and hormonal measurements. Neurobehavioral development, including autistic spectrum disorders, will be assessed at 12 years old of the child using self-reported and parent-reported instruments. Visual tests will be performed. Postnatal exposures will be assessed in the concurrent blood samples, and using questionnaire. This overall project will benefit from the robustness in the assessment of the early environmental exposures in the PELAGIE cohort, from the large expertise of the epidemiological research team in collecting longitudinal health data within birth cohort (Coord. epidemiological team of INSERM-IRSET U1085, Rennes), and from the collaboration with the hospital department (UIC Unité d’Investigation Clinique, CIC INSERM 1414, Rennes), which aims at facilitating the research activities by providing materials, medical personnel and examination centers in the Brittany area.

Arterial diseases have engendered growing interest do their unfavorable impact on cardiovascular morbidity and mortality. Arterial hypertension is a common health problem that affects 25% of the adult population in industrialized societies and a major risk factor of myocardial infarction and stroke. Hypertension is associated with a number of physiological and biochemical changes in the vessel wall, characterized by excessive contraction, growth and proliferation of smooth muscle cells. Mechanisms and signaling pathways that are involved in these structural and functional vessel wall changes are the subject of intensive research because they may permit identification of potential therapeutic targets for the development of novel pharmacological strategies. However, the pathogenesis of hypertension, as well as the basic mechanisms of blood pressure control, are still insufficiently understood. The aim of my project is to identify the molecular mechanisms involved in the pathogenesis of vascular diseases and to identify new therapeutic targets. Rho proteins (RhoA, Rac1') are major regulators of essential vascular smooth muscle cell functions. Increasing evidence has accumulated to implicate over-activation of Rho proteins as a common component for the pathogenesis of several cardiovascular disorders including hypertension. Recently, genetic analyses revealed that Rac1 could be involved in human arterial pathologies. However, the role of Rac1 in vivo and in the cardiovascular system is still poorly understood. By using both experimental models and developing approaches in human, the specific aim of my project is to determine the role of Rac1 in the cardiovascular system and to establish its involvement in hypertension. Specifically, the main objectives that I would like to carry out are: (a) To perform signaling and gain/loss-of function studies investigate the role of Rac1 GTPase in vitro in the vascular smooth muscle and endothelial cells and to identify its regulators in each type of cell. (b) To dissect genetically, through the use of genetically modified mouse strains, the role of Rac proteins and Rac-dependent routes in the normal function and physiology of the cardiovascular system and its pathological alterations. (c) To conduct genomic and proteomic approaches to determine if GTPase proteins and their regulators could be new biological markers of hypertension in human. These studies will allow us to demonstrate the role of Rac1 protein in cardiovascular processes and diseases. Furthermore, I hope that the consecution of these research aims will make it possible the identification of new diagnostic and prognostic factors for cardiovascular disease and the development of new drug targets with which to treat hypertension and may be diverse other cardiovascular dysfunctions. This project will be developp in the Institute of the thorax. This cardiovascular research is identified as one of the excellent areas of research in Nantes. The objective of my installation and the development of my team in this structure is to bring new skills and my expertise in vascular physiopathology and to strengthen this area of research.

Several vector systems allow efficient gene transfer and transgene expression in vivo. Recombinant adeno-associated vector (rAAV) is one of them and an increasing number of reports demonstrate that rAAV administered in situ in many different target organs can sustain permanent gene expression after one single administration. However, it appears that when translational studies are initiated in larger animal species, the expression of heterologous and even autologuous proteins is systematically followed by a deleterious and often destructive immune reaction from the host. This was shown in cat, dog and nonhuman primate models. More recently, the involvement of the immune system towards the rAAV capsid itself was also reported in a small cohort of factor IX-deficient patients. Therefore, if rAAV provides a clinically relevant platform for efficient and sustained gene therapy, understanding what factors trigger the host immune system remains a major issue before moving to humans. Also, the development of appropriate and relevant immunosuppression protocols is warranted to optimize clinical translation. Long-term use of immunosuppressive regiments is often associated with severe adverse effects, which may be an important limitation. Aside from investigating the clinical impact of new immunosuppressor generations, it appears necessary to understand the mechanisms responsible for triggering the immune system in the context of gene transfer in the skeletal muscle as well as to develop strategies inducing tolerance to the transgene and/or visual capsid. One of the difficulties is to address this question in a relevant animal model that mimics to some extent the patient's immune system. We believe that in that respect, the non-human primate (NHP) is a unique model that is complementary to what the murine model provides. Combining the expertise of Inserm UMR 649 in rAAV-based gene transfer in NHP with the one of Inserm UMR643 in tolerance induction strategies, we identified the following aims: 1) Given that the human and primate immune systems are different from that of the mouse, we propose to characterize the priming events responsible for the initiation of the host immune response against the transgene and/or the capsid following intramuscular (IM) rAAV delivery in the clinically relevant macaque model. 2) As an alternative to the immunogenic IM route, we have developed in NHP a strategy based on the regional perfusion (RI) of an isolated limb. Our preliminary data demonstrate that the RI route is less immunogenic against the transgene than the IM route. We propose therefore to dissect the mechanisms of transgene tolerance following RI rAAV delivery. 3) Also, we aim to develop a protocol to induce targeted and specific immune transgene tolerance following rAAV IM delivery in order to achieve sustained transgene expression without compromising the patient's immune status. Recent studies, performed by Inserm UMR 643 and Inserm UMR 649 together, have demonstrated that immature dendritic cells (DC) could be potential reagents to promote antigen-specific tolerance in vivo. We propose to test the tolerogenic effect of these cells in the macaque model of following rAAV-delivery. Altogether, the data emerging from the overall project will contribute to the development of optimal and safe rAAV-based gene therapy strategies for patients with genetic muscular disorders, as well for the treatment of several genetic indications requiring the secretion of therapeutic levels of circulating factors.