L’incidence des cancers testiculaires a fortement augmenté au cours des 50 dernières années. Des études épidémiologiques in vitro et in vivo, ont montré que l’exposition des hommes aux perturbateurs endocriniens est impliquée dans l’initiation et la progression du cancer du testicule (Beiki et al., 2010). L’exposition fœtale à ces produits pourrait provoquer des altérations de la différenciation des cellules germinales (CGs) au cours leur développement pendant la vie fœtale. Au cours de cette thèse, nous nous sommes intéressés à déterminer le rôle des perturbateurs endocriniens œstrogéno-mimétiques en mélange sur la prolifération des cellules séminomateuses TCam-2. Nous avons identifié les voies de signalisation impliquées dans la réponse à ces produits. Nous nous sommes focalisés sur les effets d’un mélange d’alkylphénols appelé M4 (4-tert-octylphénol et le 4-nonylphénol), in vitro sur la prolifération des cellules séminomateuses TCam-2, et in vivo sur la croissance de tumeurs obtenues à partir des cellules de carcinome embryonnaire NT2/D1 xénogreffées chez la souris Nude. Dans notre étude, nous avons mis en évidence l’implication d’ERα36, une isoforme tronquée du récepteur ERα, dans la réponse au mélange d’alkylphénols, M4. Ce mélange stimule la prolifération des cellules du cancer testiculaire et l’expression du récepteur ERα36. Ces effets sont médiés par la phosphorylation de CREB via la voie PI3K-ERα36-dépendante, et aboutissent à la diminution de l’expression des gènes de la famille Dnmt3, limitant le niveau de méthylation de l’ADN. ERα36 pourrait donc constituer une cible thérapeutique dans le traitement des cancers testiculaires / In vitro and in vivo epidemiological studies showed that the exposure of men to endocrine disruptors triggers the initiation and the progression of the testis cancer (Beiki et al., 2010). The fetal exposure to these products is able to alter the differentiation of germ cells in the timing of their development during the fetal life. The aim of our study was to determine the role of an endocrine disruptor mix on the proliferation of seminoma cells TCam-2. We also identified the pathways involved. In this study, we focused on the estrogen-like effects of an alkylphenol mixture called M4 (4-tert-octylphenol and 4-nonylphenol): in vitro we measured the proliferation of TCam-2 seminoma cells after M4 exposure and in vivo, we measured the tumor growth of NT2/D1 cells xenografted into Nude mice. The results highlighted the implication of ERα36 receptor, a truncated isoform of ERα, in the response to the alkylphenol mixture M4. This mixture stimulates the cell proliferation of testicular cancer cells and the expression of the ERα36 receptor. These effects are mediated by CREB phosphorylation via the PI3K-ERα36-dependant pathway, and end by downregulation of Dnmt3 genes, thus limiting the level of DNA methylation. Therefore ERα36 appears to be a promising therapeutic target for testis cancer treatment

Our laboratory is working with the human NTera2/D1 (NT2) cell line which have properties similar to progenitor cells in the CNS. These neural-like precursors cells can differentiate into all three major lineages - neurons, astrocytes, andoligodendrocytes. The pure neuronal population, called the hNT cells, possess characteristics of dopamine (DA) cells. In this dissertation, we performed various experiments to examine the neuronal and dopaminergic development of this cellline. We first cultured our hNT neurons with cells from the developingnigrostriatal (NS) pathway, the ventral mesencephalon and striatum, to determine their influence on survival, neuritic outgrowth, and DA phenotype. The survival ofhNT neurons was substantially greater when they were cultured with embryonicday (E) 18 cells, compared to monocultures or cocultures with either E14 orpostnatal day (P) 1 cells. The neuritic outgrowth of hNT neurons as assessed by the number of primary neurites per cell was increased when cultured with theareas of the brain from E14 and P1. The DA phenotype, as determined by the expression of the rate-limiting enzyme of DA synthesis was not increased in hNTneurons when they were cultured with primary rat cells from the NS pathway.Next we analyzed if the retinoic acid (RA)-treated hNT neurons and the NT2 precursor cells expressed three transcription factors required for development ofthe DA phenotype. We report that NT2 cells endogenously expressed Engrailed-1, Ptx3, and Nurr1 while RA treatment increased Nurr1 but down-regulated Engrailed-1 and Ptx3. Finally, lithium has been shown to stimulate neurogenesisin adult hippocampal precursors as well as influence the Wnt pathway known to be important for the induction of the DA phenotype.

Cell fusion is an important technique that is used in the field of medicine and biomedical research. For instance, fusion can be used to create hybridomas and novel types of secretory hybrid cells. It may also be used to engineer cultured insulin-secreting pancreatic B-cell lines for the treatment of diabetes. Historically, the applications listed above have been accomplished by a number of methodologies including dielectrophoresis, centrifugation, polyethylene glycol (PEG) and viral fusion proteins. However, these approaches often fail to produce the desired results due to poor cell viability, lack of 1:1 fusion, and use of non-physiological environments. It is proposed that the application of an electrical field generated by corona charge (corona fields) and subsequent treatment with direct current (DC) pulse technology will overcome these deficiencies. Isolated and pre-labeled neuronally committed human teratocarcinoma (NT2) cells in monoculture or co-culture, were seeded in chambers, constructed in the laboratory, and allowed to adhere to the chamber bottom prior to corona treatment. A corona generator, also constructed in the laboratory, was used to expose cells to positive and negative electrical charges to induce cell-cell contact. The cells were then pulsed with DC voltage to induce fusion. During the experiments, cells were photographed sequentially to record cell movement/contact and fusion. The project was designed to identify optimal corona-based electrofusion parameters for viable, 1:1 cell fusion. Optimal results for cell-cell contact were obtained using a cell density of 2.35 times ten to the fourth power cells per microliter Dulbecco's Modified Eagle Medium (DMEM) in a grounded circular plate corona chamber following at least 3 minutes of settling time. Corona charges from (+) 6.1 kilivolt and (-) 5.5 kilivolt potentials were determined as being most favorable for cell movement and viability. Fusion was best achieved by first exposing either a circular or square ungrounded corona chamber configuration to 3 minutes (+) corona charge followed by 3 minutes (--) corona charge; disturbing the cells in the chamber with mechanical force; and then exposing them to 8-15 sequences of a 2,500 Volts per centimeter DC pulse at 100 microseconds.