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Phthalates are industrial plasticizers with a wide range of applications. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most highly produced and frequently studied phthalates. Its metabolite, mono-(2-ethylhexyl) phthalate (MEHP) is known as a testicular toxicant. The objective of this study was to examine expression of the genes of interest in testicular germ cells exposed to MEHP in a dose- and time-dependent manner at concentrations of 1µM, 10µM, and 100µM at 24, 48, 72 and 96hr time points. The genes consisted of Testisin, GSPT1, and MGMT genes which are a tumor suppressors, phase II xenobiotic metabolizing enzyme and DNA repair gene respectively. These genes were analyzed by Quantitative Real Time PCR (RT-PCR). The results revealed an overall down-regulation for each gene as the concentration and/or time increased. Testisin was the focus of the gene expression analysis. Testisin is epigenetically silenced in testicular germ cell tumors (TGCT) by DNA methylation at the 5’CpG island of the gene. To investigate if MEHP is capable of DNA hypermethylation, a co-exposure with 5-azacytidine (demethylating agent) was conducted. Compared with the 5-azacytidine treatment alone, there was a significant down-regulation of the Testisin gene in the co-exposure. This suggests that MEHP may down-regulate Testisin gene expression by DNA methylation. These findings provide evidence that MEHP can alter the expression of Testisin, GSTP1 and MGMT, genes that are associated in the risk of developing testicular germ cell tumors. In addition, results indicated that MEHP may cause DNA methylation leading to the down-regulation/silencing of genes such as Testisin.

La détermination du sexe et la différenciation des cellules germinales est un processus très organisé qui commence au stade embryonnaire et se termine à la vie adulte. Dans les gonades embryonnaires l'expression de Sry suivie par l'expression de Sox9 initie le développement testiculaire tandis que, en l'absence de l'expression de Sry, les gènes associés au sexe féminin initient le développement des ovaires. Les cellules germinales qui ont migré vers les gonades nouvellement formées continuent leur prolifération intense jusqu'à ce qu'ils s'engagent dans la voie le mâle ou femelle. La décision de devenir des cellules germinales mâle ou femelle ne dépend pas seulement du chromosome sexuel des cellules germinales, mais aussi du microenvironnement des gonades. Si les cellules germinales entrent dans la gonade femelle, ils doivent arrêter la prolifération, dépasser l'arrêt mitotique et entrer en méiose et alors s'arrêter en prophase I. Alors que si les cellules germinales entrent dans la gonade mâle, ils doivent arrêter la prolifération et entrer en arrêt de la mitose. Ici, nous montrons que, lors de la détermination du sexe embryonnaire, la prostaglandine D2 (PGD2) produite par chacune des deux enzymes: la L-PGDS et H-PGDS dans les cellules somatiques et les cellules germinales du testicule mâle participe au programme de différenciation des cellules germinales. La voie de signalisation PGD2 entraine l'arrêt de la mitose par l'activation de l'expression et de la localisation nucléaire de l'inhibiteur du cycle cellulaire p21Cip1 et en réprimant les marqueurs de pluripotence et aussi Stra8. En outre, PGD2 est responsable de l'activation du gène spécifique au mâle Nanos2. Par conséquent, ces données suggèrent que la signalisation par le récepteur de PGD2 DP2 est requise pour la différenciation correcte de la cellule germinale mâle. / The sex determination and subsequent germ cell differentiation is highly ordered process that starts at embryonic stage and completes at adult life. In the embryonic gonads Sry expression followed by Sox9 expression initiates testis development while in the absence of Sry expression, genes associated to female fate initiate ovary development. The germ cells that migrated towards newly formed gonads continue extensive proliferation until they commit to the male or female pathway. The fate decision of germ cells as male or female does not depend only on germ cell chromosomal sex but also on gonadal micro-environment. If germ cells enter into female gonad, they have to stop proliferation, pass through mitotic arrest and enter into meiosis; then arrest into prophase I. While if germ cells enter into male gonad, they have to stop proliferation and enter into mitotic arrest. Here we show that during embryonic sex determination, Prostaglandin D2 (PGD2) produced by each of the two enzymes: L-Pgds and H-Pgds in somatic cells and germ cells of testis participates in male germ cell differentiation program. PGD2 signalling supports mitotic arrest by activating the expression and nuclear localization of cell cycle inhibitor P21cip1 and by repressing pluripotency markers and PGD2 has negative effects on Stra8 expression. In addition PGD2 supports activation of male specific gene Nanos2. Hence these data suggest that PGD2 signalling through DP2 receptor is required for proper male germ cell differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21663号 / 医博第4469号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 万代 昌紀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22880号 / 医博第4674号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 近藤 玄, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The role of national education systems is changing, and many drivers of this phenomenon have been identified (Green, 1997). On the one side, there is a growing convergence in global education policy developments given by globalisation processes, on the other side, a political and ideological discourse has spread that promotes education as essential to the achievement of a model of economic productivity and competitiveness (Ball, 2013). The current research aims to shed light not only on the degree to which national education policy in the last two decades have been influenced by this movement, referred to as the Global Education Reform Movement (GERM) by Sahlberg (2016), but also on its possible consequences on equity. In order to do so, three western and European countries have been chosen: England, Sweden, and Italy. Through a mixed-method approach, in which the analysis of policy reforms is combined with PISA secondary data, each country’s specific political landscape and variation in socio-economic inequalities in the period between 2000 and 2020 is discussed. The final comparison between the countries allows seeing that even if all three countries present features of the GERM in the policy reforms enacted from the 2000s on, the consequences on equity are ambiguous. While the three countries present divergent trends both in PISA results and indicators of socio-economic inequalities, one common phenomenon worth deepening considering the increasing focus on standards is the steep increment in scores’ variation.

Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d’anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l’hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l’environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l’Homme. Dans cette étude, nous avons analysé les effets d’un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l’organe. Nous avons tout d’abord démontré que le MEHP (10-4M) n’altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l’exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l’effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l’exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d’action des phtalates. Différentes études, notamment dans le foie, démontrent l’implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRα est très certainement impliqué ces effets puisque l’expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l’implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l’effet sur les cellules germinales mâles ai pu être mis en évidence dans l’ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras. / Since the last decades, an increase in several abnormalities of the male reproductive function has been progressively evidenced in industrialized countries. According to these observations, it was hypothesized that exposure to some environmental pollutants may impair the fetal testis development, and therefore be at the origins of those abnormalities. Among incriminated compounds, phthalates are molecules highly produced worldwide. These compound are classified as reprotoxic molecules, as they disrupt the development of the germ cell lineage in different species and lead to a decrease in testosterone production in rat. Nevertheless, very few data are available concerning their effects in human. In this study we analyzed the effects of one phthalate, the MEHP, on the human fetal testis development during the first trimester of pregnancy. It was performed using an organotypic culture system that allows the preservation of the different testis structures. We first demonstrated that MEHP (10-4M) does not affect testosterone production of the human fetal testis, in opposition to the results described in rat. We also have demonstrated that MEHP exposure triggers apoptosis in the fetal germ cells, leading to a quick decrease in the total number of these cells. Following those results, we tested the effects of lower doses of MEHP that are close to the highest doses measured in human biological fluids. We therefore demonstrated that fetal germ cells are altered by exposure to this dose of MEHP (10-5M). Finally, in the third part of this work, we focused on the mechanisms of action of phthalate toxicity. Different studies, mostly in the liver, report the involvement of the nuclear receptor superfamilly in the effect of those compounds. Thus, it seemed important to investigate their implication in the effect of phthalates on the human fetal testis. We demonstrated that LXRα is certainly implicated in these effects as its transcriptional level is increased. Moreover, this nuclear receptor regulates two metabolic pathways: Cholesterol and fatty acid synthesis pathways, that seemed to by both modulated by phthalate exposure in the human fetal testis. We also showed that the modulation of these two metabolic pathways is a common process to both the male and female gonads. Nevertheless, the germ cell decrease we evidenced in the human fetal testis was never observable in the fetal ovary. In conclusion, this work contributed to improve our knowledge about the effects of phthalate exposure on the establishment and the development of the human fetal reproductive system. We also have evidenced a new mechanism of these compounds that involves members of the nuclear receptors superfamilly, as well as cholesterol and fatty acid synthesis.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Cytokine induced transcription of the germline immunoglobulin heavy chain gene directs isotype switch recombination to that gene. Therefore, understanding the regulation of germline transcription is an important first step in understanding the class switching process. Treatment of human B cells with IL-4 results in germline epsilon transcription. Subsequent activation of a second signal is necessary for these cells to undergo class switch recombination and express surface IgE. In contrast, treatment of splenic murine B-cells with IL-4 alone does not induce germline epsilon transcription. However, treatment with IL-4 plus LPS does induces germline epsilon transcription, followed by class switching to the IgE isotype. In both human and mouse, IL-4 is absolutely required for induction of germline transcripts and expression of IgE. Therefore, IL-4 is considered to be an IgE switch factor. The murine B lymphoma line, I.29μ is an IgM+ B cell line which can be induced to switch to the IgE isotype by treatment with IL-4 plus LPS. In these cells, germline epsilon transcription is constitutive and can be further induced 5-20 fold with IL-4, whereas LPS has no effect at the RNA level. Thus, the I.29μ cell line provides a model system to study the regulatory effects of IL-4 on the murine germline epsilon promoter. The aim of this thesis is to characterize the murine germline epsilon promoter and identify the minimal DNA elements necessary and sufficient for IL-4 induction. To identify the promoter elements, two kb of the 5' flanking region to the first exon (Iε) of the germline epsilon transcript was cloned into a Luciferase reporter plasmid and assayed for promoter activity. Assay of successive 5' deletion mutations by transfections into two B cell lines, I.29μ and M12.4.1, identified the 213 bp promoter construct, -162Luc, as containing sufficient sequence to confer full promoter function. Assay of the linker scanning mutations in the -162Luc plasmid localized the IL-4 responsive effect to a 46 bp region of the promoter. This region contains three nuclear factor binding elements: a C/EBP site, a recently identified NF-IL-4 site and a NFкB/p50 site. In order to detect protein complexes that specifically interact with this active region of DNA, electrophoretic mobility shift assays were performed using double stranded, oligonucleotide probes of this IL-4 responsive region. An IL-4 inducible complex was identified in nuclear extracts of I.29μ as well as murine splenic B-cells. Competition experiments with mutant probes mapped this inducible complex to the NF-IL-4 site. Constitutive binding of both C/EBP and NFкB/p50 was demonstrated by cold competition and supershift experiments. Transfection experiments using a series of linker scanning mutations allowed identification of DNA elements necessary for IL-4 induction. In order to test if these elements are sufficent for IL-4 induction, double stranded oligonucleotides containing these elements were transfered to a minimal fos promoter plasmid and assayed for IL-4 responsiveness. A 27 bp fragment containing two DNA elements, a C/EBP and a NF-IL-4 site were sufficient to confer IL-4 inducibility to a minimal c-fos promoter. This study defined a different IL-4 response element in the murine germline epsilon promoter from that previously published. This IL-4 response element is identical to the IL-4 response element in the human germline epsilon promoter. The NF-IL-4 site is also present in the promoter of the IL-4 responsive gene, CD23b (FcεRII), and this element binds an IL-4 inducible complex present in the human monocytic cell line U937. Various reports demonstrate the presence of an IL-4 inducible complex by gel shift assays and indeed the binding activity of NF-IL-4 has been mapped to a 9 bp consensus sequence within a 19 bp fragment. However, the transfer of IL-4 inducibility has not been reported using fragments smaller than 123 bp, the importance of which is underscored by the fact that more than one factor is involved in this induction. The contribution of this thesis to the understanding of transcriptional induction by IL-4 is in the delineation of the factors involved - namely, a member of the C/EBP family and NF-IL-4 are required for IL-4 induction and NFкB/p5O modulates this induction.

Proteins, which are T cell-dependent antigens, preferentially induce antibodies of the IgG1 class in mouse, whereas LPS, which is a T-independent antigen, preferentially induces IgG3 and IgG2b. Interaction between CD40 on B cells and CD40 ligand (CD40L) on T cells has been shown to mediate T cell contact help for B cell proliferation, differentiation and immunoglobulin isotype switching. In addition, it has been shown that membranes from activated T cells induce germline γ1 transcripts, and that CD40 signaling induces germline γ1 transcripts. These results indicate that T cell contact help mediated by CD40 ligand (CD40L)-CD40 interaction may contribute to this preferential IgG1 isotype selection in response to T-dependent antigens by inducing transcription of germline Ig γ1 transcripts. Here we show that signaling via CD40 increases expression of a transiently transfected luciferase reporter plasmid driven by the germline γ1 promoter in M12.4.1 B lymphoma cells. By linker scanning mutation analysis of the promoter, we have identified a CD40 responsive region (CD40RR) which is able to confer inducibility by CD40L to a minimal c-fos promoter. The CD40RR contains three NF-кB-binding sites, each of which is required for maximal induction of the γ1 promoter activity by CD40L. Binding of the NF-кB/Re1 proteins p50, Re1A, c-Re1 and Re1B to the CD40RR can be induced by CD40 signaling in M12.4.1 cells or in splenic B cells. Co-transfection of expression plasmids for p50 together with Re1A or Re1B, but not p50 alone or p50 and c-Re1, transactivates the CD40RR in transient transfection assays in M12.4.1 cells. These data demonstrate NFкB/Re1 proteins activated by CD40 engagement play an important role in regulation of the germline γ1 promoter. Further support for this conclusion is provided by the finding that treatment of splenic B cells with NF-кB inhibitors prevents induction of germline γ1 transcripts by CD40L. Although LPS also induces NF-кB activation, it poorly induces germline γ1 promoter activity in M12.4.1 cells and it also poorly induces germline γ1 transcripts in splenic B cells and in the mouse B cell line, 1B4.B6. Western blot analyses show that LPS predominantly activates p50 and c-Re1, whereas CD40L induces all NF-кB/Re1 proteins (Re1A, Re1B, c-Re1 and p50). Likewise, in nuclear extracts from LPS-treated cells, p50/cRe1 and p50/p50 dimers are the major NF-кB/Re1 proteins which bind to the promoter for germline γ1 transcripts in electrophoretic mobility shift assays, whereas in nuclear extracts from CD40L-treated cells, p50/Re1A and p50/Re1B dimers are the major complexes. Reporter gene assay by over expressing NF-кB/Re1 fusion proteins indicates that p50/Re1A and p50/Re1B dimers, but not p50/c-Re1 or p50/p50 dimer, can transactivate the germline γ1 promoter. Despite their inability to activate the promoter, p50/c-Re1 and p50/p50 can bind to the promoter and suppress the transactivation activity of p50/Re1A and p50/Re1B. Therefore, the effect of NF-кB activation on the germline γ1 promoter depends on the Relative amounts of transactivating and non-trans activating NF-кB/Re1 dimers. The inability of LPS to induce germline γ1 transcripts can be explained by induction of non-transactivating NF-кB/Re1 dimers and the ability of CD40L to activate the promoter by a greater induction of Re1A and Re1B Re1ative to c-Re1.