The binding of CD80/CD86 on the APC to CD28 on the T cell surface provides a second signal for T cell activation. While it was once believed that this interaction represented a one-way signal, resulting in T cell activation, recently, it has been investigated as a bidirectional signaling process. CD80/86 activation produces IL-6 in DCs, but its role in macrophage activation is unknown. Dysregulation of CD80/86 expression has been observed in autoimmune disorders and cancer, and may also influence the development of immune responses including production of cytokines in response to stimulation with TLR-4 ligand, LPS. Therefore, the focus of my project was twofold: 1) to investigate the role of CD80/86 as signaling receptors capable of transmitting extracellular signals, and 2) to determine the TLR-4 activated pathways that regulate CD80/86 expression in human monocyte-derived macrophages (MDMs). Since I demonstrated that activation of CD80/86 alone did not induce expression of the four cytokines investigated, I hypothesized that CD80/86 synergizes with other signaling pathways. I show for the first time that CD80/86 activation synergizes with TLR-4 signaling to produce IL-27 and IL-10 in human MDMs. Since cIAPs play a key role in TLR-4-mediated signaling, I investigated their role in TLR-4- and CD80/86-activated production of IL-10 and IL-27. Degradation of IAPs by SMAC mimetics inhibited LPS-induced IL-10 and IL-27 production in MDMs. However, it did not alter the TLR-4 and CD80/86 synergistic effect on IL-10 and IL-27 production suggesting that IAPs may not play a role in CD80/86 activation of macrophages. Since I have demonstrated this role for IAPs, I extended my studies by examining the involvement of IAPs and other upstream signaling molecules such as SHP-1, RIP1, TRAF2, in modulating the LPS-induced CD80/86 expression. I showed that cIAP2, SHP-1, RIP1, TRAF2 co-localize to form a complex that regulates the LPS-induced CD80 and CD86 expression through AKT-activated p38 MAPK in human macrophages. These findings may lead to the development of novel therapeutic interventions in the treatment of autoimmune diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20976号 / 医博第4322号 / 新制||医||1026(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 髙折 晃史, 教授 岩田 想, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Résumé : La néphropathie diabétique (ND) est une complication microvasculaire du diabète évoluant ultimement en insuffisance rénale et l’hyperglycémie est connue comme étant l’un des facteurs de risques. De larges études cliniques, tel que le DCCT et l’UKPDS, ont montré que si le contrôle intensif de la glycémie se faisait de façon précoce, il serait possible de retarder le développement de la ND. Cependant, les résultats de l'EDIC montrent que si ce contrôle intensif se faisait plus tardivement, suite à une période d’hyperglycémie, il n’empêcherait plus sa progression. Les podocytes ont un rôle critique dans le maintien des fonctions rénales et leur apoptose corrèle de façon très spécifique avec la progression de la ND. Récemment, nous avons rapporté que SHP-1, une protéine tyrosine phosphatase, était augmentée en concentrations élevées de glucose (HG), menant à une inhibition des voies de signalisation de l'insuline. Notre hypothèse est que l’augmentation de l’expression de SHP-1 causée par l’hyperglycémie persiste même après réduction des niveaux de glucose, phénomène de mémoire hyperglycémique, causant une résistance à l'insuline, la mort des podocytes et une absence de réversibilité liée à la progression de ND. Les résultats in vivo montrent que la fonction et la pathologie rénale continuent de progresser et ce en dépit de la normalisation des niveaux de glucose avec implants d’insuline de 5 à 7 mois d’âge La progression de la pathologie corrèle avec le maintien de l’augmentation de l’expression de SHP-1, contribuant au maintien de l’inhibition des voies de l’insuline. En culture, des podocytes murins exposés en HG pendant 96 h et ensuite exposés en condition normale de glucose(NG) pour les dernières 24 h montrent une persistance de l’inhibition des voies de signalisation de l’insuline qui corrèle avec l’augmentation persistante de l’expression et l’activité phosphatase de SHP-1. L’activité des caspases 3/7 dans les podocytes est plus élevée lorsque ceux-ci sont exposés en HG qu’en NG. Le retour en NG pour les dernières 24 h n’a aucun effet bénéfique à réduire l’activité des caspases 3/7. Finalement, l’analyse épigénétique a été suggérée comme étant une explication du phénomène de mémoire hyperglycémique. La monométhylation de la lysine 4 de l’histone 3 (H3K4me1), un marqueur d’activation génique, est augmentée sur le promoteur de SHP-1 en HG et demeure élevée malgré le retour en NG pendant les dernières 24 h. En conclusion, l’hyperglycémie engendre une augmentation persistante de SHP-1 due possiblement à des modifications épigénétiques, causant le maintien de l’inhibition les voies de signalisation de l’insuline même après un retour à des niveaux normaux de glucose, contribuant à la progression de la ND. / Abstract : Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Renal podocytes apoptosis induced by hyperglycemia is an early event of DN. Clinical studies have shown that intensive blood glucose control reduced the development of DN but is not sufficient, if started late, to prevent its progression, introducing the concept of “hyperglycemic memory”. We have recently published that the tyrosine phosphatase SHP-1 is elevated in renal cortex of type 1 diabetic mice (Akita), contributing to insulin unresponsiveness and DN. We hypothesized that SHP-1 expression remains elevated regardless of systemic blood glucose normalization, and is responsible for hyperglycemic memory in podocytes leading to DN progression. In vivo contribution of SHP-1 in hyperglycemic memory was evaluated using Akita mice treated with insulin implants after 4 months of diabetes. Both urinary albuminuria and glomerular filtration rate were significantly increased in diabetic mice compared to non-diabetic mice and remained elevated despite normalization of blood glucose levels. Renal dysfunction was associated with a persistent increase of SHP-1 expression in renal cortex and inhibition of insulin action that were not normalized following insulin implants. Mouse podocytes were cultured in normal (5.6mM; NG), high glucose concentrations (25mM; HG) for 120 h or HG (96 h) followed by NG for an additional 24 h (HG+NG). We observed that Akt and ERK phosphorylation induced by insulin was inhibited in HG and were not restored despite returning glucose level to 5.6 mM after the HG period. This inhibition was associated with persistent increase of SHP-1 expression and phosphatase activity, leading to insulin signaling pathway inhibition. Moreover, caspase 3/7 activity in podocytes exposed to HG was higher than in podocytes cultured in NG and returning glucose concentrations to normal range for the last 24 h after the 96 h HG exposure had no effect on reducing caspase 3/7 activity. Epigenetic changes were studied to explain the hyperglycemic memory effect. On SHP-1 promoter, H3K4me1 levels, an activation mark, tended to be more elevated in podocytes exposed to HG and were maintained despite returning to NG levels after the HG conditions. In conclusion, hyperglycemia induces persistent and epigenetic changes of SHP-1 causing insulin unresponsiveness in the podocytes contributing to DN progression.

Il existe différents groupes de cellules lymphoïdes innées (ILC) qui ont été caractérisées en fonction des facteurs de transcriptions indispensables à leur différenciation et des cytokines qu’elles sécrètent. Les ILC1, dont font partie les cellules Natural Killer (NK), expriment T-bet et produisent de l’IFN-γ. Les ILC2 sont caractérisées par GATA-3 et sécrètent de l’IL-5 et de l’IL-13. Quant aux ILC3, elles ont été identifiées par leur sécrétion d’IL-17 et d’IL-22 ainsi que par l’expression de RORγt.Mon travail de thèse m’a amené à étudier différents aspects de la biologie des cellules NK et ILC3 : leur tolérance, leur homéostasie et leur plasticité.Les cellules NK jouent un rôle dans l’élimination de cellules cancéreuses et des cellules infectées par des bactéries et des virus. J’ai mis en évidence le rôle de la phosphatase SHP-1 (Src homology region 2 domain-containing phosphatase-1) dans les mécanismes de tolérance et d’activation des cellules NK. J’ai également montré que la protéine anti-apoptotique Bcl2 (B-cell lymphoma 2) est importante pour l’homéostasie des cellules NK. Seules les cellules en cycle cellulaire peuvent compenser l’absence de Bcl2, notamment du fait de l’augmentation de l’expression d’une autre protéine anti-apoptotique, Mcl1 (Myeloid Cell Leukemia 1). Les ILC3 sont des cellules principalement localisées dans l’intestin et qui peuvent être classées en différents groupes en fonction des marqueurs qu’elles expriment. J’ai montré qu’il existe une plasticité entre les différentes populations d’ILC3, et que cette plasticité est régulée par des facteurs environnementaux tel que le TGF-β et le ligand de Notch, DL1. / There are three groups of innate lymphoid cells (ILC), defined notably by the transcriptions factors essential to their differentiation and their cytokines secretion. ILC1, including natural killer (NK) cells, express T-bet and secrete IFN-γ. ILC2 are characterized by GATA3 expression and the production of IL-5 and IL-13. ILC3 secrete IL-17 and IL-22 and express RORγt.My PhD work dealt with different aspects of NK cells and ILC3: their tolerance, homeostasis and plasticity.NK cell are involved in killing tumor cells and bacteria- or virus-infected cells. I found that the phosphatase SHP-1 (Src homology region 2 domain-containing phosphatase-1) has a role in NK cell tolerance and activation.I also showed that the anti-apoptotic Bcl2 protein (B-cell lymphoma 2) is important for NK cell homeostasis. Only cycling NK cells could compensate the Bcl2 deficiency, due to the increase expression of another anti-apoptotic protein, Mcl1 (Myeloid Cell Leukemia 1).ILC3 are mainly located in the gut and are classified in different groups, depending on the markers that they expressed. I showed that there is plasticity between ILC3 populations and that this plasticity is regulated by environmental factors, including TGF-β and the Notch ligand, DL1.

No description available.

The thesis deals with the concept of corporate philanthropy and Corporate Social Responsibility. Our goal is to review the state of corporate philanthropy and CSR in SHP Harmanec, JSC, to explore, by a questionnaire research, the attitudes of young people to 30 years and to draw recommendations for the streamlining of corporate philanthropy in the company. Using the synthesis of the research`s results, it aims to design communication activities, which could present the philanthropic activities to this target group. The work is divided into 9 chapters in two parts. The first theoretical and methodological part summarizes available literature on corporate philanthropy and Corporate Social Responsibility. The second analytical part analyzes the company SHP Harmanec, JSC, and examines the attitudes, opinions and motivations of the young people to 30 years on the issue of corporate philanthropy and Corporate Social Responsibility. Proposal on streamlining corporate philanthropy and suggestions of the best communication activities, which could present the philanthropic activities of the company to the young, are the result.

SF-1 e LRH-1 são fatores de transcrição que exercem um papel fundamental na produção de esteroides nas gônadas e na suprarrenal, além de estarem envolvidos no processo tumorigênico desses órgãos. Por outro lado, POD-1 apresenta menor expressão em carcinomas adrenocorticais, e parece regular Sf-1. Nesse trabalho foi analisado o papel de POD-1 na regulação de SF-1 e de LRH-1 em células de tumores adrenocorticais. A hiperexpressão de POD-1 resultou em redução da expressão SF-1/SF-1. Em contraste, houve um aumento da expressão gênica de LRH-1, devido à diminuição da expressão de SHP, um regulador negativo de LRH-1. Nas células transfectadas com siRNA-POD-1, os níveis de POD-1 foram reduzidos e de SF-1 aumentado, reforçando o mecanismo regulatório entre os fatores. No ChIP assay, POD-1 se ligou a sequência E-box do promotor de SF-1. Por outro lado, não foi caracterizado a ligação de POD-1 no promotor LRH-1, embora POD-1 tenha se ligado ao E-box do promotor SHP. A redução de SF-1 diminuiu a expressão de StAR, mas não modulou a proliferação das células tumorais. Em resumo, POD-1 pode ter um papel mais amplo como regulador da transcrição de fatores que controlam o processo tumorigênico, e é um candidato a gene supressor de tumor nas células adrenocorticais. / SF-1 and LRH-1 have played a critical role in steroid production, adrenal and gonads. Moreover, there are evidences that they have acted in tumorigenesis process in these organs. POD-1 is downregulated in adrenocortical carcinoma (ACC) it seems to regulate Sf-1. In this work, it has been to analyse the role of POD-1 in SF-1 and LRH-1 regulation in adrenocortical tumor cells. The POD-1 overexpression has reduced SF-1/SF-1 expression. However, there was an increase of LRH-1 gene expression due to SHP expression decrease which is negative regulate of LRH-1. The POD-1 and SF-1 gene expression in transfected cells with siRNA-POD-1 has shown POD-1 decrease and SF-1 increase emphasizing a regulatory mechanism between POD-1 and SF-1. By ChIP assay it was shown that POD-1 binded in SF-1 promoter E-box sequence. It was not characterized that POD-1 binded in LRH-1 promoter, although POD-1 can bind in SHP promoter E-box sequence. The reduction of SF-1 expression by POD-1 has decreased the StAR expression, however, it was not enough to change tumor cell proliferation. In summary, POD-1 must have a wider role as regulator of fator transcription which controls tumorigenese process being a possible candidate as tumor supressor gene in adrenocortical cells.

In dieser Arbeit sollen die maßgeblichen nationalen Zugbeeinflussungssysteme der Länder Polen (SHP, Radio-Stop, KHP), Slowakei und Tschechien (LS, MIREL VZ1) vorgestellt und miteinander verglichen werden. Der Fokus liegt dabei auf einer funktionellen Betrachtung, wobei auch der technische Hintergrund nicht unberücksichtigt bleibt. Ein Ausblick auf die zukünftigen Bemühungen hinsichtlich ETCS rundet den Überblick ab. / This paper compares the major national train protection systems of Poland (SHP, Radio-Stop, KHP), the Slovak Republic and Czechia (LS, MIREL VZ1). The emphasis is placed on a functional description, while mentioning the relevant technical background where necessary. An outlook on future developments regarding ETCS in the respective countries concludes each chapter.

SF-1 e LRH-1 são fatores de transcrição que exercem um papel fundamental na produção de esteroides nas gônadas e na suprarrenal, além de estarem envolvidos no processo tumorigênico desses órgãos. Por outro lado, POD-1 apresenta menor expressão em carcinomas adrenocorticais, e parece regular Sf-1. Nesse trabalho foi analisado o papel de POD-1 na regulação de SF-1 e de LRH-1 em células de tumores adrenocorticais. A hiperexpressão de POD-1 resultou em redução da expressão SF-1/SF-1. Em contraste, houve um aumento da expressão gênica de LRH-1, devido à diminuição da expressão de SHP, um regulador negativo de LRH-1. Nas células transfectadas com siRNA-POD-1, os níveis de POD-1 foram reduzidos e de SF-1 aumentado, reforçando o mecanismo regulatório entre os fatores. No ChIP assay, POD-1 se ligou a sequência E-box do promotor de SF-1. Por outro lado, não foi caracterizado a ligação de POD-1 no promotor LRH-1, embora POD-1 tenha se ligado ao E-box do promotor SHP. A redução de SF-1 diminuiu a expressão de StAR, mas não modulou a proliferação das células tumorais. Em resumo, POD-1 pode ter um papel mais amplo como regulador da transcrição de fatores que controlam o processo tumorigênico, e é um candidato a gene supressor de tumor nas células adrenocorticais. / SF-1 and LRH-1 have played a critical role in steroid production, adrenal and gonads. Moreover, there are evidences that they have acted in tumorigenesis process in these organs. POD-1 is downregulated in adrenocortical carcinoma (ACC) it seems to regulate Sf-1. In this work, it has been to analyse the role of POD-1 in SF-1 and LRH-1 regulation in adrenocortical tumor cells. The POD-1 overexpression has reduced SF-1/SF-1 expression. However, there was an increase of LRH-1 gene expression due to SHP expression decrease which is negative regulate of LRH-1. The POD-1 and SF-1 gene expression in transfected cells with siRNA-POD-1 has shown POD-1 decrease and SF-1 increase emphasizing a regulatory mechanism between POD-1 and SF-1. By ChIP assay it was shown that POD-1 binded in SF-1 promoter E-box sequence. It was not characterized that POD-1 binded in LRH-1 promoter, although POD-1 can bind in SHP promoter E-box sequence. The reduction of SF-1 expression by POD-1 has decreased the StAR expression, however, it was not enough to change tumor cell proliferation. In summary, POD-1 must have a wider role as regulator of fator transcription which controls tumorigenese process being a possible candidate as tumor supressor gene in adrenocortical cells.

SHP-2 est une tyrosine phosphatase impliquée dans la signalisation intracellulaire déclenchée par des facteurs de croissance, des cytokines pro-inflammatoires et des produits bactériens. Bien que cette phosphatase soit exprimée de manière ubiquiste et donc dans l’épithélium intestinal, son rôle dans ce tissu n'était pas connu. Afin de mieux comprendre les rôles joués par cette phosphatase dans l’intestin, nous avons généré un modèle murin de délétion conditionnelle de Shp-2 spécifiquement dans les cellules épithéliales intestinales (SHP-2[indice supérieur CEI-KO]). Nos résultats montrent que dès l'âge de 1 mois, toutes les souris expérimentales ont développé spontanément de l'inflammation au niveau du côlon. En fait, dans les cellules épithéliales intestinales, SHP-2 contrôle le niveau d’activation d’effecteurs de signalisation importants tels que les kinases ERK1/2 de même que les facteurs de transcription NFκB, STAT3 et β-caténine. En modulant ces voies de signalisation, SHP-2 contrôle des processus cellulaires primordiaux pour le maintien de l’homéostasie intestinale: la détermination des cellules à mucus et des cellules de Paneth, la composition de la flore, la perméabilité paracellulaire et la restitution épithéliale. La dérégulation de ces processus cellulaires peut expliquer l'apparition rapide d'inflammation colique chez les souris SHP-2C[indice supérieur EI-KO]. De plus, l'inflammation chronique observée chez les souris SHP-2[indice supérieur] CEI-KO entraîne avec l'âge le développement de cancer colorectal associé à la colite. Finalement, nos résultats chez l'humain montrent qu'il y a une diminution significative d'expression de SHP-2 chez les patients atteints de maladies inflammatoires intestinales comparativement aux patients témoins. Également, deux polymorphismes de PTPN11 sont retrouvés préférentiellement chez les patients atteints de colite ulcéreuse. En conclusion, nos résultats démontrent que la phosphatase SHP-2 protège l'épithélium intestinal contre l'inflammation et le cancer colorectal associé à la colite.