One of the most frequently reported deficits seen in individuals with Fetal Alcohol Spectrum Disorder (FASD) is impairments in learning and memory, which is likely attributed to the teratogenic effects of ethanol on the developing hippocampus. TGF-β (transforming growth factor-β), hedgehog and Wnt signaling pathways have been identified as high probability candidate pathways associated with brain deficits seen in FASD. Increasing evidence indicates that ethanol may induce changes in DNA methylation that could alter transcription regulating factors within signaling pathways critical in brain development. The purpose of this study was to test the hypotheses that prenatal ethanol exposure during i) the first trimester-equivalent period, or ii) throughout the entire gestational period induces changes in DNA methylation and alters the transcription/translation of TGF-β1, SHH (sonic hedgehog) and Wnt3a within the developing hippocampus. Pregnant Dunkin-Hartley-strain guinea pigs were assigned to one of three groups: ethanol (4 g/kg maternal body weight), isocaloric-sucrose/pair-feeding, or no treatment. Embryonic telencephalon tissue (which gives rise to the hippocampus) and fetal hippocampus were collected at gestational day (GD) 23 or GD 65, respectively. GD 23 ethanol-exposed and nutritional control embryos exhibited decreased crown-rump and head lengths. GD 65 ethanol-exposed fetuses exhibited decreased body and brain weights compared with the control groups. Ethanol exposure during the first trimester-equivalent period, but not during the entire gestational period, resulted in an increase in global DNA methylation. First trimester-equivalent ethanol exposure did not alter TGF-β1, SHH and Wnt3a gene expression within the GD 23 telencephalon. However, ethanol exposure throughout the entire pregnancy led to an increase in the expression of all three genes within the GD 65 hippocampus. No change in TGF-β1 protein was seen in the hippocampus of ethanol-treated fetuses. Post-translationally modified (ptm) SHH, but not unmodified SHH protein, was decreased in the hippocampus of ethanol-exposed fetuses. A decrease in unmodified, but not ptm Wnt3a protein, was observed in both ethanol-exposed and nutritional control hippocampus. These results suggest that prenatal ethanol exposure may affect hippocampal development through alterations in i) DNA methylation as shown at early gestation and ii) the expression of transcription regulating factors, especially SHH, as shown at term. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-03 12:36:33.035

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23768号 / 医博第4814号 / 新制||医||1056(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 武藤 学, 教授 松田 道行, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sleep fragmentation induces acute inflammation and increases glucocorticosteroids in vertebrates. Obesity and sleep fragmentation are often concurrent pro-inflammatory conditions in patients with obstructive sleep apnea. Despite the association between the two, their simultaneous effects on immune and endocrine profiles have not been explored. In the first experiment, we investigated changes in proinflammatory (IL-1β, TNF-α) and anti-inflammatory (TGF-β1) cytokine gene expression in the periphery (liver, spleen, fat, and heart) and brain (hypothalamus, prefrontal cortex, and hippocampus) in mice exposed to various intervals of sleep fragmentation. Serum corticosterone concentration was also assessed. Sleep was disrupted in male C57BL/6J mice using an automated sleep fragmentation chamber (Lafayette Industries), which involved movement of a sweeping bar at specified intervals. Mice were exposed to bar sweeps every 20 sec (high sleep fragmentation; HSF), 120 sec (low sleep fragmentation; LSF), or the bar remained stationary (control). Trunk blood and tissue samples were collected after 24 h of SF. It was found that HSF is a potent inducer of inflammation in the periphery (IL-1β: adipose, heart, and hypothalamus), but leads to upregulation of antiinflammatory cytokines in the brain (TGF-β1: hypothalamus and hippocampus), despite elevated serum corticosterone. Due to the association between obesity and SF, this experiment was replicated in male C57BL/6J mice (lean) and ob/ob KO mice (obese) using the previously described methods. We predicted the acute inflammatory response resulting from HSF would be different for the lean and the obese mice, with the greatest cytokine gene expression levels in the OB HSF group, due to a summative effect of the pro-inflammatory conditions. Obesity was the factor that most affected cytokine gene expression profiles. Additionally, the pro- vs anti-inflammatory gene expression profile varied with tissue type. While obesity resulted in neuroinflammation (hypothalamus, prefrontal cortex, hippocampus), it led to decreases in pro-inflammatory cytokine gene expression in the periphery (spleen, fat, heart). Serum corticosterone concentration was significantly elevated due to SF, but was not affected by obesity. As a result, the obese mice likely had neuroendocrine adaptations to combat the pre-existing pro-inflammatory condition of obesity, which impacted the acute inflammatory response to sleep loss.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2015-10-19T12:42:59Z No. of bitstreams: 1 Dissertação_ICS_ Patricia Seixas Auad Brandão.pdf: 3513376 bytes, checksum: 9371f172f7be035f0e53b69538f14768 (MD5) / Made available in DSpace on 2015-10-19T12:42:59Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Patricia Seixas Auad Brandão.pdf: 3513376 bytes, checksum: 9371f172f7be035f0e53b69538f14768 (MD5) / A hepatite crônica C é um processo inflamatório persistente, de duração superior a seis meses e o seu tratamento é realizado com o uso de interferons associados à ribavirina. Fatores como o genótipo do vírus, a carga viral e características do hospedeiro estão envolvidos com o sucesso da terapia. Os fatores do hospedeiro, especialmente, os imunológicos e genéticos parecem ter um papel importante no resultado do tratamento da hepatite C. Algumas proteínas como a citocina TGF-β1 tem sido associada com as complicações decorrentes de produção excessiva de fibrose, associada com condições inflamatórias crônicas. Objetivo: O objetivo deste estudo foi avaliar a associação de polimorfismos genéticos da citocina TGF-β1 e o estágio da fibrose na hepatite crônica C. Métodos: Este é um estudo de caso-controle não pareado, onde foram avaliados pacientes monoinfectados com vírus da hepatite C (VHC) tratados com terapia combinada com interferon convencional e/ou peguilado associado a ribavirina, os quais foram divididos em dois grupos, de acordo com os estágios de fibrose: fibrose hepática leve a moderada < F2 e fibrose hepática avançada F3 e F4 e um grupo controle de uma amostra histórica de doadores de sangue. Sendo realizada a genotipagem da TGF-β1 pelo método PCR-SSP (Kit One Lambda). Resultados: Foram encontradas diferenças estatisticamente significantes entre o grupo de indivíduos com hepatite crônica C e doadores de sangue. Houve maior frequência do alelo G (p= 0,0021) nos indivíduos com hepatite crônica C como também observamos maior frequência do genótipo GG (p=0,0029) entre os indivíduos com hepatite crônica C. Conclusões: Os dados obtidos sugerem que os polimorfismos do gene TGFB1 nos códons 10 e 25 não estão associados com a fibrose hepática em indivíduos com hepatite crônica C e que o polimorfismo do gene TGFB1 no códon 25 tem maior frequência nos indivíduos com hepatite crônica C quando comparados com indivíduos sadios, podendo ser um marcador de susceptibilidade a infecção pela hepatite crônica C.

Submitted by Israel Vieira Neto (israel.vieiraneto@ufpe.br) on 2015-03-05T18:31:18Z No. of bitstreams: 2 Dissertação sergio ferreira.pdf: 495221 bytes, checksum: 34587eb69a01f6ac2b83ff92569f588d (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T18:31:18Z (GMT). No. of bitstreams: 2 Dissertação sergio ferreira.pdf: 495221 bytes, checksum: 34587eb69a01f6ac2b83ff92569f588d (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / CAPES, CNPq / O câncer cervical (CC) é o segundo tipo de câncer mais comum a afetar mulheres em todo mundo. O Papillomavírus humano (HPV) é encontrado em 99% dos casos de CC e a infecção por esse vírus é considerado um fator de risco para o desenvolvimento do câncer. Muitos estudos tem demonstrado uma relação entre polimorfismos nos genes de citocinas e doenças infecciosas. Polimorfismos nos genes da Interleucina-6 (IL-6) e o Fator de Crescimento Transformador (TGF) β1, importantes mediadores do sistema imunológico, tem sido associados com níveis séricos elevados destas citocinas e no desenvolvimento de muitas doenças e tipos de cânceres. O objetivo desse estudo foi verificar se o SNP -174G/C do gene da IL-6 e T869C e G915C do gene do TGF-β1 estão relacionados com o desenvolvimento de Neoplasias Intraepiteliais Cervicais (NIC). 115 amostras de pacientes saudáveis e 115 de pacientes com lesões foram analisadas. As análises dos SNP foram realizadas através do sequenciamento automático de DNA utilizando o “MEGABACE 1000”. Os genótipos do polimorfismo -174G/C da IL-6 que possuem pelo menos um alelo C parecem estar envolvidos no desenvolvimento de NIC induzida pelo HPV (p=0.05232). Nenhuma diferença significativa foi encontrada entre as frequências alélicas e genotípicas dos polimorfismos da TGF-β1 nos dois grupos analisados. Além disso, polimorfismos nos genes da IL-6 e TGF-β1 não estão envolvidos na progressão do CIN. Este estudo sugere que o polimorfismo -174G/C do gene da IL-6 pode ser usado como um gene marcador da susceptibilidade a infecção pelo HPV, mas não como um marcador de progressão de NIC na população Pernambucana.

Activation of cardiac fibroblasts is a key event in the progression of cardiac fibrosis that leads to heart failure. However, the molecular mechanisms underlying mechanical stress-induced cardiac fibroblast activation are complex and poorly understood. This study demonstrates that Pellino1, an E3 ubiquitin ligase, was activated in vivo in pressure overloaded rat hearts and in cultured neonatal rat cardiac fibroblasts (NRCFs) exposed to mechanical stretch in vitro. Suppression of the expression and activity of Pellino1 by adenovirus-mediated delivery of shPellino1 (adv-shpeli1) attenuated pressure overload-induced cardiac dysfunction and cardiac hypertrophy and decreased cardiac fibrosis in rat hearts. Transfection of adv-shpeli1 also significantly attenuated mechanical stress-induced proliferation, differentiation and collagen synthesis in NRCFs. Pellino1 silencing also abrogated mechanical stretch-induced polyubiquitination of tumor necrosis factor-alpha receptor association factor-6 (TRAF6) and receptor-interacting protein 1 (RIP1) and consequently decreased the DNA binding activity of nuclear factor-kappa B (NF-κB) in NRCFs. In addition, Pellino1 silencing prevented stretch-induced activation of p38 and activator protein 1 (AP-1) binding activity in NRCFs. Chromatin Immunoprecipitation (ChIP) and luciferase reporter assays showed that Pellino1 silencing prevented the binding of NF-κB and AP-1 to the promoter region of transforming growth factor-β1 (TGF-β1) thus dampening TGF-β1 transactivation. Our data reveal a previously unrecognized role of Pellino1 in extracellular matrix deposition and cardiac fibroblast activation in response to mechanical stress and provides a novel target for treatment of cardiac fibrosis and heart failure.

Les cellules souches de l’épiderme interfolliculaire humain, appelées cellules souches kératinocytaires (CSK), assurent l’homéostasie et le renouvellement du tissu durant toute la vie d’un individu grâce à leur importante capacité d’autorenouvellement. Ma thèse de doctorat a porté sur l’étude des effecteurs moléculaires impliqués dans la balance entre prolifération et quiescence dans un modèle in vitro de CSK, isolées de manière clonale et définies par le terme holoclone. Je me suis tout d’abord intéressée à la réponse des holoclones à l’effet d’un régulateur important de la prolifération et de la quiescence des cellules souches adultes : le facteur de croissance TGF β1. Mon travail s’est ensuite focalisé sur l’étude d’un gène agissant en aval de la voie de signalisation TGF β, le facteur de transcription Klf4, et dont le rôle dans la biologie des cellules souches adultes reste largement méconnu. Klf4 est en effet surtout décrit pour son rôle dans la reprogrammation des cellules somatiques en cellules iPS. Le maintien de la sensibilité des holoclones aux signaux inhibiteurs de la croissance constitue un gage de la normalité des CSK. Notre étude de la réponse des holoclones à l’effet antiprolifératif du TGF β1 montre que les holoclones, dotés d’un fort potentiel de croissance, caractéristique des CSK, conservent leur sensibilité à l’effet du TGF β1. Ces résultats nous ont permis de valider les holoclones comme constituant un modèle pertinent pour caractériser la biologie normale des CSK et décrypter les contrôles moléculaires de l’état souche. Le modèle des holoclones a été exploité dans le cadre d’une approche de génomique fonctionnelle visant à déterminer le rôle du facteur de transcription Klf4 dans les CSK. L’utilisation de vecteurs lentiviraux exprimant un shARN dirigé contre l’ARNm de Klf4 nous a permis d’étudier l’impact d’une modulation fine du niveau d’expression de Klf4 sur les propriétés des holoclones. La répression transcriptionelle de Klf4, d’environ un facteur 2, favorise de manière significative l’expansion du compartiment clonogénique au sein des holoclones. Ce gain de fonction concerne à la fois les potentiels de croissance et de reconstruction épidermique des holoclones. Un aspect important de ce travail a concerné la recherche des réseaux moléculaires régulés par Klf4 dans les holoclones. Une analyse du transcriptome nous a permis de montrer que Klf4 participe au contrôle des mécanismes de cycle cellulaire et de différenciation. Klf4 interviendrait également dans la régulation des voies de signalisation TGF β/BMP et Wnt, connues pour exercer des rôles clés dans la biologie des cellules souches. Klf4 constituerait donc un censeur de l’activité du compartiment immature dans l’épiderme interfolliculaire. Il participerait aux mécanismes de régulation du cycle cellulaire et serait susceptible d’intervenir dans le contrôle de l’autorenouvellement du compartiment souche. / Stem cells present within the human interfollicular epidermis, which are defined as keratinocytes stem cells (KSC), ensure the homeostasis and renewal of the tissue throughout the whole individual life. These functions are related to their important self-renewal capacity. My PhD project was focused on the knowledge of the molecular effectors involved in the control of the balance between proliferation and quiescence in KSC. This scientific question was investigated in an in vitro model of KSC which were clonally derived and characterized as holoclones. Holoclones are controlled by mitogenic growth factors and also by antiproliferative signals. One of these regulators is the growth factor TGF β1 which plays an important role in the control of quiescence and cell proliferation within several adult stem cell systems. In the context of growth inhibition by TGF β1, I have studied the role of a downstream gene of the TGF β pathway, the transcription factor Klf4, whose role in adult stem cell biology remains unclear. In fact, Klf4 is mostly described for its involvement in the reprogramming process of somatic cells into iPS cells. The maintenance of holoclone sensitivity to cell growth inhibitors is a critical parameter of KSC normal physiology. Holoclones possess an extensive growth capacity, which is characteristic of KSC. Despite this high proliferation rate, holoclones are still responsive to the antiproliferative effect of TGF β1. These results allowed us to validate the use of holoclone as a relevant model of non-transformed KSC suitable for the characterization of the role of candidate stemness genes in KSC biology, such as Klf4. The holoclone model was exploited to perform a functional genomic approach to investigate the role of Klf4 in KSC. We have developed a shRNA-based gene knock-down method using lentiviral vectors to assess the impact of Klf4 down-modulation on holoclone functional properties. Our results show that Klf4 down modulation controls the expansion of the clonogenic compartment present within holoclone progeny. This gain-of-function, which is maintained at the long term level, leads to an increase in holoclone 3D epidermis reconstruction capacity. A major point of this project was to elucidate the molecular networks controlled by Klf4 in holoclones. Microarray data show that Klf4 regulates the expression of several genes related to pathways involved in the control of stem cell fate. In particular, we identified many transcripts related to TGF β/BMP and Wnt signallings. Interestingly, the majority of the modulated transcripts are involved in the regulation of cell cycle and in keratinocyte differentiation process. All together these results suggest a critical role Klf4 as a stemness censor of the most immature compartment activity. Klf4 is likely to be involved in cell cycle regulation of KSC compartment and in the control of KSC self-renewal process.

La myélofibrose primitive (MFP) est un néoplasme myéloprolifératif chromosome Philadelphie négatif rare, mais de pronostic sévère. Elle se caractérise par une prolifération clonale et une mobilisation des cellules souches et progéniteurs hématopoïétiques (CSH/PH) de la moelle osseuse vers la rate et le foie. Cette anomalie de l’hématopoïèse est associée à une pathologie du stroma (myélofibrose, ostéosclérose et néoangiogenèse). L’existence d’anomalies moléculaires de la CSH/PH telles que les mutations de Jak2, Mpl, TET2 ou CALR ne permet pas à elle seule d’expliquer la physiopathologie de la maladie. Les résultats obtenus dans le laboratoire suggèrent que le microenvironnement médullaire au sein des niches hématopoïétiques et en particulier les cellules stromales mésenchymateuses (CSM), participe vraisemblablement à cette dérégulation de l’hématopoïèse, favorisant le développement du clone pathologique. Cependant, aucune preuve tangible d’une altération des CSM médullaires n’a été jusqu’à présent apportée.Dans ce travail, nous avons isolé les CSM de la moelle de patients atteints de MFP et réalisé une caractérisation « complète » de ces cellules : prolifération, phénotype, soutien de l’hématopoïèse, sécrétome, transcriptome, miRNome et capacités de différenciation. Nos résultats ont permis de dégager un faisceau d’arguments en faveur d’une dérégulation de leur différenciation ostéoblastique (DOB). (i) Les cytokines BMP2, RANTES, PDGF, TGF-β1, VEGF et Il-6 sont significativement produites en plus grande quantité par ces cellules. (ii) L’étude du transcriptome a révélé une expression significativement différente d’un ensemble de gènes impliqués dans la DOB tels que RUNX2, DLX5, TWIST1 et NOGGIN. (iii) De nombreux micro-ARN, dont certains sont connus pour être impliqués dans la DOB comme miR-210 ou dans le nichage des cellules souches hématopoïétiques comme miR-34a, sont dérégulés à l’état basal et au cours de cette DOB. (iv) Enfin, l’étude de leurs capacités de différenciation ostéoblastique in vitro et in vivo chez la souris immunodéprimée est en faveur d’une augmentation de ces capacités. Nous avons étudié l’impact du TGF- β1 dans cette DOB. Nous avons mis en évidence que les CSM de malades présentent un état basal d'activation de la voie de signalisation pSmad significativement augmenté, confirmant l’expression endogène de TGF-β1. En utilisant des inhibiteurs spécifiques du récepteur de type I au TGF- β, nous avons montré l’implication de cette cytokine dans les altérations de la DOB. En conclusion, notre travail montre pour la première fois que les CSM des malades de MFP sont anormales et ce indépendamment de la stimulation par le clone hématopoïétique pathologique, suggérant la présence d'anomalies constitutives ou acquises. Ces anomalies impliquent deux acteurs majeurs de la pathologie : le TGF-β1 et l'ostéogenèse. / Primary myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm, rare but associated with a poor prognosis. Its features are a clonal proliferation and an egress of hematopoietic stem cells (HSC) from bone marrow to spleen. These abnormalities of hematopoiesis are in relation with a pathological stroma (myelofibrosis, osteosclerosis and neoangiogenesis). Molecular abnormalities present in HSC partially explain the physiopathology of the disease. Results from our lab suggest that the bone marrow micro-environnement, especially mesenchymal stromal cells (MSC), are involved in the deregulation of hematopoiesis, promoting the clonal cells. However, there is no strong evidence of bone marrow MSC alterations reported for now.In our study, we isolated MSC from bone marrow of patients suffering from PMF and performed a broad characterization: proliferation, phenotype, hematopoiesis supporting capacities, secretome, transcriptome and miRNome analysis. Our results highlight arguments in favor of a deregulation of their osteogenic capacities. (i) Cytokines NMP2, RANTES, PDGF, TGF-β1, VEGF and Il-6 were significantly overproduced by MSCs. (ii) Transcriptome analysis revealed a specific signature involving genes participating in osteogenic differentiation such as RUNX2, DLX5, TWIST1 and NOGGIN. (iii) Many micro-RNAs, some know to be involved in osteogenic differentiation regulation, as mir-34a, are deregulated in MSCs and in MSC-derived osteoblasts. (iv) Finally, study of their osteogenic potency in vitro and in vivo in nude mice showed an increasing of their osteogenic potency. We studied the impact of TGF-β1 in this process and showed that PMF MSCs showed a basal expression of Smad pathway significantly increased as compared to control. Using specific inhibitor of TGF-β1 receptor, we demonstrated the implication of this cytokine in the osteogenic impairment.To summarize, our work shows for the first time that MSCs from PMF patients are abnormal, independently from stimulation by clonal cells, suggesting intrinsic abnormalities. These abnormalities involve two main factor of the disease: TGF-β1 and osteogenesis.

L’élucidation des mécanismes permettant l’optimisation de la réponse immunitaire antitumorale correspond à un enjeu majeur pour le développement de stratégies d’immunothérapie efficace. En effet, les réponses immunitaires antitumorales se traduisent rarement par l’éradication de la tumeur. Dans ce contexte, les travaux antérieurs de mon équipe ont démontré que l’interaction de l’intégrine αE(CD103)β7, souvent exprimée par les lymphocytes infiltrant la tumeur (TIL), avec son ligand E-cadhérine, à la surface des cellules tumorales épithéliales, joue un rôle majeur dans la potentialisation de l’activité lytique des cellules T en induisant la polarisation et l’exocytose des granules cytotoxiques. Nos résultats ont indiqué aussi que le TGF-β1, souvent abondant dans les tumeurs, joue un rôle déterminant dans cette induction suite à l’engagement du récepteur des cellules T. Dans ce contexte, nous avons cherché à comprendre les mécanismes de régulation du gène ITGAE qui codent la sous-unité alphaE de l’intégrine CD103. Nos résultats ont montré que les facteurs transcriptionnels Smad2, Smad3 et NFAT-1 sont impliqués dans la régulation de l’expression de la sous-unité αE(CD103). En effet, une costimulation avec du TGF-β1 recombinant et un anticorps anti-CD3 d’un clone T CD103- induit l’expression de cette intégrine qui est accompagnée d’une translocation dans le noyau de Smad2, Smad3 et NFAT-1 qui sont cytoplasmiques à l’état basal. L’inhibition spécifique de ces facteurs transcriptionnels inhibe l’expression de CD103 et abroge le potentiel lytique du clone T vis à vis de sa cible tumorale autologue. De plus, nous avons identifié deux séquences régulatrices du gène ITGAE humain, un promoteur proximal et un enhancer. Par ailleurs, mon équipe a récemment montré que l’interaction de CD103 à la surface des TIL avec une molécule E-cadhérine recombinante est suffisante pour induire la polarisation des granules cytolytiques par un mécanisme dépendant de la PLC-g1 et ERK et que cette intégrine possède non seulement une fonction d’adhérence, mais aussi une fonction de costimulation du signal TCR des TIL antitumoraux. Nous avons cherché à mieux comprendre la signalisation de l’intégrine CD103, en identifiant les domaines intracytoplasmiques de la sous-unité αE impliqués dans son activation. Nous avons ainsi construit une protéine de fusion CD103-GFP et plusieurs mutants du domaine intracytoplasmique de la sous-unité αE qui ont été ensuite transfectés dans la lignée Jurkat Tag CD103-/beta7+. Nos résultats ont montré que le domaine intracytoplasmique de la chaîne alphaE n’est pas nécessaire à la reconnaissance du ligand, la E-cadhérine. Par contre, nous avons montré que ce domaine est impliqué dans le phénomène de clustering de l’intégrine et dans sa polarisation à la zone de contact avec des billes couvertes avec la E-cadhérine-Fc. Nous avons identifié un domaine de 8 acides aminés (ESIRKAQL), contenant une sérine en position 1163 potentiellement phosphorylable, et qui est indispensable pour la signalisation de l’intégrine. De plus, nos travaux ont montré que ce domaine ESIRKAQL, est nécessaire pour la phosphorylation de la ERK1/2 et PLC-g1. Ainsi, une meilleure compréhension des mécanismes moléculaires régulant les fonctions de CD103 pourrait contribuer au développement et à l’amélioration de la réponse antitumorale exercée par les CTL. / The elucidation of mechanisms for optimizing the antitumor immune response is a major challenge for the development of strategies for effective immunotherapy. Indeed, the anti-tumor immune responses rarely result in the eradication of the tumor. In this context, the previous work of my team have shown that the interaction of integrin αE(CD103)β7, often expressed by tumor infiltrating lymphocytes (TIL) with its ligand E-cadherin at the cell surface tumor epithelial cells, plays a major role in the potentiation of the lytic activity of T cells by inducing polarization and exocytosis of cytotoxic granules. Our results also indicated that TGF-β1, often abundant in tumors, plays a key role in the induction due to the commitment of the T cell receptor. In this context, we sought to understand the mechanisms regulating ITGAE gene encoding the subunit αE of integrin. Our results showed that the transcription factors Smad2, Smad3 and NFAT-1 are involved in regulating the expression of subunit αE(CD103)β7. Indeed, costimulation with recombinant TGF-β1 and anti-CD3 antibody induces on T cell clone CD103- the expression of this integrin ant the translocation into the nucleus of Smad2, Smad3 and NFAT-1 that are cytoplasmic at baseline. Specific inhibition of these transcription factors inhibits the expression of CD103 and repeals the lytic potential of cloned T with respect to the autologous tumor target. In addition, we identified two regulatory sequences of human ITGAE gene, proximal promoter and enhancer. In addition, my team has recently shown that the interaction of CD103 on the surface of TIL with a recombinant molecule E-cadherin is sufficient to induce the polarization of cytolytic granules by ERK and PLC-γ1 pathway thus this integrin has not only a function of adherence, but also a function of costimulatory signal TCR of TIL. We sought to better understand the signaling of integrin CD103, by identifying the cytoplasmic domains of the subunit αE involved in its activation. We have constructed a fusion protein CD103-GFP and several mutants of intracytoplasmic domain of the subunit αE which were then transfected into the Jurkat Tag cell line CD103-/ β7+. Our results showed that the intracytoplasmic domain of CD103 is not necessary for ligand recognition, E-cadherin. By cons, we have shown that this area is involved in the phenomenon of clustering of integrin and its polarization to the contact area with balls covered with E-cadherin-Fc. We have identified a range of 8 amino acids (ESIRKAQL) containing a potentially phosphorylatable serine in position 1163, which is essential for integrin signaling. In addition, our work has shown that this area ESIRKAQL is necessary for the phosphorylation of ERK1/2 and PLC-g1. Thus, a better understanding of the molecular mechanisms that regulate the functions of CD103 may contribute to the development and improvement of the antitumor response exerted by CTL .

La fibrose radio-induite est une complication sévère et tardive de la radiothérapie. Plusieurs études cliniques ont montré que la combinaison pentoxifylline-vitamine E est un traitement sûr et efficace contre la fibrose. Cependant, les mécanismes moléculaires de son efficacité restent inexplorés. Nous avons montré l’efficacité de la combinaison pentoxifylline-vitamine E dans l’entéropathie radique dans une faisabilité clinique. En parallèle, en utilisant un modèle unique, in vitro, de cellules musculaires lisses intestinales primaires isolées des personnes atteintes de l’entéropathie radique, nous avons montré une synergie entre la pentoxifylline et l’analogue hydrophile de vitamine E (trolox) qui permet à l’association d’inhiber l’expression de TGF-β1 au niveau de l’ARN messager et de la protéine. Cette action inhibitrice intervient au niveau transcriptionnel et conduit à une inhibition conséquente des cibles de la voie de signalisation TGF-β1/Smad (Col Iα1, FN1, PAI-1, CTGF), alors qu’elle semble sans effet sur la voie de signalisation Rho/ROCK. Pour la première fois, dans ces cellules issues de l’entéropathie radique, nous avons montré une surexpression de miR-210 ; microRNA induit par l’hypoxie. L’association pentoxifylline-trolox inverse la surexpression de miR-210 aussi bien dans les conditions normoxique que dans les conditions hypoxiques. L’implication de miR-210 dans l’entéropathie radique n’a pas été préalablement étudiée, néanmoins nous avons montré qu’un inhibiteur de miR-210 diminue l’expression de Col Iα1 dans ce modèle. L’effet anti-fibrosant exercé par l’association pentoxifylline-vitamine E est partiellement induit par l’inhibition de la cascade TGF-β1. L’inhibition de miR-210 est un deuxième mécanisme potentiel nécessitant d’autres investigations. Cette étude renforce les essais clinique antérieurs en montrant in vitro une synergie entre pentoxifylline et vitamine E et permettant de proposer cette association en première ligne thérapeutique dans la fibrose radio-induite. De plus, miR-210 est proposé comme une possible cible thérapeutique pour traiter la fibrose radio-induite. / Radiation-induced fibrosis is a serious late complication of radiotherapy. Pentoxifylline-vitamin E has proven effective and safe in clinical trials as treatment of fibrosis, while the molecular mechanism of its activity is yet unexplored. We showed efficacy of Pentoxifylline-vitamin E combination in radiation-induced enteropathy in a small clinical study. In parallel, using a unique in vitro model of primary smooth muscle cells isolated from intestinal samples isolated from humans with radiation enteropathy we showed that pentoxifylline and the hydrophilic analogous of vitamin E (trolox) synergize to inhibit TGF-β1 protein and mRNA expression. This inhibitory action is mediated at the transcriptional level and leads to subsequent inhibition of TGF-β1/Smad targets (Col Iα1, FN1, PAI-1, CTGF), while it has no effect on the Rho/Rock pathway. We have also demonstrated, for the first time, an overexpression of the hypoxia-induced microRNA miR-210 in the fibrotic cells. Pentoxifylline-trolox combination could reverse this miR-210 overexpression in normoxic and hypoxic conditions. While miR-210 has not been previously shown to be involved in radiation-induced enteropathy, we showed that miR-210 inhibitor could reduce mRNA expression of Col Iα1. The anti-fibrotic effect of combined pentoxifylline-vitamin E is at least in part mediated by inhibition of the TGF-β1 cascade. MiR-210 inhibition is another mechanism which needs further investigations. This study strengthens previous clinical data showing pentoxifylline-vitamin E synergy and supports its use as a first-line treatment of radiation-induced fibrosis. Also, it suggests miR-210 as a new potential therapeutic target for the treatment of this complication.