Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois marcadores próximos à mutação c.424_427del da STAT5B nos pacientes homozigotos para a mesma, em 33 indivíduos heterozigotos de sete famílias independentes e em 53 indivíduos controles. O mesmo haplótipo estava presente nos pacientes homozigotos para a mutação e em todos os heterozigotos, enquanto em apenas 9,4% dos controles (p < 0,001), apontando a probabilidade de que a mutação c.424_427del nas diferentes famílias tenha sido herdada de um antepassado em comum. Para avaliar o efeito da mutação c.424_427del da STAT5B em heterozigose, foram comparados em conjunto os 33 indivíduos heterozigotos e os 38 familiares não portadores em relação à altura e a alguns exames laboratoriais (gerais e hormonais). Os indivíduos heterozigotos foram significativamente mais baixos, com uma redução na altura de 0,6 desvios-padrão (p= 0,006). Também apresentaram redução significativa dos desvios-padrão de fator de crescimento insulina-símile 1 (IGF-1) e da proteína 3 de ligação a fator de crescimento insulina-símile (IGFBP-3), sem alteração em outros exames. Esses achados mostram que as mutações na STAT5B em heterozigose causam um impacto negativo significativo na altura, mais leve que o visto em pacientes com mutações em homozigose, com altura dentro da variação normal. Esse resultado favorece a hipótese de que variantes patogênicas raras em heterozigose contribuem para a variabilidade da altura normal / Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous for STAT5B c.424_427del mutation, in 33 heterozygous individuals from seven unrelated families and in 53 control individuals. The same haplotype was present in the homozygous boys and in all heterozygous individuals, while in only 9,4% control individuals (p < 0,001), pointing to the probability that STAT5B c.424_427del mutation in different families has been inherited from a common ancestor. To study the effects of heterozygous STAT5B c.424_427del mutation, 33 heterozygous individuals were compared to 38 non-carrier relatives on height and some laboratorial tests. Heterozygous individuals were significantly shorter than their noncarrier relatives, with a height reduction of 0.6 standard deviation scores (p= 0,006). Furthermore, they had a significant reduction in insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) standard deviation scores, without differences in the other exams. These findings show that heterozygous STAT5B mutations cause a significant negative impact on height, milder than the effect seen in patients with homozygous mutations, with height within the normal range. This result favors the hypothesis that rare pathogenic variants in heterozygous state contribute to normal height variability

Les adénomes hépatocellulaires (AHC) sont des tumeurs hépatiques bénignes rares se développant chez la femme jeune suite à la prise de contraceptifs oraux et pouvant se compliquer d’hémorragie et de transformation maligne en carcinome hépatocellulaire (CHC). Une classification génotype/phénotype a mise en évidence trois groupes d’AHC : les AHC inactivés pour le facteur de transcription HNF1A, les AHC mutés pour la β-caténine et les AHC dit « inflammatoires » ayant une activation de la voie JAK/STAT. Nous avons identifiés des mutations activatrices du gènes GNAS, codant pour la sous unité alpha de la protéine Gs, dans un sous-groupe d’AHC inflammatoires ainsi que chez des patients avec des AHC et atteints d’un syndrome de McCune Albright, une maladie rare combinant des tumeurs endocriniennes, une dysplasie fibreuse osseuse et des taches cutanés café au lait. Cette découverte confirme les interactions entre la voie de l’AMP cyclique induite par les mutations GNAS et la voie JAK/STAT. Les CHC sont les tumeurs primitives du foie les plus fréquentes, survenant souvent sur un foie cirrhotique exposé à différents facteurs de risque comme l’hépatite B chronique, l’hépatite C chronique, l’alcool ou le syndrome métabolique. Le CHC est le résultat de l’accumulation d’altérations génétiques et épigénétiques. Premièrement, nous avons identifiés les mutations du promoteur de TERT (Telomerase reverse transcriptase) comme les altérations génétiques somatiques les plus fréquentes des CHC. Ces mutations ont été aussi retrouvées dans des lésions prénéoplasiques développées sur cirrhose suggérant leurs rôles précoces dans l’initiation tumorale et la transformation maligne. A l’inverse l’étude des mutations du promoteur de TERT et la réalisation de séquençage haut-débit dans les AHC et les transformation d’adénome en CHC nous a permis de disséquer les mécanismes de transformation maligne sur foie sain avec la présence de manière précoce d’une mutation de la β-caténine et dans un second temps l’apparition d’une mutation dans le promoteur de TERT. Par la suite, nous avons mis en évidence une signature moléculaire pronostique transcriptomique chez les patients avec CHC traités par résection hépatique. Cette signature moléculaire prédisant à la fois la récidive tumorale et le décès a été validée dans des cohortes de patients à l’étranger. Enfin, nous avons mise en évidence le rôle oncogénique de l’adeno-associated virus de type 2 dans la survenue de CHC sur foie sain via un mécanisme de mutagénèse insertionnelle dans des gènes clés de la carcinogénèse comme TERT, CCNA2, MLL4 ou TNFSF10. Ces résultats ont permit de mettre en évidence de nouveaux facteurs de risque viraux de survenue du CHC, d’identifier de nouvelles altérations génétiques impliquées dans la transformation maligne sur cirrhose et sur foie sain et permit de développer une signature moléculaire pronostique qui pourrait être utiliser dans le futur comme une aide à la stratification thérapeutique chez les patients atteint de CHC. / Hepatocellular adenomas (HCA) are rare benign liver tumors occuring in young women taking oral contraception and complications as haemorrhage or malignant transformation in hepatocellular carcinomes (HCC) could occur. A genotype/phenotype classification has defined different subgroups of tumors : HCA with inactivating mutations of HNF1A, HCA with activating mutations of β-catenin and inflammatory HCA with activation of the JAK/STAT pathway. We have identified activation mutations of GNAS, that codes for the alpha subunit of the Gs protein in a subgroup of inflammatory HCA and in patients with HCA and McCune Albright syndrom, a rare disease that combined endocrine tumor, bone fibrous dysplasia and « cafe au lait » skin macula. These findings highlight the crosstalk between the cyclic AMP pathway induced by GNAS mutation with the JAK/STAT pathway. HCC are the most frequent primary liver tumors worldwide and mainly occur on cirrhosis due to various risk factor as hepatitis B and C virus, alcohol consumption and metabolic syndrome. HCC is due to the accumulation of genetic and epigenetic alterations in the malignant hepatocytes. We have identified TERT (telomerase reverse transcriptase) promoter mutations as the most frequent somatic genetic alterations in HCC. These mutations were also found in cirrhotic premalignant nodules underlying their role in tumor initiation and malignant transformation. In contrast, the study of the different steps of malignant transformation of HCA into HCC using next generation sequencing and TERT promoter screening have shown that activatiing mutation of β-catenin is an early genetic alteration whereas TERT promoter mutation is required in a second step to promote a full malignant transformation. We have also identified a prognostic molecular signature, the 5-gene score, in patients with HCC treated by liver resection. The 5-gene score predicts tumor recurrence and disease specific survival and has been validated in different cohorts of patients worldwide. Finally, we have shown that adeno-associated virus type 2 is involved in liver carcinogenesis on normal liver through insertional mutagenesis in key cancer genes as TERT, CCNA2, MLL4 and TNFSF10. These results have underlined a new oncogenic virus involved in HCC development, identified new genetic alterations involved in malignant transformation on cirrhosis and normal liver and a new prognostic molecular signature that will help to guide treatment of patients with HCC in the future.

No description available.

Les mécanismes cellulaires anti-prolifératifs, lesquels comprennent l’apoptose, aussi appelée la mort cellulaire programmée, l’arrêt transitoire du cycle cellulaire et la sénescence, permettent à la cellule de prévenir, en réponse à différents stress, l’accumulation de mutations pouvant conduire à une prolifération incontrôlée et, éventuellement, au développement d’une tumeur. La régulation de ces différents mécanismes requiert l’activation de protéines appelées des suppresseurs de tumeur, dont le principal est p53. p53 est un facteur de transcription dont la stabilisation et l’activation conduit à une hausse de l’expression de gènes directement impliqués dans l’arrêt de la prolifération. Au cours des dernières années, l’ensemble des travaux sur p53 ont permis de mettre en évidence la complexité de sa fonction, de même que la multitude de voies de signalisation et de protéines avec lesquelles il coopère pour maintenir l’intégrité du génome. De ce fait, l’étude des mécanismes d’activation de p53 est de mise pour la compréhension de sa régulation et, éventuellement, pour la prévention et l’élaboration de nouvelles stratégies de traitement contre le cancer. L’objet de cette thèse est la mise en évidence d’un mécanisme d’activation de p53 et de la sénescence par la protéine SOCS1, un suppresseur de la signalisation par les cytokines. Ce mécanisme implique une interaction directe entre les deux protéines, plus précisément entre le domaine SH2 de SOCS1 et le domaine de transactivation de p53. SOCS1 interagit également, au niveau de son SOCS Box, avec les kinases ATM et ATR de la voie du dommage à l’ADN de façon à faciliter la phosphorylation de p53 en sérine 15. Ainsi, en interagissant à la fois avec p53 et ATM/ATR, SOCS1 contribue à la stabilisation et à l’activation de p53. En accord avec ce modèle, l’inhibition de SOCS1 dans des fibroblastes humains normaux tend à diminuer le nombre de cellules sénescentes suite à l’expression de l’oncogène ca-STAT5A et à réduire l’accumulation nucléaire de p53 dans ces cellules. De la même façon, les lymphocytes T provenant de souris Socs1-/-Ifnγ-/- sont moins susceptibles d’entrer en apoptose que les lymphocytes provenant de souris Socs1+/+Ifnγ+/+, suite à une exposition à des radiations. Dans les deux contextes, on observe une baisse de l’expression des gènes cibles de p53, ce qui démontre que SOCS1 est impliquée dans l’activation de p53 in vivo. Cette thèse a également pour but de mettre en évidence l’implication de SOCS1 dans l’activation d’autres facteurs de transcription et, par le fait même, de démontrer qu’elle peut agir comme un régulateur plus général de la transcription. Une étude approfondie de l’interaction entre SOCS1 et p53 a permis de démontrer que le domaine de transactivation II de p53 (acides aminés 36-67) est suffisant pour l’interaction. Plus précisément, il semble que le tryptophane 53 (W53) et la phénylalanine 54 (F54) sont les principaux résidus impliqués. Une analyse structurale de ce domaine de p53 a conduit à l’identification d’un motif conservé dans plusieurs autres facteurs de transcription pourvus d’un domaine de transactivation acide, dont p63, p73 et E2F1. En accord avec ces résultats, SOCS1 est en mesure d’interagir avec chacune des deux protéines. Ainsi, la capacité de SOCS1 d’interagir et de réguler l’activité de p53 peut s’étendre à d’autres facteurs de transcription. En terminant, le mécanisme présenté dans cette thèse contribue à la compréhension de la régulation de p53, le principal suppresseur de tumeur de la cellule. De plus, il met en évidence une nouvelle fonction de SOCS1, laquelle était jusqu’alors essentiellement connue pour inhiber la voie de signalisation JAK/STAT. Ce nouveau rôle pour SOCS1 permet d’expliquer de quelle manière une activation aberrante de la signalisation par les cytokines peut déclencher la sénescence ou l’apoptose. Enfin, le fait que SOCS1 puisse réguler différents facteurs de transcription permet de la qualifier de régulateur général des facteurs de transcription composés d’un domaine de transactivation acide. / In response to different stress, three anti-proliferative mechanisms, namely apoptosis, also called programmed cell death, transient growth arrest and senescence, prevent the cells from cumulating mutations that can lead to uncontrolled proliferation and, eventually, to tumor development. Regulation of these mechanisms requires the activation of proteins called tumor suppressors. One of them, p53, is a transcription factor whose stabilization and activation lead to an increase in expression of genes directly implicated in cell cycle arrest. In the past years, studies about p53 showed how much its function is complex and with how many signaling pathways and proteins it cooperates to maintain genome integrity. Thus, studying the activation mechanisms of p53 is essential to understand its regulation and, thereby, to prevent tumor development and to elaborate new strategies for cancer treatment. The first aim of this thesis is to show a new activation mechanism of p53 and of senescence by the protein SOCS1, a suppressor of cytokine signaling. This mechanism implies a direct interaction between the two proteins, specifically between the SH2 domain of SOCS1 and the N-terminal transactivation domain of p53. SOCS1 also interacts with the DNA damage-regulated kinases ATM and ATR via its C-terminal domain, which contains a SOCS Box, to facilitate the phosphorylation of p53 on its serine 15. Thus, by interacting at the same time with p53 and ATM, SOCS1 contributes to stabilization and activation of p53. In accordance with this model, SOCS1 inhibition in human normal fibroblasts decreases the number of senescent cells in which the activated oncogene STAT5A is expressed and reduces p53 nuclear accumulation in these cells. In the same way, T cells from Socs1-/-Ifnγ-/- mice are less likely to undergo apoptosis than T cells from Socs1+/+Ifnγ+/+ mice, after exposure to γ radiation. In both contexts, the expression of p53 target genes is decreased, which indicates that SOCS1 is implicated in p53 activation in vivo. This thesis also aims to show the role of SOCS1 in the activation of other transcription factors and, thereby, to show that it can act as a more general regulator of transcription. A detailed study of the interaction between SOCS1 and p53 showed that the transactivation domain II of p53 (amino acids 36-67) is sufficient for the interaction. Specifically, it seems that tryptophan 53 (W53) and phenylalanine 54 (F54) are essential for the interaction. A structural analysis of this p53 region highlights an acid transactivation domain actually conserved in many others transcription factors, such as p63, p73 and E2F1. In accordance with this observation, SOCS1 is able to interact with both proteins. Thus, the capacity of SOCS1 to interact with p53 and to regulate its activity may extend to other transcription factors. The mechanism showed in this thesis contributes to the understanding of p53 regulation and highlights a new function for the SOCS1 protein. Indeed, until now, SOCS1 was mostly known to be a negative regulator of the JAK/STAT pathway. Moreover, this new role for SOCS1 explains how an aberrant cytokine signaling can trigger senescence or apoptosis. Finally, the fact that SOCS1 can regulate different transcription factors allows us to consider it as a general regulator of transcription factors containing an acid transactivation domain.

Le stroma inflammatoire joue un rôle primordial lors de la carcinogénèse. Dans ce contexte, nous montrons que la cytokine LIF est à l'origine d'une population de fibroblastes capable de remodeler la matrice extracellulaire de manière à la rendre permissive à l'invasion collective des cellules tumorales. En effet, nous montrons que la production de LIF par les cellules tumorales et fibroblastiques, après une stimulation au TGFβ, va réguler les capacités contractiles et pro-invasives de ces dernières via la régulation du cytosquelette d'acto-myosine et de manière indépendante de l'expression de α-SMA. En effet, l'inhibition pharmacologique des kinases JAKs permet de bloquer l'environnement fibrotique des tumeurs et d'ainsi bloquer l'invasion des cellules tumorales in vitro et in vivo. Nous montrons ensuite que LIF est à l'origine d'un switch épigénétique responsable de l'activation constitutive de la voie de signalisation JAK1/STAT3. Ce processus, régulé par la forme acétylée de STAT3, et son interaction avec l'ADN methyltransférase DNMT3b permet l'hypermethylation du promoter de la phosphatase SHP1 et donc la phosphorylation constitutive de JAK1. Une fois mis en place, ce nouveau profil de méthylation est maintenu par DNMT1. La surexpression de LIF dans les carcinomes humains corréle avec un environnement fibrotique, la présence de nodules invasifs et un mauvais pronostic clinique. De même, il existe une forte corrélation négative entre l'acétylation de STAT3 et l'expression de SHP1 dans le stroma tumoral. Nos résultats montrent qu'inhiber l'activité des DNMT et des kinases JAK permet de reprogrammer les capacités pro-invasive des fibroblastes associés aux carcinomes. / Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. We identify LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion. Indeed, pharmacological inhibition of JAK activity by counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. We next unveil that LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signaling, which results in sustained pro-invasive activity of fibroblasts. The process is mediated by p300-histone acetyltransferase acetylation of STAT3, and DNA methyltransferase DNMT3b, which induce the hypermethylation of SHP1 phosphatase promoter and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signaling is maintained by DNMT1. Accordingly, carcinomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Moreover, we show that STAT3 acetylation and phosphorylation are inversely correlated with SHP1 expression in tumors stroma. Combined inhibition of DNMT activities and JAK signaling results in long-term reversion of CAF-associated pro-invasive activity and restoration of the wild-type fibroblast phenotype.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:22:54Z No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:23:08Z (GMT) No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Made available in DSpace on 2015-05-12T13:23:08Z (GMT). No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O adenoma pleomórfico (AP), o carcinoma mucoepidermóide (CME) e o carcinoma adenóide cístico (CAC) representam tumores frequentes em glândula salivar. A via de sinalização Sonic Hedgehog (Hh) e o Transdutor de sinal e ativador da transcrição 3 (STAT3) desempenham funções importantes na proliferação celular, favorecendo o desenvolvimento tumoral e a proteína MCM3 tem sido considerada uma nova classe de marcadores de proliferação celular. Portanto, o presente trabalho propõe-se a estudar componentes da via Hh, bem como o STAT3 e o MCM3 em neoplasias de glândula salivar, na tentativa de adicionar informações sobre as características biológicas dessas neoplasias. Foram utilizados 9 casos de AP, 17 casos de CAC e 20 casos de CME e, por meio da técnica imunoistoquímica, realizou-se a detecção das seguintes proteínas: SHH, GLI1, SUFU, HHIP, STAT3 e MCM3. No AP, observou-se alta expressão citoplasmática de SHH e SUFU, e baixa expressão de STAT3 e MCM3. No CAC, observou-se alta expressão de GLI1, HHIP e STAT3 e baixa expressão de SHH, SUFU e MCM3. No CME, observou-se alta expressão de SHH, GLI1, SUFU e HHIP e baixa expressão de STAT3 e MCM3. Quando comparado entre os tipos tumorais, observou-se diferença estatisticamente significante para expressão de SHH (p=0.0064), STAT3 (p=0.0003) e MCM3 (p=0.0257). Ao comparar a marcação parenquimal e estromal, observou-se maior expressão em parênquima para todos os tumores (p<0.05). Em glândula salivar normal, observou-se marcação citoplasmática das células ductais para SHH, GLI1, SUFU e HHIP, uma discreta marcação citoplasmática para STAT3 e ausência de marcação para MCM3. Não foi observada correlação entre a via Hh e STAT3 nos tumores de glândula salivar (p>0.05). Os resultados desse trabalho apontam para uma possível participação da via Hh na morfogênese e no desenvolvimento do AP, CAC e CME, assim como a participação do STAT3 no desenvolvimento do CAC. Em adição, o MCM3 não parece ser um bom marcador de proliferação para tumores de glândula salivar. Sugerimos que novos estudos sejam realizados visando compreender o mecanismo pelo qual a via Hh e o STAT3 promovem o crescimento e progressão desses tumores e identificar inibidores dessas vias. / The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC) and the adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands. The Sonic Hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The MCM3 protein has been considered as a novel class of cell proliferation markers. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 and MCM3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC and 20 cases of MEC. Using immunohistochemistry, were investigated: SHH, GLI1, Sufu, HHIP, STAT3 and MCM3. In PA, there was high expression of cytoplasmic SHH and Sufu, and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP and STAT3 and low expression of SHH, SUFU and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p=0.0064), STAT3 (p=0.0003) and MCM3 (p=0.0257) when all tumors were compared. And a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p<0.05). In normal salivary gland, ductal segment showed imunolabeling for SHH, GLI1, SUFU and HHIP, a discrete cytoplasmic labeling for STAT3 and MCM3 was negative. No correlation was observed between the Hh pathway and STAT3 in salivary gland tumors (p>0.05). The findings suggest a possible role of Hh pathway in the morphogenesis and development of AP, CAC and CME, as well as the participation of STAT3 in the development of ACC. In addition, the MCM3 did not seem to be a good marker of proliferation for salivary gland tumors. It is important that further studies be conducted to understand the mechanism by which the Hh pathway and the STAT3 promote the growth and progression of these tumors and inhibitors of these pathways might be identified.

Mutações inativadoras em homozigose no gene do transdutor de sinal e ativador de transcrição 5B (STAT5B) causam insensibilidade ao hormônio de crescimento associada a disfunção imunológica grave que se manifesta na forma de infecções exacerbadas e de repetição, pneumonia intersticial linfocítica e outros eventos autoimunes. A caracterização do fenótipo destas mutações em heterozigose não foi realizada previamente. Dois pacientes descritos com mutação em homozigose na STAT5B (c.424_427del / p.L142RfsX19) são irmãos brasileiros naturais de Criciúma - Santa Catarina, sem consanguinidade conhecida na família. Houve também o relato de dois outros casos semelhantes na cidade, já falecidos, sugerindo que mutações na STAT5B pudessem ser relativamente frequentes nesta região. Os objetivos deste estudo foram investigar a frequência da mutação c.424_427del da STAT5B na população de Criciúma, avaliar a existência de efeito fundador e caracterizar o efeito da mutação c.424_427del da STAT5B em heterozigose sobre o fenótipo antropométrico e hormonal. Para investigar a frequência desta mutação em Criciúma, 1192 indivíduos da população foram genotipados. Foram identificados sete indivíduos heterozigotos, caracterizando uma frequência alélica mínima de 0,29% (intervalo de confiança 95%: 0,08 a 0,5%), significativamente mais alta que a frequência de outras variantes patogênicas da STAT5B descritas em bases de dados públicas. Utilizando-se o equilíbrio de Hardy-Weinberg, foi possível estimar a incidência de casos de homozigotos para o alelo mutado em um a cada 40 anos. No entanto, utilizando-se a maior frequência possível de acordo com o intervalo de confiança, esta incidência poderia atingir um a cada 13 anos. Além disso, foram estudados os pais dos dois casos relatados como semelhantes aos pacientes homozigotos para mutações na STAT5B e estes pais eram portadores da mutação c.424_427del da STAT5B em heterozigose. Para avaliar o efeito fundador, foram analisados dois marcadores próximos à mutação c.424_427del da STAT5B nos pacientes homozigotos para a mesma, em 33 indivíduos heterozigotos de sete famílias independentes e em 53 indivíduos controles. O mesmo haplótipo estava presente nos pacientes homozigotos para a mutação e em todos os heterozigotos, enquanto em apenas 9,4% dos controles (p < 0,001), apontando a probabilidade de que a mutação c.424_427del nas diferentes famílias tenha sido herdada de um antepassado em comum. Para avaliar o efeito da mutação c.424_427del da STAT5B em heterozigose, foram comparados em conjunto os 33 indivíduos heterozigotos e os 38 familiares não portadores em relação à altura e a alguns exames laboratoriais (gerais e hormonais). Os indivíduos heterozigotos foram significativamente mais baixos, com uma redução na altura de 0,6 desvios-padrão (p= 0,006). Também apresentaram redução significativa dos desvios-padrão de fator de crescimento insulina-símile 1 (IGF-1) e da proteína 3 de ligação a fator de crescimento insulina-símile (IGFBP-3), sem alteração em outros exames. Esses achados mostram que as mutações na STAT5B em heterozigose causam um impacto negativo significativo na altura, mais leve que o visto em pacientes com mutações em homozigose, com altura dentro da variação normal. Esse resultado favorece a hipótese de que variantes patogênicas raras em heterozigose contribuem para a variabilidade da altura normal / Homozygous inactivating mutations in signal transducer and activator of transcription 5B gene (STAT5B) cause growth hormone insensitivity associated with signs of severe immune dysfunction, such as recurrent infections, lymphoid interstitial pneumonia and other autoimmune events. The phenotypic characterization of these mutations in heterozygous state has not been accomplished previously. Two patients with a homozygous STAT5B mutation (c.424_427del / p.L142RfsX19) are Brazilian brothers born in the city of Criciúma, Santa Catarina, and there is not known consanguinity in their family. Moreover, there was a report about two similar cases in this city, already deceased, suggesting that STAT5B mutations could be relatively frequent in this region. The objectives of this study were to evaluate the frequency of STAT5B c.424_427del mutation in Criciúma, to assess the existence of the founder effect and to characterize the effect of heterozygous STAT5B c.424_427del mutation on anthropometric and hormonal phenotypes. To evaluate the frequency of this mutation in Criciúma, 1192 individuals from the population were genotyped. Seven heterozygous individuals were identified, which characterized a minimum allele frequency of 0.29% (95% confidence interval: 0.08 to 0.5%), significantly higher than the frequency of other pathogenic variants described in public databases. By using the Hardy-Weinberg law, it was possible to estimate the incidence of cases of individuals homozygous for this mutation at one every 40 years. However, by using the highest possible frequency according to the confidence interval, this incidence could reach one every 13 years. Additionally, the parents of the two reported cases who were similar to patients with homozygous STAT5B mutations were genotyped and these parents were heterozygous for STAT5B c.424_427del mutation. To assess the founder effect, two markers near the mutation were analyzed in the two boys homozygous for STAT5B c.424_427del mutation, in 33 heterozygous individuals from seven unrelated families and in 53 control individuals. The same haplotype was present in the homozygous boys and in all heterozygous individuals, while in only 9,4% control individuals (p < 0,001), pointing to the probability that STAT5B c.424_427del mutation in different families has been inherited from a common ancestor. To study the effects of heterozygous STAT5B c.424_427del mutation, 33 heterozygous individuals were compared to 38 non-carrier relatives on height and some laboratorial tests. Heterozygous individuals were significantly shorter than their noncarrier relatives, with a height reduction of 0.6 standard deviation scores (p= 0,006). Furthermore, they had a significant reduction in insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) standard deviation scores, without differences in the other exams. These findings show that heterozygous STAT5B mutations cause a significant negative impact on height, milder than the effect seen in patients with homozygous mutations, with height within the normal range. This result favors the hypothesis that rare pathogenic variants in heterozygous state contribute to normal height variability

Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes rares qui se développent le plus souvent chez la femme jeune suite à la prise de contraceptifs oraux. Les complications principales sont l’hémorragie et plus rarement, la transformation maligne en carcinome hépatocellulaire (CHC). Des travaux récents ont permis d’identifier 3 groupes moléculaires principales d’AHC qui se définissent par (1) l’inactivation du facteur de transcription HNF1A (H-HCA), (2) l'activation de la voie Wnt/ß-caténine (bHCA) ou (3) la présence d’infiltrats inflammatoires (IHCA).Afin d’identifier les voies de tumorigenèse associées au développement d’AHC inflammatoires (IHCA), une analyse transcriptomique comparant des IHCA à des foies non tumoraux a été réalisée au laboratoire, ce qui a permis d’identifier dans ce groupe tumoral une activation de la voie IL-6/JAK/STAT3. Nous avons recherché de nouvelles altérations géniques et nous avons caractérisé le mécanisme d'activation de la voie IL-6/JAK/STAT dans les IHCA. Les conséquences fonctionnelles sur la voie STAT3 des différents mutants ont été analysées par une modélisation de leur expression dans des lignées hépatocellulaires. Par ailleurs, nous avons réalisé des études génomiques intégrées (analyse CGH-SNP, méthylome et séquençage exome) sur une large série de 250 AHC avec pour objectif d’affiner la classification moléculaire des AHC, d’identifier de nouveaux gènes altérés dans ces tumeurs et d’élucider les mécanismes de transformation maligne des AHC en CHC.Dans le groupe des IHCA, ces analyses nous ont permis d’identifier de nombreux oncogènes activés par mutation somatique ; de plus, trois de ces gènes n’avaient jamais été décrits comme étant mutés dans des tumeurs humaines. Nous avons identifié des mutations activatrices du récepteur à l’IL-6, gp130 dans 60% des IHCA. Nous avons aussi retrouvé des mutations de FRK, une src-like kinase, dans 10% des IHCA, du facteur de transcription STAT3 dans 5% des IHCA, du gène GNAS dans 5% des cas, et de la tyrosine kinase JAK1 dans 1% des cas. Toutes les mutations identifiées étaient somatiques, monoalléliques et mutuellement exclusives. Nous avons pu montrer, dans des systèmes de lignées cellulaires hépatocellulaires, que l'expression des formes mutées de ces gènes est capable d’activer la voie IL-6/STAT3 en absence du ligand IL-6, contrairement aux protéines sauvages. Nous avons identifié des inhibiteurs pharmacologiques qui permettent d’inhiber de façon spécifique ces mutants et qui pourraient être utilisés en clinique pour le traitement des IHCA.Grâce à une technique de CGH-SNP, nous avons identifié des événements récurrents de pertes et gains de chromosomes associés aux groupes moléculaires d’AHC. De façon similaire, l’étude de la méthylation dans les AHC a permis de mettre en évidence un pattern spécifique à chaque sous groupe. Nous avons montré que l’instabilité chromosomique augmente progressivement dans les lésions borderline et dans les CHC développés sur AHC comparés aux AHC classiques. Le séquençage exome de 5 transformations malignes de AHC en CHC a identifié un nombre plus important de mutations dans les AHC qui ont transformé comparé aux AHC classiques ; ce nombre est significativement augmenté dans la partie CHC des tumeurs. La comparaison de la partie bénigne et maligne des tumeurs a mis en évidence l'activation de ß-caténine comme un évènement précoce dans le processus de transformation et a révélé la présence de mutations somatiques fréquentes dans le promoteur de la télomèrase (TERT), identifiées principalement dans la partie maligne des tumeurs.En conclusion, cette étude a permis d’identifier des mécanismes distincts conduisant à l'activation de STAT3 dans les IHCA, renforçant le rôle de la voie JAK-STAT3 dans la tumorigenèse bénigne hépatocellulaire ainsi que le lien entre Src kinases et inflammation. Ces travaux ont permis d’affiner la classification moléculaire des AHC avec des corrélations étroites... / Hepatocellular adenomas (HCA) are rare benign tumors that develop most often in young women after taking oral contraception. The main complications are hemorrhage and rarely, malignant transformation to hepatocellular carcinoma (HCC). Recent work in the laboratory identified three main HCA molecular groups that are defined by (1) inactivation of the transcription factor HNF1A (H-HCA), (2) activation of the Wnt/ß-catenin pathway (bHCA) or (3) the presence of inflammatory infiltrates (IHCA).To identify tumorigenesis pathways associated with the development of inflammatory HCA (IHCA), a transcriptome analysis comparing IHCA to non-tumor liver was performed in the laboratory, leading to the identification of an activation of the IL-6/JAK/STAT3 pathway in these tumors. We sought new gene alterations and we characterized the activation mechanism of the IL-6/JAK/STAT pathway in IHCA. The functional consequences of the different mutants on the STAT3 pathway were analyzed by modeling their expression in hepatocellular cell lines. In addition, we performed integrated genomic studies (CGH-SNP analysis, methylome and exome sequencing) on a wide range of 250 HCA with the aim to refine the molecular classification of HCA, to identify new genes altered in these tumors and to elucidate the mechanisms of malignant transformation of HCA to HCC.In the group of the IHCA, we identified many oncogenes activated by somatic mutation; in addition, three of these genes were never been described as mutated in human tumors. We identified activating mutations in the IL-6 receptor gp130 in 60% of IHCA. We also found mutations in FRK, a src-like kinase, in 10% of IHCA, of the transcription factor STAT3 in 5% of IHCA, of the GNAS gene in 5% of cases, and of the tyrosine kinase JAK1 in 1% of the cases. All identified mutations were somatic and monoallelic and were mutually exclusive. We have shown in hepatocellular cell lines that the expression of mutated forms of these genes is able to activate the IL-6/STAT3 pathway in the absence of the IL-6 ligand, in contrast to wild-type proteins. We have identified pharmacological inhibitors that specifically inhibit the mutants and that could be used for the clinical treatment of IHCA.Using a CGH-SNP technique, we identified recurrent chromosomes gains and losses associated with the HCA molecular groups. Similarly, the study of methylation in HCA highlighted a specific pattern in each subgroup. We showed that chromosomal instability increases gradually in borderline lesions and in HCC developed on HCA compared to classical HCA. Exome sequencing of 5 malignant transformation of HCA to HCC identified a large number of mutations in the transformed HCA compared to classical HCA; and this number is significantly increased in HCC tumors counterpart. Comparison of benign and malignant tumors highlighted the activation of ß-catenin as an early event in the transformation process and revealed frequent somatic mutations in the promoter of the telomerase gene (TERT), identified mainly in the malignant part of tumors.In conclusion, this study has led to the identification of distinct mechanisms leading to the activation of STAT3 in IHCA, strengthening the role of the JAK-STAT3 pathway in benign hepatocellular tumorigenesis and the relationship between Src kinases and inflammation. This work helped to refine the molecular classification of HCA with tight correlations between genotype and phenotype, and led to advances in the identification of major genetic determinants involved in the process of malignant transformation.

Les cytokines jouent un rôle essentiel dans la réponse immunitaire ; elles modulent les propriétés et la survie de nombreuses cellules immunitaires. Les lymphocytes T (LT) sont régulés par un large spectre de cytokines. Néanmoins certaines sous-populations de LT restent peu définies à ce jour tout comme l’impact de cytokines sur ces cellules en conditions physiologiques et pathologiques. Une population de LT caractérisée par la double expression des corécepteurs CD4 et CD8 a été identifiée en périphérie chez des donneurs sains et sa fréquence est augmentée chez les patients atteints de certaines pathologies. Cependant, peu d’études ont déterminé le phénotype et les fonctions de ces cellules. Nous avons caractérisé le phénotype ex vivo des LT CD4+CD8+ issus de sujets sains et étudié la réponse de ces cellules à plusieurs cytokines importantes dans l’homéostasie et l’activation des lymphocytes. Notre étude révèle que les LT CD4+CD8+ forment une population hétérogène présentant de nombreuses caractéristiques des cellules mémoires. De plus, notre étude montre que ces cellules ont une capacité accrue de produire des cytokines et enzymes lytiques comparée aux LT CD4 et CD8. Finalement, nous avons observé qu’une plus grande proportion de LT CD4+CD8+ répond aux cytokines IL-2, IL-15 et IL-7 comparée aux LT CD4 et CD8 simples positifs. Plusieurs études ont révélé que certaines cytokines participent à la pathobiologie de maladies auto-immunes notamment la sclérose en plaques (SEP). La SEP est une maladie inflammatoire chronique du système nerveux central (SNC) caractérisée par une destruction de la gaine de myéline et une perte axonale à l’origine des symptômes cliniques. L’impact des médiateurs immunitaires dans la destruction et/ou la réparation reste néanmoins à éclaircir. Plusieurs études suggèrent que l’interleukine-27 (IL-27) joue un rôle central dans la SEP : l’IL-27 diminue la sévérité de la maladie dans un modèle murin de la SEP ; la réponse des patients à certaines thérapies corrèle avec des niveaux périphériques élevés d’IL-27. Notre étude avait pour but de déterminer comment l’IL-27 module les LT des patients atteints de SEP. Nous avons observé des niveaux d’IL-27 augmentés à la fois en périphérie et dans le SNC des patients SEP. Nous avons mis en évidence que les effets induits par l’IL-27 sont altérés dans les LT des patients SEP ; notamment l’induction de PD-L1 et la réponse à l’IL-12 sont plus faibles comparées aux LT de sujets sains. La voie de signalisation STAT3 est induite plus fortement en réponse à l’IL 27 dans les cellules de patients SEP en comparaison à celles de sujets sains. Finalement, nous avons démontré que la forme soluble du récepteur à l’IL-27 est présente en plus grande quantité dans le sérum des patients SEP et ces niveaux sont suffisants pour bloquer l’effet de l’IL-27 sur ses cellules cibles. En conclusion, nos résultats permettent de mieux comprendre comment certaines cytokines participent à la régulation des populations lymphocytaires. En condition pathologique, la modulation de ces cytokines pourrait s’avérer efficace dans un but thérapeutique afin de promouvoir et/ou inhiber certaines réponses immunitaires notamment dans la SEP. / Cytokines play an essential role in modulating the immune response. They act on cells from both innate and adaptive immunity by modulating their phenotype and influencing their survival. T cells are highly regulated by a broad spectrum of cytokines. Nevertheless, some subsets of T cells remain ill-defined and a better understanding of cytokine-mediated responses in physiological and pathological conditions requires further investigation. A T cell population characterized by the dual expression of CD4 and CD8 co-receptors has been identified in the periphery of healthy donors and its frequency is notably increased in several pathologies. However, very few studies have determined the phenotype and functions of these cells under physiological conditions. We performed an ex vivo phenotypic characterization of CD4+CD8+ T cells from healthy subjects and analyzed the response of these cells to several important cytokines implicated in homeostasis and activation of immune cells. Our study reveals that CD4+CD8+ T cells are heterogeneous and exhibit many characteristics of memory cells. In addition, our study shows that these cells have an enhanced ability to produce cytokines and lytic enzymes compared to CD4 and CD8 T cells. Finally, we found that a higher proportion of CD4+CD8+ T cells respond to IL-2, IL-15 and IL-7 cytokines compared to CD4 and CD8 counterparts. Numerous studies showed that lymphocyte populations and some cytokines are involved in the development of several autoimmune diseases, including multiple sclerosis (MS). MS is a chronic inflammatory disease of the central nervous system (CNS) characterized by the destruction of the myelin sheath, axonal loss and oligodendrocytic death leading to clinical symptoms. The immune mediators involved in the destruction and/or repair remain to be clarified. Several studies suggest that interleukin-27 (IL-27) plays a central role in MS: IL-27 dampens the severity of the disease in a MS mouse model and in MS patients the response to some therapies correlates with higher levels of peripheral IL-27. The purpose of our study was to determine how IL-27 modulates T cells in MS patients. We observed that IL-27 levels are increased in both periphery and CNS of MS patients. We found that IL-27-mediated effects are impaired in T cells from MS patients in particular PD-L1 induction and IL-12 response that are lower compared to T cells from healthy subjects. IL-27-triggered STAT3 signaling pathway is also enhanced in cells from MS patients compared to healthy subjects. Finally, our study showed that the soluble form of the receptor of IL-27 is present in greater levels in the serum of MS patients and the measured doses are sufficient to block the capacity of IL-27 to act on its target cells. To conclude, our results provide a better understanding of how certain cytokines participate in the regulation of lymphocyte populations. Under pathological conditions, modulation of these cytokines may be effective for therapeutic applications in order to promote and/or inhibit immune responses, particularly in MS.

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