Thesis advisor: Mary F. Roberts / Inositol-containing molecules and inositol phosphatases have diverse roles in cells. One of the inositol phospholipids phosphatases, PTEN (Phosphatase and Tensin Homolog deleted on Chromosome Ten), is a tumor suppressor and antagonizes the PI3K signaling pathway by dephosphorylating PI(3,4,5)P3 at the 3 position of the inositol ring. In testing predictions of a molecular dynamics simulation, a hydrophobic site adjacent to the active site on PTEN was identified and verified by protein kinetic studies. This hydrophobic site plays an important role in substrate and substrate analogue binding with one of residues, Arg47, critical for PTEN phosphatase activity. Mutations of Arg47 reduced enzyme activities toward both the short-chain substrate as monomers and micelles and long-chain phospholipid presented in vesicles. PI(4,5)P2 the product of PI(3,4,5)P3 dephosphorylation, activates PTEN. Studies by others suggested this occurred when the product was bound to the N-terminal region of the protein (not visible in the crystal structure). However, no direct proof of this existed. The effect of PI(4,5)P2 on PTEN enzyme activities in different substrates systems was studied. 31P NMR was used to probe the spatial location and functional role of PI(4,5)P2 binding site. The fixed field 31P NMR and high resolution field cycling 31P NMR results indicated there are discrete sites for both substrate and activator lipids on PTEN, and both of sites are spatially separate from the hydrophobic site. The active site, adjacent hydrophobic site, and N-terminal activator binding site worked synergistically to regulate PTEN interacting with the membrane. Thermophilic and hyperthermophilic archaea and bacteria thrive at high temperatures. They often accumulate small organic molecules, called compatible solutes or osmolytes, to protect proteins from thermal denaturation. The thermoprotection mechanism of compatible solutes was explored using inositol monophosphatase (IMPase) from Archaeoglobus fulgidus as the model protein. The protective effect of unusual compatible solutes, di-inositol-1,1'-phosphate (DIP) and diglycerol phosphate (DGP), as well as common compatible solutes glutamate and other anions, on the IMPase thermostability was studied. Specific binding sites of glutamate ions on the IMPase were identified by crystallography and field cycling NMR. However, mutations at these discrete binding sites did not eliminate the thermoprotection, but reduced the thermal stability (Tm) of the protein. Our results indicate the specific binding of osmolytes to the protein exists, but they do not account for the thermoprotection. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No description available.

O objetivo deste estudo foi comparar leiomiomas e leiomiossarcomas de boca por meio de análise histológica e imunoistoquímica, utilizando os marcadores Ki67, p53 e PTEN. A actina de músculo liso foi utilizada para confirmar o diagnóstico. Foram examinados 13 tumores de músculo liso disponíveis nos arquivos do Departamento de Estomatologia da Faculdade de Odontologia da USP, sendo 7 leiomiossarcomas, 4 angioleiomiomas e 2 leiomiomas sólidos. As lâminas foram avaliadas de acordo com o grau de atipia, índice mitótico e presença de necrose. Foi realizada a análise imunoistoquímica para o ki67, p53 e PTEN, por meio da contagem de células positivas em 500 células nas áreas mais representativas. A maioria dos angioleiomiomas não apresentou atipia, mitose ou necrose; atipia discreta foi encontrada nos dois leiomiomas sólidos, um deles com 1 mitose por 10 CGA. Atipia foi observada em todos os leiomiossarcomas, o índice mitótico variou de 0 a 6 mitoses por 10 CGA, e apenas um caso apresentou necrose. Houve positividade para o ki67 em apenas um angioleiomioma e em 5 leiomiossarcomas, o restante dos casos foram negativos. Em relação ao p53, ocorreu leve positividade para um caso de leiomioma sólido e moderada para o outro; a maioria dos angioleiomiomas foram levemente positivos e apenas um foi negativo; todos os leiomiossarcomas foram positivos, 2 com marcação leve, 4 moderada e um intensa. A expressão do PTEN foi negativa em um leiomioma sólido e intensamente positiva em outro; todos os casos de angioleiomioma foram positivos, sendo que 2 demonstraram positividade leve e 2 moderada; 3 leiomiossarcomas apresentaram positividade moderada e 3 intensa, apenas um foi negativo. Nossos resultados sugerem que a marcação para ki67 e p53 são úteis na diferenciação entre leiomiomas e leiomiossarcomas. O mesmo não foi observado para o anticorpo PTEN. / This study aimed to compare oral leiomyomas and leiomyosarcomas by means of histological and immunohistochemical analysis. Cases from the Stomatology Department, School of Dentistry at University of São Paulo were retrieved. For immunohistochemistry Ki67, p53 and PTEN markers were used. Smooth muscle actin was used to confirm the diagnosis. There were 13 smooth muscle tumors: 7 leiomyosarcomas, 4 angioleiomyomas and 2 solid leiomyomas. For morphological analysis, sections were evaluated for atypia, mitotic index and presence of necrosis. Immunohistochemical analysis of Ki67, p53 and PTEN expression was performed by counting 500 positive cells in the most representative areas. Most angioleiomyomas did not present atypia, mitosis or necrosis; mild atypia was found in two solid leiomyomas, one with 1 mitosis per 10 HPF. Atypia was found in all leiomyosarcomas, the mitotic index varied from 0 to 6 mitosis per 10 HPF, and necrosis was found in only one case. Ki67 expression was positive in one angioleiomyoma and 5 leiomyosarcomas, the remaining cases were negative. For p53, one solid leiomyoma was mildly positive and the other showed moderate positivity; most of angioleiomyomas were mildly positive and only one was negative; all leiomyosarcomas were positive, 2 with mild expression, 4 moderate and one intense. PTEN expression was negative in one solid leiomyoma and intensely positive in the other; all angioleiomyomas were positive, with 2 mildly positive and 2 moderately positive; 3 leiomyosarcomas presented moderate positivity and 3 intense, only one was negative. Our results suggest that ki67 and p53 are useful in the differentiation between leiomyoma and leiomyosarcoma. The same was not found for PTEN.

O câncer é uma doença potencialmente letal. Os tumores se desenvolvem em células que estão se dividindo. Sua iniciação ou progressão está associada com o acúmulo de alterações genéticas. Essas alterações podem ser aberrações cromossômicas, mutações, polimorfismos e modificações epigenéticas. O câncer se desenvolve quando mecanismos de defesa do organismo sofrem alterações. Os tumores do sistema nervoso representam aproximadamente 2% de todos os tipos de câncer. O papel central do sistema nervoso e as conseqüências funcionais da perda de neurônios podem explicar a severidade dos tumores cerebrais. A formação dos tumores do cérebro humano é um processo complexo, envolvendo um acúmulo de alterações genéticas. Os genes supressores de tumor estão envolvidos na formação de tumores. Sua perda, inativação ou disfunção leva a célula a se dividir desordenadamente, surgindo assim tumores e neoplasias no local onde elas ocorrem. O objetivo deste trabalho foi fazer uma triagem de ocorrência de polimorfismos conformacionais no gene supressor de tumor PTEN em 50 amostras de tumores de Sistema Nervoso. Nenhuma alteração mutacional foi encontrada. Nossos resultados se assemelham aos da literatura em relação à ausência de alterações no gene PTEN em tumores benignos; em relação aos glioblastomas, a literatura cita uma alta freqüência de alterações no gene PTEN, mas não encontramos nenhuma alteração nas 9 amostras estudadas. Outro mecanismo como por exemplo a metilação da região promotora, poderia estar envolvido na inativação desse gene nos tumores analisados. / Cancer is a potentially lethal illness. Tumors develop in cells that are dividing. Its initiation or progression is associated with the accumulation of genetic alterations. These alterations can be chromosome aberrations, mutations, polimorphisms and epigenetic modifications. The cancer develops when mechanisms of defence of the organism are altered. The tumors of the nervous system represent approximately 2% of all the types of cancer. The central role of the nervous system and the functional consequences of the loss of neurons can explain the severity of the cerebral tumors. The formation of the tumors of the human brain is a complex process, involving an accumulation of genetic alterations. The tumor suppressor genes are involved in the formation of tumors. If there is a loss, inactivation or disfunction, the cell will divide disorderly, and tumors and other neoplasias will appear in the place where they occur. The objective of this work was to make a selection of the occurrence of conformacional polymorphisms in the tumor suppresor gene PTEN in 50 samples of tumors of Nervous System. No mutacional alteration was found. Our results if are similar to the ones of the literature in relation to the absence of alterations in gene PTEN in benign tumors; in relation to glioblastomas, literature cites a high frequency of alterations in gene PTEN, but we did not find any alteration in the 9 studied samples. Another mechanism as, for example, the metilation of the promoterl region, could be involved in the inativação of this gene in the analyzed tumors.

Submitted by Carvalho Dias João Paulo (joao.dias@famerp.br) on 2018-04-05T17:26:15Z No. of bitstreams: 1 gracieledtenani_dissert.pdf: 2576225 bytes, checksum: 0b7b634cafdb9080cbe9b93e41c2e195 (MD5) / Made available in DSpace on 2018-04-05T17:26:15Z (GMT). No. of bitstreams: 1 gracieledtenani_dissert.pdf: 2576225 bytes, checksum: 0b7b634cafdb9080cbe9b93e41c2e195 (MD5) Previous issue date: 2016-12-07 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Background - Hepatocellular carcinoma (HCC) is highlighted as the most aggressive malignant liver tumor. The identification of candidate genes to become biomarkers may help to clarify the pathophysiology of HCC, as well as the diagnosis of the disease at early stage, leading to new therapeutic interventions. Objectives -To evaluate the association of genetic variants and the gene expression involved in the cell signaling process, apoptosis, and angiogenesis with HCC, to characterize risk subgroups and identify biological markers for early diagnosis, prognosis and treatment of the disease. Casuistics and Methods – We studied 343 subjects, 102 with HCC (SG = study group) and 215 controls (CG = control group) for the analysis of PTEN polymorphisms (rs10490920, rs532678 and rs701848) and VEGF-A (rs3025039 and rs1570360). For gene expression analysis of PTEN and PIK3CA, 24 patients with HCC were selected (SGge = Study Group of gene expression), 16 with cirrhosis (CiGge = Cirrhosis Group of gene expression) and 10 controls who underwent bileo and digestive surgery (CGge = Group control of gene expression). The polymorphisms of related genes were analyzed by PCR/RFLP (polymerase chain reaction/restriction fragments length polymorphism), while the gene expression (fresh liver tissue) by qPCR (quantitative/polymerase chain reaction). Data from the clinical profile, lifestyle and comorbidities were obtained from medical records and questionnaire. Alpha error level was set at 5%. Results - Male gender, advanced age, smoking, alcohol consumption and diabetes mellitus (DM) prevailed in the group with HCC compared to the control (P<0.05). Genetic polymorphisms: PTEN- rs10490920 - The T/T genotype was noted in both groups followed by T/C, and the T allele (P>0.05). PTEN- rs532678- The genotype T/C was the most common in both groups, followed by C/C, and the C allele was predominant in GE compared to control group (P>0.05). PTEN- rs701848- The genotype C/C was highlighted in SG compared to CG, followed by T/T, and the T allele (P>0.05). VEGF-A- rs3025039- The C/C genotype is distinguished in both groups (P>0.05), the same occured for the C allele (P = 0.4226). VEGF-A- rs1570360 – G/G genotype prevailed in both groups (P>0.05), as well as the G allele (P=0.6387). Although similarity between the groups for genotypic and allelic distribution was observed, mutant PTEN and VEGF-A alleles prevailed in patients with HCC and tobacco and alcohol consumption, compared to the control group (P <0.05). PTEN and VEGF haplotype analysis was similar among the groups (P>0.05). Gene expression - PTEN expression levels was decreased in patients with HCC (median= 0,908) compared to cirrhotic patients (median = 5.93, P=0.0347). PIK3CA expression levels (median- HCC= 0,108; cirrhosis= 0,493) were similar between groups (P> 0.05). Conclusion – PTEN and VEGF-A genetic variants, as well as their haplotypes were not associated to HCC. Reduced gene expression of PTEN in tumor tissue can be associated with HCC, while PIK3CA does not differentiate between patients with HCC from those with cirrhosis. It stands out as independent risk factors for HCC, smoking, alcohol consumption, male sex, advanced age and DM. PTEN and VEGF-A mutant alleles, particularly in the presence of smoking and alcohol consumption may enhance the risk for HCC. / Introdução - O carcinoma hepatocelular (CHC) destaca-se como o mais agressivo tumor maligno do fígado. A identificação de genes candidatos a biomarcadores pode contribuir para esclarecer a fisiopatologia do CHC e auxiliar no diagnóstico precoce da doença com novas intervenções terapêuticas. Objetivos - Avaliar a associação de variantes genéticas e expressão gênica envolvidas no processo de sinalização celular, apoptose e angiogênese com CHC, visando caracterizar subgrupos de risco e identificar marcadores biológicos para diagnóstico precoce, prognóstico e tratamento da doença. Casuística e Métodos – Foram estudados 343 indivíduos, sendo 102 com CHC (GE= Grupo de estudo) e 215 controles (GC= Grupo controle) para a análise dos polimorfismos de PTEN (rs10490920, rs532678, rs701848) e VEGF-A (rs3025039 e rs1570360). Para a análise de expressão gênica de PTEN e PIK3CA, foram selecionados 24 pacientes com CHC (GEeg= Grupo de estudo da expressão gênica), 16 com cirrose (GCieg= Grupo cirrose da expressão gênica) e 10 controles submetidos a cirurgias bileo-digestivas (GCeg= Grupo controle da expressão gênica). Os polimorfismos dos referidos genes foram analisados por PCR/RFLP (polymerase chain reaction/restriction fragments lengh polymorphism), enquanto a expressão gênica (tecido hepático fresco) por qPCR (quantitative/polymerase chain reaction). Dados do perfil clínico, hábitos de vida e comorbidades foram obtidos em prontuário médico e questionário. Admitiu-se erro α de 5%. Resultados - O gênero masculino, idade avançada, tabagismo, etilismo e diabetes mellitus (DM) prevaleceram no grupo com CHC, comparado ao controle (P<0,05). Polimorfismos genéticos: PTEN- rs10490920- O genótipo T/T destacou-se em ambos os grupos, seguido de T/C, assim como o alelo T (P>0,05). PTEN- rs532678- O genótipo T/C foi o mais frequente em ambos os grupos, seguido de C/C, assim como o alelo C predominou em GE comparado a GC (P>0,05). PTEN- rs701848- O genótipo T/C destacou-se em GE compado a GC, seguido de T/T, assim como o alelo T (P>0,05). VEGF-A- rs3025039- O genótipo C/C destacou-se em ambos os grupos (P>0,05), o mesmo ocorreu para o alelo C (P=0,4226). VEGF-A- rs1570360 – O genótipo G/G prevaleceu em ambos os grupos (P>0,05), assim como o alelo G (P=0, 6387). Embora detectada semelhança entre os grupos para distribuição genotípica e alélica, alelos mutantes de PTEN e VEGF-A prevaleceram em pacientes com CHC e hábitos tabagista e etilista, comparado ao controle (P<0,05). A análise de haplótipos de PTEN e VEGF-A mostrou semelhança entre os grupos (P>0,05). Expressão gênica- Houve diminuição dos níveis de expressão de PTEN em pacientes com CHC (mediana= 0,908) comparado aos cirróticos (mediana= 5,93; P= 0, 0347). Níveis de expressão de PIK3CA (mediana- CHC=0,108; cirrose= 0,493) foram semelhantes entre os grupos (P>0,05). Conclusão – Variantes genéticas de PTEN e VEGF-A, assim como seus haplótipos, não se associam ao CHC. Expressão gênica reduzida de PTEN no tecido tumoral hepático pode estar associado a CHC, enquanto PIK3CA não diferencia pacientes com CHC daqueles com cirrose. Destacam-se como fatores de risco independentes para CHC tabagismo, etilismo, sexo masculino, idade avançada e DM. Alelos mutantes de PTEN e VEGF-A, particularmente na presença de tabagismo e etilismo podem potencializar o risco para CHC.

O objetivo deste estudo foi comparar leiomiomas e leiomiossarcomas de boca por meio de análise histológica e imunoistoquímica, utilizando os marcadores Ki67, p53 e PTEN. A actina de músculo liso foi utilizada para confirmar o diagnóstico. Foram examinados 13 tumores de músculo liso disponíveis nos arquivos do Departamento de Estomatologia da Faculdade de Odontologia da USP, sendo 7 leiomiossarcomas, 4 angioleiomiomas e 2 leiomiomas sólidos. As lâminas foram avaliadas de acordo com o grau de atipia, índice mitótico e presença de necrose. Foi realizada a análise imunoistoquímica para o ki67, p53 e PTEN, por meio da contagem de células positivas em 500 células nas áreas mais representativas. A maioria dos angioleiomiomas não apresentou atipia, mitose ou necrose; atipia discreta foi encontrada nos dois leiomiomas sólidos, um deles com 1 mitose por 10 CGA. Atipia foi observada em todos os leiomiossarcomas, o índice mitótico variou de 0 a 6 mitoses por 10 CGA, e apenas um caso apresentou necrose. Houve positividade para o ki67 em apenas um angioleiomioma e em 5 leiomiossarcomas, o restante dos casos foram negativos. Em relação ao p53, ocorreu leve positividade para um caso de leiomioma sólido e moderada para o outro; a maioria dos angioleiomiomas foram levemente positivos e apenas um foi negativo; todos os leiomiossarcomas foram positivos, 2 com marcação leve, 4 moderada e um intensa. A expressão do PTEN foi negativa em um leiomioma sólido e intensamente positiva em outro; todos os casos de angioleiomioma foram positivos, sendo que 2 demonstraram positividade leve e 2 moderada; 3 leiomiossarcomas apresentaram positividade moderada e 3 intensa, apenas um foi negativo. Nossos resultados sugerem que a marcação para ki67 e p53 são úteis na diferenciação entre leiomiomas e leiomiossarcomas. O mesmo não foi observado para o anticorpo PTEN. / This study aimed to compare oral leiomyomas and leiomyosarcomas by means of histological and immunohistochemical analysis. Cases from the Stomatology Department, School of Dentistry at University of São Paulo were retrieved. For immunohistochemistry Ki67, p53 and PTEN markers were used. Smooth muscle actin was used to confirm the diagnosis. There were 13 smooth muscle tumors: 7 leiomyosarcomas, 4 angioleiomyomas and 2 solid leiomyomas. For morphological analysis, sections were evaluated for atypia, mitotic index and presence of necrosis. Immunohistochemical analysis of Ki67, p53 and PTEN expression was performed by counting 500 positive cells in the most representative areas. Most angioleiomyomas did not present atypia, mitosis or necrosis; mild atypia was found in two solid leiomyomas, one with 1 mitosis per 10 HPF. Atypia was found in all leiomyosarcomas, the mitotic index varied from 0 to 6 mitosis per 10 HPF, and necrosis was found in only one case. Ki67 expression was positive in one angioleiomyoma and 5 leiomyosarcomas, the remaining cases were negative. For p53, one solid leiomyoma was mildly positive and the other showed moderate positivity; most of angioleiomyomas were mildly positive and only one was negative; all leiomyosarcomas were positive, 2 with mild expression, 4 moderate and one intense. PTEN expression was negative in one solid leiomyoma and intensely positive in the other; all angioleiomyomas were positive, with 2 mildly positive and 2 moderately positive; 3 leiomyosarcomas presented moderate positivity and 3 intense, only one was negative. Our results suggest that ki67 and p53 are useful in the differentiation between leiomyoma and leiomyosarcoma. The same was not found for PTEN.

De nombreuses études épidémiologiques associent le cholestérol avec l’incidence et le développement du cancer de la prostate. Parmi les acteurs impliqués dans le métabolisme du cholestérol, les récepteurs nucléaires LXRα et LXRβ sont identifiés comme d’importants régulateurs intra-cellulaires capables d’ajuster les niveaux d’accumulation de cette molécule. En parallèle, ces récepteurs peuvent exercer des effets anti-prolifératif et pro-apoptotique sur des cellules tumorales prostatiques. L’ensemble de ces données suggère un rôle protecteur des LXRs dans le cadre du cancer de la prostate. Les objectifs de ces travaux ont été d’étudier les mécanismes moléculaires reliant les LXRs au cycle cellulaire et de comprendre le rôle de ces récepteurs nucléaires dans le processus de la carcinogenèse prostatique. Nous avons ainsi montré que les LXRs exerçaient leur effet anti-prolifératif sur des cellules murines épithéliales prostatiques (MPECs) notamment via des modulations des voies de transduction PI3K/AKT et MAPK. Ces résultats font de ces cellules un modèle d’étude des mécanismes moléculaires impliqués dans cet effet. Les souris Lxrαβ-/- nourries avec un régime riche en cholestérol présentent un phénotype de néoplasie prostatique associé à un profil tanscriptomique proche de celui de souris modèles de cancer de la prostate démontrant l’effet protecteur des LXRs dans une condition d’hypercholestérolémie. Enfin, l’expression et l’activité des LXRs et leur gènes cibles ont été étudiées dans un modèle murin de carcinogenèse prostatique, les souris Pten-/-, dont l’invalidation du gène suppresseur de tumeur Pten est spécifique de l’épithélium prostatique. De manière intéressante, nous montrons que les LXRs sont activés au cours de la carcinogenèse prostatique et que la perte des LXRs dans les souris Pten-/- entraîne une accélération de la progression tumorale. Au final, ces résultats mettent en lumière que les LXRs peuvent exercer un rôle de barrière limitant la progression tumorale, notamment par l’intermédiaire de leurs gènes cibles codant les cassettes d’efflux de cholestérol, Abca1 et Abcg1. / Several epidemiological studies associate cholesterol to prostate cancer incidence and development. Among the actors involved in cholesterol metabolism, the nuclear receptors LXRα and β have been identified as important intracellular regulators, able to adjust cholesterol accumulation. In parallel, these receptors exert anti-proliferative and proapoptotic effects on prostate tumor cells. Altogether, these data suggest a protective role of LXRs against prostate cancer. This work aimed at understanding the molecular mecanisms linking LXRs to cell cycle regulation and prostate carcinogenesis. We showed that LXRs are able to exert their anti-proliferative effect on murine prostate epithelial cells (MPECs) via modulation of PI3K/AKT and MAPK pathways. These results demonstrate the value of these cells to study the molecular mecanisms involved in this LXR effect. Lxrαβ-/- mice fed a high cholesterol diet, display prostate neoplasia and a transcriptomic profil close to the one of a mouse model of prostate cancer demonstrating the protective effect of LXRs in hypercholesterolemia condition. Expression and activity of LXRs and their target genes have been studied in a mouse model of prostate cancer, the Pten-/- mice with a knock-out of the tumour supressor Pten specifically in the prostate epithelium. Interestingly, we showed that LXRs are activated during prostate carcinogenesis and that the loss of LXRs in Pten-/- mice leads to an acceleration of tumor progression. In summary, these results highlight the role of LXRs as a barrier to constrain tumor progression in particular through their target genes Abca1 and Abcg1.

La leucémie aiguë lymphoblastique (LAL-T) est une hémopathie maligne qui représente 10 à 15% des LAL pédiatriques et 25% des LAL de l’adulte. Bien que la prise en charge et le pronostic (rémission dans 80-85% des cas) des LAL se soient améliorés au cours des 10 dernières années en partie dû à une meilleure stratification thérapeutique de ces entités malignes, le tableau clinique et le devenir des patients atteints de LAL-T restent péjoratif avec environ 30% de rechute dans les 2 années qui suivent le diagnostic. Au cours de ces dernières années, des sous-types spécifiques de LAL-T associés à une valeur pronostique ont été décrits et des thérapies ciblées devraient pouvoir être proposées à l’avenir. Dans ce contexte, mon travail de thèse a permis de définir et de mieux comprendre les différents niveaux de dérégulation de MYC dans les LAL-T à travers l’analyse moléculaire et biochimique de MYC et de ses principaux régulateurs sur une large cohorte protocolaire de LAL-T pédiatriques et adultes. Tout d’abord, nous montrons que l'expression de MYC est très variable et que des niveaux d'expression élevés sont observés dans de nombreux cas en absence de mutations NOTCH1/FBXW7. De plus, nos travaux mettent en évidence que la dérégulation post-traductionnelle de MYC, via l'axe PI3K/AKT à travers l'inactivation de PTEN, constitue une voie majeure d'activation de MYC dans les LAL-T. Ainsi, l'ensemble de ces résultats confirment la pertinence d’envisager des stratégies thérapeutiques ciblant MYC pour le traitement des LAL-T. Mon projet de thèse a également consisté en la génération d’un modèle murin original permettant de suivre les clones tumoraux surexprimant Myc depuis les étapes de développement (pré-)tumoral les plus précoces jusqu’aux étapes finales de progression maligne. / T-cell Acute Lymphoblastic Leukemia (T-ALL) are malignant proliferations of thymocytes, which represent 10-15% of pediatric and 25% of adult ALL. Despite indisputable therapeutic progress, T-ALLs remain of poor prognosis. Patients often present with a high tumor load accompanied by a rapid disease progression, and about 30% of cases relapse within the first 2 years following diagnosis. It is now clear that significant improvements in therapy will require a more accurate knowledge of the oncogenes involved, as well as their oncogenic role within complex functional networks. In this context, my PhD project was focused on the understanding of the regulation of MYC in T-ALL. We demonstrate that MYC expression is highly variable and that high MYC expression levels can be generated independently of NOTCH1 pathway. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down regulation of PTEN. Altogether, our results lend further support to the significance of therapeutic targeting of MYC in T-ALL pathogenesis. The second part of my project was to generate an original transgenic mouse model designed to “track” inducible MYC+ clones from the earliest steps of (pre-)malignant development to the onset of leukemia.

O câncer é uma doença potencialmente letal. Os tumores se desenvolvem em células que estão se dividindo. Sua iniciação ou progressão está associada com o acúmulo de alterações genéticas. Essas alterações podem ser aberrações cromossômicas, mutações, polimorfismos e modificações epigenéticas. O câncer se desenvolve quando mecanismos de defesa do organismo sofrem alterações. Os tumores do sistema nervoso representam aproximadamente 2% de todos os tipos de câncer. O papel central do sistema nervoso e as conseqüências funcionais da perda de neurônios podem explicar a severidade dos tumores cerebrais. A formação dos tumores do cérebro humano é um processo complexo, envolvendo um acúmulo de alterações genéticas. Os genes supressores de tumor estão envolvidos na formação de tumores. Sua perda, inativação ou disfunção leva a célula a se dividir desordenadamente, surgindo assim tumores e neoplasias no local onde elas ocorrem. O objetivo deste trabalho foi fazer uma triagem de ocorrência de polimorfismos conformacionais no gene supressor de tumor PTEN em 50 amostras de tumores de Sistema Nervoso. Nenhuma alteração mutacional foi encontrada. Nossos resultados se assemelham aos da literatura em relação à ausência de alterações no gene PTEN em tumores benignos; em relação aos glioblastomas, a literatura cita uma alta freqüência de alterações no gene PTEN, mas não encontramos nenhuma alteração nas 9 amostras estudadas. Outro mecanismo como por exemplo a metilação da região promotora, poderia estar envolvido na inativação desse gene nos tumores analisados. / Cancer is a potentially lethal illness. Tumors develop in cells that are dividing. Its initiation or progression is associated with the accumulation of genetic alterations. These alterations can be chromosome aberrations, mutations, polimorphisms and epigenetic modifications. The cancer develops when mechanisms of defence of the organism are altered. The tumors of the nervous system represent approximately 2% of all the types of cancer. The central role of the nervous system and the functional consequences of the loss of neurons can explain the severity of the cerebral tumors. The formation of the tumors of the human brain is a complex process, involving an accumulation of genetic alterations. The tumor suppressor genes are involved in the formation of tumors. If there is a loss, inactivation or disfunction, the cell will divide disorderly, and tumors and other neoplasias will appear in the place where they occur. The objective of this work was to make a selection of the occurrence of conformacional polymorphisms in the tumor suppresor gene PTEN in 50 samples of tumors of Nervous System. No mutacional alteration was found. Our results if are similar to the ones of the literature in relation to the absence of alterations in gene PTEN in benign tumors; in relation to glioblastomas, literature cites a high frequency of alterations in gene PTEN, but we did not find any alteration in the 9 studied samples. Another mechanism as, for example, the metilation of the promoterl region, could be involved in the inativação of this gene in the analyzed tumors.

La mélanomagenèse est un processus complexe sous-tendu par des mécanismes cellulaires et moléculaires variés. L’ensemble de ces mécanismes moléculaires est impliqué dans les réseaux moléculaires permettant une signalisation coordonnée au sein de la cellule. De nombreuses publications montrent que les voies de signalisation MAPK et PI3K/AKT ont un rôle important dans la mélanomagenèse. NRAS et BRAF sont des oncogènes de la voie MAPK mutés respectivement dans 20% et 50% des mélanomes. PTEN est un gène suppresseur de tumeur inhibant la voie PI3K/AKT, dont la perte est souvent associée aux mutations de BRAF. Le traitement récent des mélanomes métastatiques avec les inhibiteurs spécifiques de BRAFV600E donne des résultats exceptionnels, mais ces résultats sont limités aux patients dont le mélanome est porteur de la mutation BRAFV600E, et il existe naturellement des échappements thérapeutiques, parfois lié à l’apparition de mutations NRAS. Nous avons choisi d’étudier le rôle de NRAS et de PTEN, qui sont des protéines majeures des voies MAPK et PI3K. Le but de ce travail est d’évaluer la coopération de NRAS et PTEN au cours de la mélanomagenèse. L’expression de PTEN est fréquemment altérée au cours du mélanome, mais le rôle de PTEN est mal connu. Au cours de ce travail, nous décrivons pour la première fois une mutation de NRAS concomitante à une perte de PTEN dans des prélèvements humains de mélanome et dans des lignées cellulaires humaines. Afin de comprendre l’effet de cette double mutation sur la mélanomagenèse, nous avons étudié des souris transgéniques avec expression d’une forme oncogénique de NRAS et/ou inactivation de PTEN dans le lignage mélanocytaire. L’inactivation isolée de PTEN n’a aucun effet sur la mélanomagénèse. En revanche, en association avec la mutation oncogénique de NRAS, la perte de PTEN accélère le développement des mélanomes, en réduisant le temps de latence et en provoquant l’apparition de métastases plus nombreuses en comparaison aux mélanomes présentant uniquement la mutation oncogénique de NRAS. Nous avons également démontré que la perte de PTEN induit un échappement au phénomène de sénescence. En conclusion, l’inactivation de PTEN coopère avec les mutations de NRAS pour l’initiation et la progression des mélanomes. / Melanomagenesis is a multistep process involving various cellular and molecular changes. The phospho-inositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways have important roles in melanoma development. Treatment with BRAF inhibitors can be highly effective against metastatic melanomas, but the effects are limited to BRAFV600E patients, and resistance often develops. PTEN and NRAS, both major components of the PI3K and MAPK pathways, are potential alternative targets for melanoma therapy. The aim of this study was to evaluate the cooperation between NRAS and PTEN during melanomagenesis. PTEN expression is frequently altered in melanoma, but the role of PTEN is not yet fully understood. We found concomitant NRAS mutation and loss of PTEN in human melanoma samples and cell lines. Then, we studied transgenic mice with inactivation of PTEN and/or expression of an oncogenic form of NRAS in the melanocyte lineage. PTEN inactivation alone had no discernible effect on melanomagenesis. However, in melanomas initiated by oncogenic NRAS, inactivation of PTEN caused more rapid melanoma development and higher metastatic rate. We demonstrate that PTEN loss induces a senescence bypass. Thus, PTEN inactivation cooperated with oncogenic NRAS to promote melanoma initiation and progression