CHAPTER I: Leishmania spp are obligate intracellular protozoan that can cause a complex of known infectious diseases such as leishmaniasis, affecting millions of people worldwide. The host-pathogen interaction involves the parasite surface molecules and cellular receptors that culminate in phagocytosis. This internalization process involves changes in cytoskeleton through interaction with proteins related to actin polymerization, as AFAP, Arp 2/3 complex, Galectin-3, Septins and WASp, as well as activation and signaling pathways. It is known that L. amazonensis promastigotes after entry into phagocytes are present in phagosomes, and these vesicles undergo a maturation process, forming phagolysosome, important for differentiation of the promastigote forms to amastigotes. This study aimed to understand which proteins related to actin cytoskeleton exert role in the process of invasion and multiplication of L. amazonensis and which signaling pathways are involved in the formation of phagolysosome, the invasion and multiplication of this protozoan process the host cell. For this, we did invasion and multiplication assays with macrophages C57 BL/6 and BALB/c peritoneal treated or not with cytochalasin D, invasion assay with macrophages C57 BL/6 previously fixed in formaldehyde (0.01%), assay with macrophages C57 BL/6 knockdown for AFAP-1L1, Arp2, Galectin-3, Septin 4, 14 and WASP proteins compared to wild type cells. And yet, assays phagolysosome kinetics of formation and proliferation and invasion assays using inhibitors of Akt, ERK2, MEK1, MEK1/2, mTOR, PI3K and Ras signaling pathways. The results show that L. amazonensis invaded cells defective in actin polymerization, as well as fixed cells as well as decreased expression of Arp2, Galectin-3 and WASp proteins increased internalization of these parasites. The signaling pathways MEK1/2, ERK2 and AKT delayed the recruitment of the lysosomes to phagosome, and inhibition of PI3K, MEK1/2 and ERK2 pathways drastically decreased internalization of the parasite in the cells. We concluded that L. amazonensis were able to actively invade phagocytic cells, and that the Arp2, Galectin-3 and WASp proteins are involved in the process of entry into host cells. In addition, the MEK1/2, AKT and ERK2 signaling pathways are involved in the formation of phagolysosome as well as PI3K, MEK1/2 and ERK2 pathways seem to favor infection with L. amazonensis. CHAPTER II: The flagellate protozoan Trypanosoma cruzi is the causative agent for Chagas disease. It is estimated that there are eight million people infected worldwide. T. cruzi has different surface proteins related to cell invasion. In this context, our research group identified in T. cruzi, a protein of 21 kDa (P21) showed that pro-phagocytic and chemotactic activities, playing an important role in the internalization of this parasite in vitro. This study aimed to evaluate the role of recombinant protein P21-His6 based on native P21 of T. cruzi in Leishmania amazonensis in order to seek greater understanding of the mechanism of action of this protein and its relationship with the host in vivo. To this end, the footpad infect BALB/c mouse treated with 40μg of active and denatured recombinant P21, BSA and PBS for 6 weeks and assess the footpad size, parasitic load (real time PCR) and cytokine production of IL-1β in the paws, the popliteal lymph nodes and spleen. The results showed an increase in the footpad area, as well as increased parasite load in the infected and treated animals with P21-His6. Also, increased production of IL-β in the footpad and the popliteal lymph nodes also in animals treated with P21-His6. We conclude that the P21 protein plays a role in pro-phagocytic also in vivo experiments, favoring infection. / CAPÍTULO I: Leishmania spp são protozoários intracelulares obrigatórios, que podem causar um complexo de doenças infecciosas conhecidas como leishmanioses, que afetam milhões de pessoas mundialmente. A interação entre patógeno-hospedeiro envolve moléculas de superfície do parasito e receptores celulares que culminam na fagocitose. Este processo de internalização envolve modificações no citoesqueleto celular por meio da interação com proteínas relacionadas à polimerização de actina, como AFAP, Complexo Arp2/3, Galectina-3, Septinas e WASp, bem como a ativação de vias de sinalização. Sabe-se que formas promastigotas de Leishmania (Leishmania) amazonensis após a entrada nos fagócitos, estão presentes em fagossomos, e essas vesículas sofrem um processo de maturação, formando o fagolisossomo importante para a diferenciação da forma promastigota em amastigota. Este trabalho teve como objetivo, entender quais proteínas relacionadas ao citoesqueleto de actina exercem papel no processo de invasão e na multiplicação de L. amazonensis, bem como, quais vias de sinalização estão envolvidas no processo de formação do fagolisossomo, na invasão e na multiplicação deste protozoário na célula hospedeira. Para isso, fizemos ensaios de invasão e multiplicação em macrófagos imortalizados C57 BL/6 e peritoneais de BALB/c tratados ou não com citocalasina D, ensaio de invasão em macrófagos C57 BL/6 previamente fixadas em formaldeído, ensaio com macrófagos C57 BL/6 knockdown para as proteínas AFAP-1L1, Arp2, Galectina-3, Septina 4 e 14 e WASp, comparando com células Wild Type. E ainda, ensaios de cinética de formação do fagolissomo e ensaios de invasão e multiplicação utilizando inibidores das vias de sinalização envolvendo Akt, ERK2, MEK1, MEK1/2, mTOR, PI3K e Ras foram realizados. Os resultados mostram que L. amazonensis invadiu células deficientes na polimerização de actina, e também células fixadas, bem como a diminuição da expressão das proteínas Arp2, Galectina-3 e WASp aumentaram a internalização desses parasitos. As vias de sinalização MEK1/2, ERK2, AKT atrasaram o recrutamento de lisossomos para o fagossomo, e a inibição das vias PI3K, MEK1/2 e ERK2 diminuíram drasticamente a internalização do parasito nas células. Concluímos que L. amazonensis foi capaz de invadir ativamente células fagocíticas, e que as proteínas Arp2, Galectina-3 e WASp estão envolvidas no processo de entrada na células hospedeira. Além disso, as vias de sinalização MEK1/2, ERK2 e AKT estão envolvidas na formação do fagolisossomo, bem como as vias PI3K, MEK1/2 e ERK2 parecem favorecer a infecção por L. amazonensis. CAPÍTULO II: O protozoário flagelado Trypanosoma cruzi é o causador da Doença de Chagas. Estima-se a existência de 8 milhões de pessoas infectadas em todo o mundo. T. cruzi apresenta diferentes proteínas de superfície relacionadas ao processo de invasão celular. Nesse contexto, nosso grupo de pesquisa identificou em T. cruzi, uma proteína de 21 kDa (P21) que apresentou atividades pro-fagocíticas e quimiotáticas, exercendo papel importante na internalização desse protozoário in vitro. O presente trabalho teve como finalidade avaliar o papel da proteína recombinante P21-His6 baseada na P21 nativa de T. cruzi na infecção por Leishmania amazonensis, a fim de buscar maior entendimento no mecanismo de ação dessa proteína e sua relação com o hospedeiro in vivo. Para isso, infectamos as patas de animais BALB/c e tratamos os animais com 40μg de P21 recombinante ativa e desnaturada, PBS e BSA por 6 semanas e avaliamos o tamanho da pata, a carga parasitária (PCR em tempo real) e a produção da citocina IL-1β nas patas, linfonodos do poplíteo e baços. Os resultados mostraram aumento na área da pata, bem como aumento da carga parasitária nos animais infectados e tratados com a P21-His6. E ainda, aumento da produção da citocina Il-1β na pata e no linfonodo do poplíteo também nos animais tratados com a P21-His6 também foram observados. Concluímos que a proteína P21 exerce papel pro-fagocítico também em experimentos in vivo, favorecendo a infecção. / Mestre em Imunologia e Parasitologia Aplicadas

A proteína P21 do Trypanosoma cruzi participa no processo de infecção da célula hospedeira, desse modo é de grande importância: elucidar as vias de sinalização induzidas pela proteína, bem como caracterizar a nível molecular e estrutural a P21. O que vai auxiliar no entendimento da função biológica da P21 e sua participação no processo de infecção. A P21 recombinante é expressa Escherichia coli em sua maioria na fração insolúvel. Visando aumento de proteína na fração solúvel, foi realizada a clonagem do gene da P21 em vetor pSMT3, bem como testes de expressão subsequentes em diferentes cepas de expressão de E. coli, em vetor pET-28 e pSMT3 e variadas condições de expressão e lise celular. Desse modo obtiveram-se as condições que permitissem uma maior concentração da P21 na fração solúvel. A expressão foi realizada no vetor pET-28, cepa BL21, a 37°C com meio 2xYT a 0.5 mM de IPTG, utilizando a técnica de sonicação como lise. A P21 foi purificada em cromatografia de afinidade e posteriormente em coluna de exclusão molecular. Foram realizados estudos de modelagem por homologia levaram a elaborar a hipótese de que a P21 tem a função de inibidor de serinoprotease do tipo kunitz. Posteriormente essa hipótese foi confirmada com ensaios de avaliação da atividade inibitória da P21 frente à tripsina, quimiotripsina e elastase neutrofílica, o qual a P21 mostrou capaz de inibir a elastase neutrofílica. Estudos com espalhamento dinâmico da luz (DLS) revelaram que as amostras testadas de P21 contém diferentes concentrações de agregados de alto peso molecular em todos os pHs avaliados. Outras medidas foram realizadas para avaliar o estado de agregação da P21 de acordo com a temperatura e verificou-se que entre 32-52 °C, a proteína apresenta menor raio hidrodinâmico, indicando menor agregação nesse intervalo. Estudos de dicroísmo circular revelaram que a P21 apresenta por volta de 20% de α hélice, 32% de folha-β, 22% de volta e 23 % estrutura randômica. De acordo com a curva de desnaturação referente ao espectro de CD obtido, a P21 se mostra desnaturada a partir de 64°C. / The Trypanosoma cruzi protein P21 participates in the host cell infection process, but its specific function is poorly described. Thus it is important to elucidate the signaling pathways induced by the protein and characterize the P21 at the structural and molecular levels, as a contribution towards the understanding of the P21 biological function and its role in the infection process. The Escherichia coli recombinant P21 is expressed mostly in the insoluble fraction. Aiming to increase protein in the soluble fraction, we performed the cloning of the P21 gene in pSMT3 vector and subsequent expression tests in different expression strains of E. coli in pET-28 and pSMT3 vectors and varied expression conditions and cell lysis. Thus we obtained conditions that allow a greater concentration of the P21 in the soluble fraction. The expression vector was performed using vector pET-28, in Bl21 strain at 37°C in 2xYT culture medium with 0.5 mM IPTG, using the sonication technique in cell lysis. The recombinant P21 was purified by Ni affinity chromatography and subsequently a molecular exclusion column. We performed homology modeling studies which led to assume that P21 can be a serinprotease inhibitor of Kunitz type. Furthermore, this hypothesis was confirmed in the experiments testing the P21 inhibitory activity against the trypsin, chymotrypsin and elastase. We found the P21 exclusively inhibit neutrophil elastase. Studies using dynamic light scattering (DLS) revealed that the samples containing P21 contained large molecular weigth aggregates in different concentrations at all evaluated pHs. Others measurements were performed to assess the P21 aggregation state according to the temperature and we found that between 32-52 °C the protein had a smaller hydrodynamic radius, indicating less aggregation in this range. Circular dichroism (CD) studies revealed that P21 has about 20% α-helix, 32% β, -sheet, 22% turn and 23% of random structure. According to the denaturation curve for the CD spectrum obtained, the P21 was denatured from 64°C.

A resistência tubular renal tem sido estudada a fim de se ampliar a compreensão da fisiopatologia da Insuficiência renal aguda (IRA). A isquemia renal induz à resistência a um subseqüente insulto isquêmico sendo que os mecanismos de resistência parecem depender de alterações celulares. O p21 é um inibidor do ciclo celular, o qual pode ser induzido por radicais livres de oxigênio e parece ter um efeito protetor na IRA isquêmica. O objetivo deste estudo é avaliar o papel do p21 e do estresse oxidativo em modelo de resistência adquirida após episódio de IRA isquêmica, e em túbulos proximais isolados após isquemia. Ratos Wistar foram divididos em 3 grupos: grupo 1- sham, grupo 2- submetido a procedimento sham e após 2 dias submetido à isquemia de 45 min e grupo 3- submetido à isquemia de 45 min e após 2 dias submetido à segunda isquemia de 45 min. Os valores de uréia plasmática (114±60 vs. 136±44 mg/dL, n.s.), a creatinina sérica (0,86±0,2 vs. 0,98±0,1mg/dL, n.s.) e o clearance de creatinina (0,21±0,1vs. 0,24±0,1mL/min/100g, n.s.), avaliados 48 h após o segundo procedimento (Dia 4), foram semelhantes entre os grupos 2 e 3. O tempo de recuperação da IRA também foi semelhante entre os grupos 2 e 3. A histologia mostrou necrose tubular aguda aparentemente de grau semelhante entre os grupos 2 e 3. O infiltrado linfocitário foi semelhante entre os 3 grupos, entretanto houve aumento no infiltrado de macrófagos no grupo 3. Foi observado aumento na proliferação celular no grupo 2 e grupo 3, quando comparados ao grupo 1(125±28 cél./mm2, p<0,05), entretanto, a proliferação foi mais intensa no grupo 2 (1.262±440 cél /mm2) que no grupo 3 (653±300 cél /mm2, p<0,05 vs. group 2). O grau de apoptose encontrado foi semelhante entre o grupo 2 e o grupo 3. Houve aumento na expressão do p21 apenas no grupo 3 sendo que esta expressão foi semelhante nos grupos 1 e 2. Foi estudada também a resistência celular em túbulos proximais (TP) isolados de ratos normais (grupo Controle) e ratos submetidos à isquemia de 35 min, 24 h antes do estudo (grupo Isquemia). TP do grupo Isquemia foram susceptíveis à hipóxia, porém, resistentes à lesão de reoxigenação. Além disto, apresentaram menor produção de hidroperóxidos. Portanto, a resistência renal isquêmica aparentemente está associada a mecanismos celulares, o estresse oxidativo e o aumento na expressão do p21 são possíveis mediadores destes mecanismos. / Renal tubular resistance has been studied for the understanding of ischemic acute renal failure (ARF). Subsequent ischemic episodes may induce renal resistance whose mechanisms seem to be related to cell alterations. P21 is a cell cycle inhibitor that may be induced by oxygen free radicals and may have a protective effect in ischemic ARF. This study aimed at evaluating the role of oxidative stress and p21 on tubular resistance in isolated renal tubules and in a model of acquired resistance after renal ischemia. Wistar rats were divided into 3 groups: group 1 - sham; group 2 - submitted to sham procedure and after 2 days submitted to 45 min ischemia and group 3 - submitted to ischemia of 45 min followed by a second 45 min ischemia after 2 days. Plasma urea levels (114±60 vs. 136±44 mg/dL), serum creatinine (0.86±0.2 vs. 0.98±0.1mg/dL) and creatinine clearance (0.21±0.1vs. 0.24±0.1mL/min/100g.) evaluated at 48 hours after the second procedure were similar between groups 2 and 3 (all NS). ARF recovery time was also similar between groups 2 and 3. Histology disclosed the same degree of acute tubular necrosis between groups 2 and 3. Lymphocytes infiltrate was similar among all groups whereas macrophages infiltrate was greater in group 3. Enhanced cell proliferation was observed in groups 2 and 3 when compared with group 1 (125±28 cel/mm2, p<0.05), however it was greater in group 2 (1,262±440 cel/mm2) than group 3 (653±300 cel/mm2, p<0.05 vs. group 2). Degree of apoptosis was similar between groups 2 and 3. The p21 expression was increased only in group 3 whereas it was similar in groups 1 and 2. Cell resistance was also evaluated in isolated renal proximal tubules (PT) from control and ischemia groups. In the latter group, animals were submitted to 35 min ischemia and PT were isolated one day later. PT from the ischemia group were sensitive to hypoxia but resistant to reoxygenation injury which was followed by lower hydroperoxides production. In conclusion, renal resistance obtained by an ischemia was associated with cell mechanisms involving oxidative stress and increased p21 expression as mediators of this protection.

Purpose: To enhance prognostic information of protein biomarkers for clear cell renal cell carcinomas (ccRCCs), we analyzed them within prognostic groups of ccRCC harboring different tumor characteristics of this clinically and molecularly heterogeneous tumor entity. Methods: Tissue microarrays from 145 patients with primary ccRCC were immunohistochemically analyzed for VHL (von Hippel-Lindau tumor suppressor), Ki67 (marker of proliferation 1), p53 (tumor protein p53), p21 (cyclin-dependent kinase inhibitor 1A), survivin (baculoviral IAP repeat containing 5), and UEA-1 (ulex europaeus agglutinin I) to assess microvessel-density. Results: When analyzing all patients, nuclear staining of Ki67 (hazard ratio [HR] 1.08, 95% confidence interval [CI] 1.04–1.12) and nuclear survivin (nS; HR 1.04, 95% CI 1.01–1.08) were significantly associated with disease-specific survival (DSS). In the cohort of patients with advanced localized or metastasized ccRCC, high staining of Ki67, p53 and nS predicted shorter DSS (Ki67: HR 1.07, 95% CI 1.02–1.11; p53: HR 1.05, 95% CI 1.01–1.09; nS: HR 1.08, 95% CI 1.02–1.14). In organ-confined ccRCC, patients with high p21-staining had a longer DSS (HR 0.96, 95% CI 0.92–0.99). In a multivariate model with stepwise backward elimination, tumor size and p21-staining showed a significant association with DSS in patients with 'organ-confined' ccRCCs. The p21-staining increased the concordance index of tumor size from 0.75 to 0.78. In patients with 'organ-confined' ccRCC, no disease-related deaths occurred in the group with p21-expression below the threshold of 32.5% p21-positive cells (log rank test: P=0.002). Conclusion: The prognostic information of the studied protein biomarkers depended on anatomic tumor stages, which displayed different acquired biological tumor characteristics. Analysis of prognostic factors within different clinical ccRCC groups could help to enhance their prognostic power. The p21-staining was an independent prognostic factor and increased prognostic accuracy in a predictive model in 'organ-confined' ccRCC.

Mutation des p53 Gen ist die häufigste genetische Veränderung in allen humanen Tumoren. Weit verbreitete p53 misssense-Mutationen heben die Tumor suppressive Funktion auf und führen zu gain-of-function Eigenschaften, die Tumorproliferation, Chemoresistenz, Angiogenese, Migration, Invasion und Metastasen fördern. In dieser Arbeit haben ich für drei solche Hotspot-Mutationen, p53R245Q, p53R246S und p53R270H, eine höhere Sensitivität gegenüber Behandlung mit Piperlongumine in p53-defizienten MEFs und Eµ-myc Lymphomzellen im Vergleich zur Kontrolle und den anderen drei Hotspot-Mutationen, p53R172H, p53G242S und p53R279Q, gefunden. Nachfolgend, haben ich entdeckt, dass Piperlongumine-induzierter Zelltod durch ROS Akkumulation über die Aktivierung von p38 und JNK, vermittelt wurde. Das Antioxidans N-acetyl-L-cysteine (NAC) oder p38/JNK Inhibitoren konnten vollständig oder teilweise Piperlongumine-induzierten Zelltod unterdrücken. Nach Behandlung mit Piperlongumine, haben die p53R245Q, p53R246S und p53R270H-Mutanten die Aktivierung von p21 inhibiert und so die Aktivierung und Funktion von NRF2, durch Piperlongumine induziert, blockiert, dass zu dem massiven Zelltod in Zellen mit diesen Mutationen beiträgt. Auf ähnliche Weise, verursachte der klinisch verwendete Inhibitor von Crm1, KPT-330, schweren Zelltod in p53-/- MEFs mit den p53R245Q, p53R246S und p53R270H-Mutationen. Folglich könnte Crm1 als potenzielles Target für Lymphome mit p53R245Q, p53R246S und p53R270H-Mutationen berücksichtigt werden. Zusammenfassend bekräftigen die Daten in dieser Arbeit das Phänomen, dass oxidativer Stress oder Crm1 Inhibitoren effektiv Zellen mit p53R245Q, p53R246S und p53R270H-Mutationen eliminieren können, mit niedriger Toxizität für Kontrollzellen. Demzufolge, könnten oxidativer Stress Signalwege oder Crm1 als potenzielle Angriffsziele für Lymphome mit p53R245Q, p53R246S und p53R270H-Mutationen dienen. / Mutation of the p53 gene is the most common genetic alteration among all human cancers. Prevalent p53 missense mutations abrogate its tumor suppressive function and lead to gain-of-function properties that promote cancer cell proliferation, chemoresistance, angiogenesis, migration, invasion, and metastasis. This doctoral thesis aims to identify the metabolic vulnerabilities of six p53 hotspot mutants in lymphomas. In this work, three hotspot mutants, p53R245Q, p53R246S and p53R270H, were more sensitive to piperlongumine treatment in p53-deficient MEFs and Eμ-myc lymphoma cells than the empty control and the other three hotspot mutants, p53R172H, p53G242S and p53R279Q. Thereafter, I found piperlongumine-induced cell death was mediated by ROS accumulation via the activation of p38 and JNK. Antioxidant N-acetyl-L-cysteine (NAC) or p38/JNK inhibitors could completely or partially suppress piperlongumine-induced cell death. Upon piperlongumine treatment, p53R245Q, p53R246S and p53R270H-mutant inhibited piperlongumine-induced activation of p21 and consequently attenuated the activation and function of NRF2 induced by piperlongumine, contributing to the massive cell death in cells harboring these mutants. Similarly, KPT-330, a clinical inhibitor of Crm1, also caused severe cell death in p53-/- MEFs harboring p53R245Q, p53R246S and p53R270H-mutant. This implied that Crm1 could be also considered as a potential target for lymphomas harboring p53R245Q, p53R246S and p53R270H-mutant. Taken together, data presented in this work underscore the phenomenon that exogenous oxidative stress or Crm1 inhibitor is effective in eliminating cells harboring p53R245Q, p53R246S and p53R270H-mutant with low toxicity to cells harboring the empty control, suggesting oxidative stress pathways or Crm1 as potential targets in lymphomas with p53R245Q, p53R246S and p53R270H-mutant.

5 mutations responsables de retard mental ont été identifiées dans le gène p21-activated kinase 3 (pak3). Nous avons récemment identifiés dans pak3 deux exons alternatifs très conservés appelés b et c. Ainsi, en plus du variants PAK3a (dépourvu des inserts b ou c), le gène pak3 code pour 3 nouveaux variants d'épissage PAK3b, PAK3c et PAK3cb qui sont constitutivement actifs et insensibles aux GTPases. De plus, contrairement à PAK1 et PAK3a, leur domaine d'auto-inhibition est incapable d'inhiber un domaine kinase. Ainsi, le but de ce projet était de comprendre le mécanisme de régulation de la kinase PAK3. Un modèle de régulation a récemment été proposé dans lequel PAK1 forme des homodimères pouvant être dissociés par les GTPases, permettant ainsi l'activation de la kinase. En se basant sur ces observations j'ai cherché à identifier les dimères PAK3 et j'ai montré que les kinases PAK3a, b, c et cb forment préférentiellement des hétérodimères avec PAK1. J'ai démontré l'existence de ces dimères dans le cerveau et j'ai mis en évidence que ces hétérodimères permettent à chaque monomère de réguler l'activité kinase de son partenaire in vitro. Ce travail permet de proposer un modèle de régulation symétrique pour PAK3a qui forme des hétérodimères avec PAK1 et un nouveau modèle de régulation asymétrique pour les variants d'épissage, également basé sur leur hétérodimérisation avec PAK1. Mes résultats montrant une corégulation des kinases PAK neuronales suggèrent d'une part que leur activation puisse être synchronisée et d'autre part que dans certaines situations physiopathologiques (Cancer et maladies neurologiques) leur dérèglement puissent interférer.

Résumé : Le “Suppressor of cytokine signaling -1” (SOCS1) est une protéine de 24 kD qui fonctionne principalement comme un régulateur négatif des voies de signalisation intracellulaires. SOCS1 inhibe l’axe JAK-STAT et induit l’ubiquitylation et la dégradation de certaines protéines cibles. L’expression de SOCS1 est diminuée par l’hyperméthylation de son promoteur dans plus de 65% des cas de carcinome hépatocellulaire. Les souris déficientes en SOCS1 ne survivent que trois semaines après la naissance à cause d’une hyperinflammation induite par IFN-[gamma]. Afin d’étudier le rôle anti tumoral de SOCS1 dans le foie, nous avons utilisé les souris Socs1[indice supérieur -/-]Ifng[indice supérieur -/-], Ifng[indice supérieur -/-] et les souris de type sauvage. Nous avons démontré que le taux de la régénération du foie après une hépatectomie partielle était augmenté chez les souris déficientes en SOCS1. De plus, les souris Socs1[indice supérieur -/]-Ifng[indice supérieur -/-] étaient plus susceptibles pour le développement des nodules hépatiques suite à un traitement avec diethylnitrosamine (DEN). Par contre, les souris déficientes en IFN-[gamma] ont démontré une résistance contre le cancer du foie. Néanmoins, au contraire de nos attentes préliminaires, nous n’avons pas observé une augmentation des taux sériques d’IL-6. Pourtant, la prolifération compensatoire et la synthèse de l’ADN étaient élevées chez les souris SOCS1 KO. Afin d’expliquer cette observation, nous avons étudié l’activation de p53. Nous n’avons pas trouvé une réponse différente de stabilisation ni de phosphorylation de p53 (Ser15) après traitement au cisplatin (in vitro) ou DEN (in vivo). Par contre, nous avons observé que l’expression du gène Cdkn1a était élevée chez les hépatocytes déficients en SOCS1. De plus, l’expression ectopique de SOCS1 pouvait supprimer l’expression de p21 chez les cellules HepG2 traitées au cisplatin. Nous avons aussi constaté que la stabilité de p21 était augmentée chez les hépatocytes primaires déficients en SOCS1. En effet, SOCS1 induisait l’ubiquitylation et la dégradation de p21. SOCS1 pouvait interagir avec p21 par son domaine SH2. De plus, SOCS1 pouvait contrôler la localisation cytoplasmique de p21 en régulant l’activité d’AKT. Bien que p21 soit connu comme un inhibiteur du cycle cellulaire, il peut également participer à l’assemblage des complexes CDK4-Cyclin D. Nous avons démontré que l’expression de p21 et des cyclines de type D était augmentée chez les souris déficientes en SOCS1 après l’hépatectomie partielle. En diminuant l’expression de p21 par shRNA, nous avons empêché la réponse proliférative des hépatocytes SOCS1 KO. Finalement, nous avons trouvé que l’expression élevée de p21 chez les hépatocytes déficients en SOCS1 rendait les cellules plus résistantes contre l’apoptose. En conclusion, nos résultats suggèrent que SOCS1 protège contre le cancer du foie par la régulation des activités oncogéniques de p21. // Abstract : Suppressor of cytokine signaling - 1 (SOCS1) is an inducible 24 kD protein that principally acts as a negative regulator of different intracellular signaling pathways. SOCS1 exerts its regulatory feedback by blocking the JAK - STAT axis and inducing ubiquitylation and subsequent proteasomal degradation of target proteins. The gene coding for Socs1 has a CpG - rich promoter and can be methylated by methyltransferases. SOCS1 is silenced due to hypermethylation of its promoter in almost 65% of hepatocellular carcinoma cases. SOCS1 deficient mice cannot survive more than three weeks of age because of enhanced IFN - [gamma] induced inflammation. To better understand the tumor suppressor role of SOCS1 in the liver we used Socs1[superscript - / -]Ifng[superscript - / -], while Ifng[superscript - / -] and wild type mice served as controls. We found that SOCS1 deficient mice showed accelerated liver regeneration following standard partial hepatectomy (PH). Moreover, Socs1 null mice were susceptible to development of hepatic nodules after treatment with diethylnitrosamine (DEN). Interestingly, the IFN - [gamma] deficient mice showed reduced number of liver tumors. In contrast to our preliminary expectations, we did not observe elevated IL - 6 serum levels in SOCS1 deficient mice compared to the controls. Nevertheless, loss of SOCS1 was associated with increased compensator y proliferation and DNA synthesis after PH and DEN treatment. To find an explanation for the increased tumorigeneis in the SOCS1 deficient liver, we examined the activation of p53 and its target genes. Although we observed neither a variable phosphorylation (Ser15), nor an impaired stabilization of p53 after cisplatin ( in vitro ) or DEN treatment ( in vivo ), Cdkn1a expression was increased in the absence of SOCS1. We also found that ectopic expression of SOCS1 could suppress the mRNA levels of p21 in HepG2 cells treated with cisplatin. In addition, we found that loss of SOCS1 increased p21 stability in hepatocytes and that SOCS1 could induce p21 ubiquitylation and subsequent proteasomal degradation. We showed that SOCS1 could bind directly to p21 via its SH2 domain. Furthermore, in SOCS1 deficient hepatocytes, p21 was retained in the cytosol in an AKT dependent fashion. While classically known as a cell cycle inhibitor, p21 can promote the assembly and kinase activity of CDK4 - cyclin D complexes. We showed that D - type cyclins and p21 levels were increased in the liver of SOCS1 deficient mice following PH. Suppression of p21 by transient shRNA transfection in SOCS1 deficient primary hepatocytes could reverse their increased proliferative response to mitogens. Finally, we found that increased p21 expression in SOCS1 deficient hepatocytes renders them resistant to apoptosis. In conclusion, our findings suggest that SOCS1 protects against liver cancer via inhibiting the oncogenic potential of p21.

Geólogo / El desarrollo de la tecnología minera subterránea en los últimos años ha permitido un rápido avance en la construcción de túneles y labores, lo que ha llevado a tener una menor cantidad de tiempo para levantar información geológica relevante. Esto último ha producido la existencia de zonas sin información de estructuras, lo que constituye potencialmente un riesgo no solo a la infraestructura sino también a la vida de las personas. En este contexto se enmarca la presente memoria de título, en la cual se busca, a partir de estructuras mayores mapeadas en zonas con alta resolución de información, calcular la densidad de estructuras (P21) para así poder complementar la información de aquellas zonas con baja resolución en la mina El Teniente. Los parámetros utilizados para los cálculos de P21 fueron: densidad de mapeo, largo de estructuras, espesor típico, amplitud, ondulación y efecto de la litología. La información fue obtenida de los mapeos históricos de las minas Reservas Norte (ReNo) y Esmeralda, en sus respectivos niveles de producción y hundimiento. Para analizar los resultados de P21 obtenidos, se dividió el problema en 3 casos comparativos: entre nivel de producción y hundimiento de una misma mina, entre dos zonas de una misma mina en un mismo nivel y entre dos minas distintas del yacimiento. Los resultados muestran que entre los niveles de producción y hundimiento ubicados a una diferencia de cota de 17 metros, se encuentran las mismas familias, orientaciones y espesores de estructuras. Sin embargo, lo anterior no se traduce en que los valores de P21 sean similares, lo que sugiere que estos dependen de variables estadísticas como número de mapeos y/o densidad de mapeos y no de variables geológicas. Por otra parte, las variaciones de P21 dentro de un nivel, estarían controladas por la cercanía a los sistemas de fallas y/o a los contactos litológicos, donde aquellos en que se involucra el Complejo Máfico El Teniente (CMET) suelen poseer valores más altos. Además, las dos minas a pesar de presentar litologías y características geométricas similares poseen densidades distintas (0,08 m/m2 y 0,15 m/m2 promedio). Si a lo anterior se le suma que la mina con mayor número de estructuras mapeadas (Esmeralda) posee un menor largo interpretado, indicaría que las diferencias se deben a una interpretación que subestima o sobrestima la información existente. En cuanto a relaciones geométricas, se obtuvo una relación lineal entre espesor y largo de fallas, que mejoró aquella creada anteriormente en El Teniente y que se acerca más a la existente en la literatura. Utilizando dicha curva y ocupando la información de diferencias de P21 en distintas minas es posible concluir que mina Esmeralda subestima la información, por lo que se recomienda realizar nuevos estudios que permitan mejorar dichas interpretaciones.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.