Apoptosis represents one of the cell death mechanisms which is realized after the application of taxanes in breast cancer cell lines. Apoptosis induction can be principally triggered either by outer or inner pathway. The aim of the diploma thesis is to contribute to the elucidation of role and mechanisms of the inner mitochondrial pathway of apoptosis induction after taxane application (paclitaxel and SB-T-1216) employing a model of breast carcinoma cell lines SK- BR-3 (nonfunctional p53, functional capase-3) and MCF-7 (functional p53, nonfunctional caspase-3). Specifically, we tested the effect of both employed taxanes on mitochondrial membrane potential, ROS level and the expression and localization of proteins regulating inner mitochondrial pathway. Taxane application resulted in mitochondrial membrane dissipation in SK-BR-3 cell line. However, this was not shown in MCF-7 cell line. We found no changes in Bax and Smac/DIABLO expression after taxane application in both tested cell lines. There was a decrease of Bid expression after taxane application in SK-BR-3 line, but not in MCF-7 line. Taxane application did not lead to the translocation of Bax and Bid (tBid) proteins from cytosol to mitochondria in both tested cell lines. Similarly, there was no Smac/DIABLO release from mitochondria to...

Ziel der vorliegenden Arbeit war es, die Rolle des pro-apoptotischen Proteins Bim am endoplasmatischen Retikulum (ER) und an den Mitochondrien zu untersuchen. Für diese Untersuchungen wurden zwei Isoformen von Bim verwendet, zum einen BimL, welches an den Motor Dynein Komplex gebunden ist, zum anderen BimS, welches im Zytosol lokalisiert ist. Um eine konditionale Expression von Bim zu erreichen, wurde Myc-markierte humane cDNA unter der Kontrolle des Tet-Off Systems in einen adenoviralen Vector kloniert. Eine Überexpression von BimL und BimS induzierte in der Prostatakarzinomzelllinie DU145 Bax- und Bak-abhängigen apoptotischen Zelltod. Eine Überexpression des anti-apoptotischen Proteins Bcl-2 lokalisiert am ER zeigte eine vollständige Hemmung der Bim-induzierten Apoptose, was die Wichtigkeit des ER unterstreicht. Überexpression von Bcl-2 an den Mitochondrien führte eine partielle Hemmung herbei. Bim Expression induzierte Bax- und Bak-abhängig den Zusammenbruch des mitochondrialen Membranpotentials. Dieses wurde ebenso in mit am ER lokalisiertem Bcl-2 Zellen beobachtet. Bcl-2 lokalisiert an den Mitochondrien verminderte dagegen mitochondriale Permeabilisation. Proteinanalysen zeigten eine Hochregulierung von ER-Stress Proteinen nach Bim Überexpression. Zusätzlich wurde Cytochrom c Freisetzung aus den Mitochondrien und Aktivierung von Caspase-9, -3 und -8 beobachtet. Mit einem Breitband-Caspase Hemmer konnte der Bim-induzierte Zelltod vollständig gehemmt werden, was zeigt, dass Caspasen essentiell sind. Zusammenfassend kann gesagt werden, dass Bim, parallel zum mitochondrialen Signalweg, ER-Stress auslöst und, dass Bim eine effektive Apoptose durch die Interaktion des ER und der Mitochondrien induziert. / The aim of this thesis was to investigate the role of the pro-apoptotic BH3-only protein Bim, at the endoplasmic reticulum (ER) and the mitochondria. For this purpose, a full length human myc-tagged Bim cDNA was cloned into an adenoviral vector, which allows for the conditional expression of the transgene under the control of a Tet-Off-system. Two different Bim isoforms were used for these investigations. One was BimL, which is bound to the motor dynein complex of the microtubule and the other one was BimS, which is localized in the cytosol. The enforced expression of each of these two isoforms in the prostate cancer cell line DU145, showed the capability of BimL and BimS to induce apoptosis via either Bak or Bax. Also, Bax- and Bak-dependent breakdown of the mitochondrial membrane potential upon overexpression of either Bim isoforms was measured. This effect was also observed in cells overexpression the anti-apoptotic protein Bcl-2 at the ER. However, targeting Bcl-2 to the mitochondria partially inhibited Bim-induced mitochondrial permeabilization. These findings indicated the execution of the intrinsic apoptotic pathway upon Bim signalling. Nevertheless, expression of Bcl-2 at the mitochondria partially suppressed Bim-induced apoptosis whereas ER-targeted Bcl-2 entirely prevented cell death induction by Bim underlining the importance of the ER. Further, an upregulation of ER stress proteins upon Bim expression was seen. Cytochrome c release form the mitochondria and activation of caspase-9, -3 and -8 was observed. In addition, the complete inhibition of Bim-induced cell death by a pan caspase inhibitor revealed that caspases are crucial. In conclusion, Bim induces the mitochondrial apoptotic pathway and, in parallel, triggers ER stress. It seems that Bim mediates cell death through the interaction of the mitochondria and the ER. The ER-mitochondria cross-talk leads to the amplification of the apoptotic death signal.

INTRODUÇÃO: O carcinoma de pulmão de pequenas células (CPPC) e o carcinoma neuroendócrino (CNE) extra-pulmonar apresentam características histopatológicas e tratamentos similares, porém os desfechos encontrados nos dois grupos podem ser diferentes. Avaliamos a expressão de alguns biomarcadores e a associação destes com taxa de resposta (TR) à quimioterapia baseada em platina e sobrevida global (SG) nos dois grupos. METODOS: Realizamos estudo retrospectivo de pacientes com CPPC e CNE extra-pulmonares tratados com quimioterapia baseada em platina. Todas as amostras tumorais foram revisadas pelo mesmo patologista (R.S.S.M.) e analisadas quanto a expressão imunoistoquímica de Ki-67, ERCC1, Bcl-2 e Lin28a, a qual foi determinada através do H-escore (calculado multiplicando o produto da intensidade da coloração - 0 a 3 - com a porcentagem de células positivas - 0 a 100 -, podendo variar de 0 a 300 - positivo quando >= 200). Os biomarcadores foram analisados tanto como variáveis contínuas quanto categóricas e a TR foi determinada por RECIST 1.1. A associação entre a expressão de cada biomarcador e a TR foi avaliada através do teste de qui-quadrado ou teste exato de Fisher para variáveis categóricas e regressão logística simples para variáveis contínuas. Sobrevida global foi estimada por Kaplan-Meier e as curvas foram comparadas por log-rank. O modelo de regressão de cox foi utilizado para avaliar associação entre SG e a expressão de biomarcadores como variável contínua. RESULTADOS: Entre Julho de 2006 e Julho de 2014, 142 pacientes foram identificados: N=82 (57,7%) com CPPC e N=60 (42,3%) com CNE extra-pulmonar. As características clínicas eram semelhantes em ambos os grupos. Mediana de ki67 foi de 60% (7-100) no CPPC e de 50% (20-95%) no segundo grupo (p=0,858). Com uma mediana de 5 ciclos por paciente (N=123 elegíveis para análise de TR), a TR foi de 86,8% no CPPC, enquanto nos com CNE extra-pulmonar, foi de 44,6% (p < 0.001). A mediana de SG (N=132 elegíveis para análise da SG) foi similar entre os grupos (10,3 meses em CPPC e 11,1 meses em CNE extra-pulmonar; p=0,069). Não houve diferença no padrão de expressão do ERCC1 (p=0,277) e do Lin28a (p=0,051) entre os grupos. Bcl2 foi expresso em 38 pacientes (46,3%) com CPPC e em 17 pacientes (28,3%) com CNE extra-pulmonar (p=0,030). Apenas no grupo com CNE extra-pulmonar, a alta expressão do Bcl2 foi associada com pior prognóstico (8,0 meses vs 14,7 meses; p=0,025). A expressão dos demais marcadores em CNE extra-pulmonar e dos quatro em CPPC não apresentou influência sobre a SG, não havendo também associação entre estes e a taxa de resposta à quimioterapia. Dentre os pacientes com CNE extra-pulmonar, não houve diferença na SG ou na TR entre os pacientes com carcinoma bem diferenciado (N=13;) e com carcinoma pouco diferenciado (N=47). CONCLUSÃO: Apesar do CPPC e do CNE extra-pulmonar serem tratados de forma semelhante, nesta coorte a taxa de resposta entre os grupos foi significativamente diferente. Quando comparado com CPPC, os pacientes com CNE extra-pulmonar apresentam uma menor responsividade à quimioterapia baseada em platina, mas com tendência a maior SG. Dentre os CNE extra-pulmonares, a alta expressão de Bcl-2 foi associada a pior prognóstico. Os demais biomarcadores não apresentaram papel preditor de resposta ou prognóstico / INTRODUCTION: Small cell lung cancer (SCLC) and high-grade extrapulmonary neuroendocrine carcinomas (EPNEC) share similar histopathological features and treatment, but outcomes may differ. We evaluated the expression of biomarkers and their association with response rate (RR) to platin-based chemotherapy and overall survival (OS) in these entities. METHODS: We conducted a retrospective analysis of patients with advanced EPNEC and SCLC treated with platinum-based chemotherapy. A single pathologist (R.S.S.M.) revised all samples. Paraffin-embedded tumor samples were tested for Ki-67, ERCC1, Bcl-2 and Lin28a expression by immunohistochemistry (IHC). Final IHC score (H-score) was calculated multiplying the intensity of staining by grading (0-300, with >= 200 considered positive). Biomarkers were analyzed as both categorical and continuous variables. RR was determined by RECIST 1.1. Associations between each biomarkers expression and RR were assessed using Chi-square or Fisher\'s exact test for categorical variables and univariate logistic regression for continuous variables. OS was estimated by the Kaplan-Meier method and curves were compared by log-rank. Cox regression analysis was used to evaluate any association between biomarkers expression (continuous variables) and OS. RESULTS: From July 2006 to July 2014, 142 patients were identified: N=82 (57,7%) with SCLC and N=60 (42,3%) with EPNEC. Baseline clinical characteristics were similar. Median Ki67 was 60% (7-100) among SCLC patients and 50% (20-95%) in EPNEC (p=0,858). With a median of 5 cycles per patient in both groups (N=123 evaluable patients), the RR was significantly higher in the SCLC group (86,8% vs 44.6%; p < 0.001). Median OS (N=132 evaluable patients) was similar between the groups (10.3 months in SCLC and 11.1 months in EPNEC; p=0,069). In the EPNEC group, there wasn\'t any difference in OS or RR between the patients with welldifferentiated (N=13) and poorly differentiated carcinoma (N=47). ERCC1 (p=0.277) and Lin28a (p=0.051) were similarly expressed between the groups. Bcl2 was expressed in 38 SCLC patients (46.3%) and in 17 EPNEC patients (28.3%; p=0.030). Only in the EPNEC group, Bcl2 high expression was associated with worse survival (8.0 months vs 14.7 months; p = 0.025). RR to chemotherapy was not influenced by the expression of the ERCC1, Lin28a, Bcl-2, Ki-67 in either EPNEC or SCLC groups. CONCLUSION: Even though SCLC and EPNEC are treated similarly, in this cohort, the rate response differed significantly. When compared with SCLC, patients with EPNEC apparently had tumors less responsive to platin-based chemotherapy, but tended to live longer. In EPNEC treated with platin, high expression of Bcl2 was associated with poor prognosis. We could not identify additional predictive or prognostic biomarkers

INTRODUÇÃO: As alterações da mucosa do estômago excluso após Derivação Gástrica em Y-de-Roux (DGYR) para tratamento da obesidade mórbida não são bem conhecidas. Atualmente, pouco se sabe a respeito das consequências da cirurgia, especialmente, considerando que tal técnica necessita de vigilância para possíveis alterações de mucosa. Adicionalmente, é possível que o refluxo duodenal biliopancreático para dentro do estômago excluso, sem tamponamento pela ingestão de alimentos, pode, após décadas, danificar a mucosa gástrica e provavelmente aumentar o risco de câncer gástrico. OBJETIVO: Analisar as alterações da mucosa do estômago excluso através de: índice de proliferação celular (Ki-67), apoptose (caspase 3 e Bcl-2), função hormonal (gastrina) e infiltrado inflamatório (CD3 e CD8). MÉTODOS: Enteroscopia de duplo balão foi realizada em 35 pacientes submetidos à DGYR com mais de 36 meses de cirurgia. Foram realizadas múltiplas biópsias no coto gástrico funcional e na mucosa do estômago excluso. Biópsias gástricas de 32 pacientes obesos não operados foram utilizadas como grupo controle. Biópsias endoscópicas foram seccionadas a partir de blocos de tecidos fixados em formalina e embebidos em parafina. Amostras de 4 m de espessura foram examinadas por imuno-histoquímica pelo método de estreptavidina-biotinaperoxidase. RESULTADOS: Os dois grupos foram comparados por idade, gênero, presença de gastrite, metaplasia intestinal e de Helicobacter pylori. O número médio de células de gastrina positivas foi de 55,5 (desvio padrão (DP) = 11,7) no grupo controle e 29,6 (DP = 7,9), nos casos, p= 0,0003. Índice de proliferação (Ki-67) nos casos (corpo=24,7%, antro=24,9%) foi significativamente maior em comparação com os controles (corpo=15% e antro=17,7%), p = 0,002 e 0,01 ,respectivamente. Imunoexpressão de caspase 3 foi maior nos controles em comparação ao estômago excluso (31 x 46%), p = 0,02. Não houve diferença estatística entre as expressões de CD3 , CD8 , e Bcl- 2 nos controles e nos casos. Não houve associação entre os resultados imuno-histoquímicos e a presença de Helicobacter pylori ou alterações histológicas. CONCLUSÕES: Proliferação celular está aumentada e a apoptose está diminuída na mucosa do estômago excluso em comparação com os controles obesos não operados. Alterações na renovação celular e nas secreções hormonais nestas condições podem ser relevantes em seguimento a longo prazo / INTRODUCTION: Mucosal alterations in the excluded stomach mucosal after Roux-en-Y gastric bypass for morbid obesity have not been clearly clarified. Currently, little is known regarding the long-term consequences of the surgical specially considering that the technique hinders surveillance for possible mucosal alterations. Indeed, it is possible that duodenal reflux of bile and pancreatic secretions without any buffering offered by food intake may, after decades, damage the gastric epithelium and lead to an increase gastric cancer risk. OBJECTIVE: This study aims to analyze the mucosal alterations (proliferative status (Ki-67), apoptosis (caspase 3 and Bcl-2), inflammatory response (CD3 and CD8) and for hormonal function (gastrin)) in the excluded stomach. METHODS: Double-balloon enteroscopy was performed in 35 patients who underwent Roux-en-Y gastric bypass longer than 36 months. Multiple biopsies of the proximal pouch and the excluded gastric mucosa were collected. Gastric biopsies from 32 non-operated obese patients were utilized as controls. Endoscopic biopsies were cut from tissue blocks fixed in formalin and embedded in paraffin. Sections 4 m thick were examined for immunoexpression using the streptavidin-biotin-peroxidase method. RESULTS: The two groups were compared for age, gender, gastritis, intestinal metaplasia, and presence of Helicobacter pylori. The mean number of positive gastrin cells was 55.5 (standart deviation (SD) = 11.7) in the control group and 29.6 (SD=7.9) in the cases, p=0.0003. Ki-67 proliferative index in cases (body=24.7%, antrum=24.9%) was significantly higher compared to controls (body=15.0% and antrum=17.7%), p=0.002 and 0.01, respectively. Caspase 3 immunoexpression was higher in the controls compared to the excluded stomach (46 vs. 31%), p=0.02. There was no statistical difference between CD3, CD8, and Bcl-2 immunoexpressions among the controls and cases. CONCLUSIONS: Cell proliferation is increased and apoptosis is downregulated in the excluded gastric mucosa compared to the non-operated obese controls. Alterations in cell turnover and in hormonal secretions in these conditions may be of important in long-term follow-up

Introdução: De origem controversa e repercussões imprevisíveis, o acometimento ovariano pela endometriose é considerado importante marcador de extensão da doença, pois pode se associar a endometriose profunda. Inúmeras teorias etiopatogênicas tentam explicar a gênese da endometriose ovariana e, duas delas recentemente tem sido reativadas, como a da metaplasia celômica que justificaria o conceito atual de endometriose intra-ovariana profunda e a da menstruação retrógrada, que explica a origem tubárea dos endometriomas. Estima-se em 5% a 10% de câncer ovariano em lesões de endometriose de ovário; enquanto, a frequência total de transformação maligna foi estimada entre 0,3 a 2,5%. Objetivo: Avaliar as formas de apresentação da endometriose ovariana e possíveis associações com o quadro clínico, com outros locais de doença, com os marcadores de atividade proliferativa (Ki-67), com a expressão de alterações moleculares dos mecanismos apoptóticos consideradas importantes no processo de carcinogênese das lesões de endometriose (p53 e Bcl-2) e com os receptores de estrogênio (dependência hormonal). Métodos: Estudo de coorte retrospectivo exploratório, com 63 pacientes operadas entre 2002 a 2012, com diagnóstico de endometriose ovariana preenchendo os critérios de inclusão e exclusão. Os preparados histológicos foram reavaliados e reclassificados de acordo com o tipo histológico, com a forma de apresentação e com a presença de infiltração do parênquima ovariano, sendo divididas em endometriose ovariana peritoneal, cistica e intraparenquimatosa. Foram avaliados a expressão do Ki-67, do p53, do Bcl- 2 e dos receptores de estrogênio no epitélio e no estroma tecidual. As pacientes ainda foram avaliadas de acordo com os sintomas clínicos e locais concomitantes de doença. Resultados: A forma de apresentação da endometriose ovariana mais frequente foi a cística (72,2%), seguida pela intraparenquimatosa (22,2%) e pela forma peritoneal (5,6%). Todas podem apresentar componente infiltrativo. A endometriose ovariana infiltrativa esteve presente em 30,5% dos casos. Não se evidenciou associação entre sintomas, distribuição anatômica do doença e expressão dos marcadores com as diferentes formas de apresentação ou com a infiltração do parênquima adjacente. Conclusão: A endometriose ovariana apresenta três formas distintas de apresentação, cística, intraparenquimatosa e peritoneal. Todas podem apresentar componente infiltrativo. Apesar da clara diferenciação histológica, ainda se deve identificar o significado clínico destes achados / Introduction: Of controversial origin and unpredictable repercussions, ovarian endometriosis is an important marker of disease extensiveness, as it may be related to deep infiltrating endometriosis. Numerous theories try to explain its origin, but two of them have been recently reactivated, such as celomic metaplasia, which would justify the concept of deep ovarian endometriosis, and retrograde menstruation, which can explain the tubal origin of ovarian endometriosis. It is estimated 5% to 10% of ovarian cancer in ovarian endometriosis, but malignant transformation may occur in 0.3 to 2.5% of the cases. Objective: Identify the presenting forms of ovarian endometrisosis and its possible relations to clinical symptoms, to other sites of disease, to proliferative activity markers (Ki-67), to the molecular expression of apoptotic mechanisms, considered important to the process of malignant transformation (p53 and Bcl-2) and to estrogen receptors (hormonal dependency). Methods: This is a retrospective exploratory cohort study, done between 2002 and 2012, including 63 women with laparoscopic diagnosis of ovarian endometriosis which fullfilled inclusion and exclusion criteria. The histologic specimens were reanalysed and reclassified according to the histologic pattern, to its presenting form and to the presence of parenchyma infiltration. The expression of Ki-67, p53, Bcl-2 and estrogen receptors were evaluated in the tissue epithelium and stroma. Clinical symptoms and concomitant sites of disease were also evaluated. Results: The most frequent form of ovarian endometriosis was cystic (72.2%), followed by intra-parenchymatous (22.2%) and peritoneal (5.6%). All of them can be infiltrative. The prevalence of infiltrative ovarian endometriosis was 30.5%. No association were found between symptoms, anatomical distribution of disease, markers expression and the presenting forms of ovarian endometriosis as well as adjacent parenchymal infiltration. Conclusion: Ovarian endometriosis has three distinct presenting forms, cystic, intra-parenchymatous and peritoneal. All of them can be infiltrative. Even though there is a clear histologic differentiation, its clinical significance is still to be determined

Introdução: De origem controversa e repercussões imprevisíveis, o acometimento ovariano pela endometriose é considerado importante marcador de extensão da doença, pois pode se associar a endometriose profunda. Inúmeras teorias etiopatogênicas tentam explicar a gênese da endometriose ovariana e, duas delas recentemente tem sido reativadas, como a da metaplasia celômica que justificaria o conceito atual de endometriose intra-ovariana profunda e a da menstruação retrógrada, que explica a origem tubárea dos endometriomas. Estima-se em 5% a 10% de câncer ovariano em lesões de endometriose de ovário; enquanto, a frequência total de transformação maligna foi estimada entre 0,3 a 2,5%. Objetivo: Avaliar as formas de apresentação da endometriose ovariana e possíveis associações com o quadro clínico, com outros locais de doença, com os marcadores de atividade proliferativa (Ki-67), com a expressão de alterações moleculares dos mecanismos apoptóticos consideradas importantes no processo de carcinogênese das lesões de endometriose (p53 e Bcl-2) e com os receptores de estrogênio (dependência hormonal). Métodos: Estudo de coorte retrospectivo exploratório, com 63 pacientes operadas entre 2002 a 2012, com diagnóstico de endometriose ovariana preenchendo os critérios de inclusão e exclusão. Os preparados histológicos foram reavaliados e reclassificados de acordo com o tipo histológico, com a forma de apresentação e com a presença de infiltração do parênquima ovariano, sendo divididas em endometriose ovariana peritoneal, cistica e intraparenquimatosa. Foram avaliados a expressão do Ki-67, do p53, do Bcl- 2 e dos receptores de estrogênio no epitélio e no estroma tecidual. As pacientes ainda foram avaliadas de acordo com os sintomas clínicos e locais concomitantes de doença. Resultados: A forma de apresentação da endometriose ovariana mais frequente foi a cística (72,2%), seguida pela intraparenquimatosa (22,2%) e pela forma peritoneal (5,6%). Todas podem apresentar componente infiltrativo. A endometriose ovariana infiltrativa esteve presente em 30,5% dos casos. Não se evidenciou associação entre sintomas, distribuição anatômica do doença e expressão dos marcadores com as diferentes formas de apresentação ou com a infiltração do parênquima adjacente. Conclusão: A endometriose ovariana apresenta três formas distintas de apresentação, cística, intraparenquimatosa e peritoneal. Todas podem apresentar componente infiltrativo. Apesar da clara diferenciação histológica, ainda se deve identificar o significado clínico destes achados / Introduction: Of controversial origin and unpredictable repercussions, ovarian endometriosis is an important marker of disease extensiveness, as it may be related to deep infiltrating endometriosis. Numerous theories try to explain its origin, but two of them have been recently reactivated, such as celomic metaplasia, which would justify the concept of deep ovarian endometriosis, and retrograde menstruation, which can explain the tubal origin of ovarian endometriosis. It is estimated 5% to 10% of ovarian cancer in ovarian endometriosis, but malignant transformation may occur in 0.3 to 2.5% of the cases. Objective: Identify the presenting forms of ovarian endometrisosis and its possible relations to clinical symptoms, to other sites of disease, to proliferative activity markers (Ki-67), to the molecular expression of apoptotic mechanisms, considered important to the process of malignant transformation (p53 and Bcl-2) and to estrogen receptors (hormonal dependency). Methods: This is a retrospective exploratory cohort study, done between 2002 and 2012, including 63 women with laparoscopic diagnosis of ovarian endometriosis which fullfilled inclusion and exclusion criteria. The histologic specimens were reanalysed and reclassified according to the histologic pattern, to its presenting form and to the presence of parenchyma infiltration. The expression of Ki-67, p53, Bcl-2 and estrogen receptors were evaluated in the tissue epithelium and stroma. Clinical symptoms and concomitant sites of disease were also evaluated. Results: The most frequent form of ovarian endometriosis was cystic (72.2%), followed by intra-parenchymatous (22.2%) and peritoneal (5.6%). All of them can be infiltrative. The prevalence of infiltrative ovarian endometriosis was 30.5%. No association were found between symptoms, anatomical distribution of disease, markers expression and the presenting forms of ovarian endometriosis as well as adjacent parenchymal infiltration. Conclusion: Ovarian endometriosis has three distinct presenting forms, cystic, intra-parenchymatous and peritoneal. All of them can be infiltrative. Even though there is a clear histologic differentiation, its clinical significance is still to be determined

Les réactions allergiques telles que les réactions d’hypersensibilité de contact (HSC) sont un problème de santé publique. Il s’agit d’une réaction inflammatoire aiguë qui survient suite à des expositions répétées d’une molécule allergisante avec la peau et dans laquelle les cellules dendritiques (DC) jouent un rôle essentiel. Les composés chimiques tels que le dinitrochlorobenzène (DNCB) ou le cinnamaldéhyde (CinA), responsables d'HSC, sont capables d’induire un stress chimique et de produire des espèces réactives de l’oxygène (ERO). Parmi les voies de détoxication en réponse aux xénobiotiques, la voie Nrf2/Keap1 est une voie centrale connue pour la détection de composés électrophiles. A l’état basal et en absence de stress, Nrf2 est couplé à son répresseur cytosolique Keap1 qui assure sa dégradation via le protéasome. En présence d’un stress chimique, Nrf2 transloque dans le noyau et induit l’expression des gènes antioxydants [hème-oxygénase 1 (ho-1), NADPH quinone oxydoréductase (nqo1), glutathione-s-transférase (gst)]. En absence de Nrf2, nous avons montré que le DNCB et le CinA induisent la mort cellulaire des DC via l'activation des caspases impliquées dans la voie mitochondriale ou intrinsèque de l'apoptose. Cette mort cellulaire induite par le DNCB est ERO dépendante tandis que celle induite par le CinA est moins sensible à la production des ERO. En présence de Nrf2, la survie des DC est régulée par l'expression de bcl-2, un gène antiapoptotique, et des gènes antioxydants. Nrf2 semblerait activer ou réprimer la transcription des gènes et ce en fonction de la molécule testée, du temps de traitement. Par ailleurs, nous avons également montré que Nrf2 joue un rôle clef dans les phases de sensibilisation et d'élicitation de la réaction d'HSC mais également au cours de l'irritation. Des transferts adoptifs de DC ont permis de montrer le rôle clef de Nrf2 dans la DC au cours de l'HSC. Enfin, notre étude montre que Nrf2 régule les Treg au niveau du tissu cutané et participe à la tolérance cutanée. / Allergic reactions such as contact hypersensitivity (CHS) are a problem of public health occurring after repeated exposures to contact sensitizers. CHS is a common skin disease involving dendritic cells (DC) playing a key role in this pathology. Contact sensitizers, like dinitrochlorobenzene (DNCB) or cinnamaldehyde (CinA) are known to induce reactive oxygen species (ROS) production. The Nrf2/Keap1 pathway is central for detoxification. In the absence of a chemical stress, Keap1 associates with Nrf2 and leading to its degradation. In the presence of an electrophilic compound like contact sensitizers, Keap1’s conformation is modified leading to Nrf2 translocation to the nucleus and transcription of its target genes [heme-oxygénase 1 (ho-1), NADPH quinone oxydoreductase (nqo1), glutathione-s-transferase (gst)]. We showed, for the first time, that Nrf2 controls the loss of mitochondrial membrane potential and caspase-3/7 activity in DC activated by contact sensitizers. In the absence of Nrf2, DNCB and CinA induced DC apoptosis via caspase activation involved in intrinsic pathway of apoptosis also called ‘mitochondrial pathway’. This apoptosis was mainly mediated by the production of ROS in response to DNCB. However, ROS faintly control CinA-induced cell death. We also showed that Nrf2 controls the transcription of the anti-apoptotic gene bcl-2 in response to DNCB or CinA and also the transcription of immune related and antioxidant genes that could be implicated in DC apoptosis.Otherwise, we also showed that Nrf2 plays a key role in sensitization and elicitation phases of CHS and even in the irritation phase. Adoptive transfer experiments showed that Nrf2 plays a crucial role in DC during CHS.Finally, we showed that Nrf2 regulates skin Treg and participates to skin tolerance.

Résumé en français : Notre équipe étudie les mécanismes de l’apoptose induite par la nutline-3, une molécule capable de se fixer sur MDM2 et d’activer la p53, dans différents types de lymphomes associés au virus d’Epstein-Barr (EBV) comme le lymphome de Burkitt (LB) ou syndromes lymphoprolifératifs post-transplantation (PTLD). Nos résultats montrent que la nutline-3 induit l’apoptose des cellules de LB EBV (-) alors que les cellules EBV (+) en latence de type III sont résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans ce phénomène de résistance afin de mettre en place des stratégies pour les contourner. Une première étude initiée par les résultats d’une analyse transcriptomique, effectuée après traitement avec la nutline-3 de deux lignées qui ne diffèrent que par leur statut EBV, nous a permis de montrer que : 1) l’autophagie est induite en réponse au traitement dans les cellules EBV (+) en latence de type III ; 2) ces cellules expriment fortement Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie contribue à la résistance de ces cellules à l’apoptose. Par ailleurs, nos résultats indiquent que la protéine anti-apoptotique Bcl-2 est également impliquée dans la résistance de ces cellules et que l’utilisation d’ABT-737, un inhibiteur de Bcl-2, restaure leur sensibilité à la nutline-3. L’efficacité de ce composé a donc été évaluée in vivo, seul ou en combinaison avec des traitements conventionnels (Cyclophosphamide pour le LB et Rituximab pour les PTLD). Les résultats obtenus lors de ces études pré-cliniques montrent que : 1) ABT-737 réduit considérablement la croissance tumorale et augmente la survie de souris xénogreffées avec des cellules d’une lignée lymphoblastoïde (LCL, utilisées comme modèle pour les PTLD) alors qu’il n’a pas d’effets chez les souris xénogreffées avec une lignée de LB ; 2) la combinaison BT-737/Cyclophosphamide permet de limiter la croissance tumorale durant le traitement mais n’améliore pas la survie des souris xénogreffées avec une lignée de LB ; 3) l’association ABT-737/Rituximab est très efficace et induit une rémission complète chez 70% des souris xénogreffées avec la lignée de LCL / - Résumé en anglais : Our team is working on the mechanisms of apoptosis induced by nutlin-3, a small molecule which binds to MDM2 and activates p53, in different lymphomas associated with Epstein-Barr virus such as Burkitt lymphoma (BL) or Post-transplant lymphoproliferative disorder (PTLD). Our results show that nutlin-3 strongly induce apoptosis in EBV (-) cells whereas EBV (+) latency III cells are much more resistant. The aim of my PhD project was to study the mechanisms involved in the resistance of EBV (+) latency III cells to apoptosis and to develop new therapeutic strategies to bypass these mechanisms. A transcriptomic analysis was realized after treatment with nutlin-3 of two cell lines which only differs by their EBV status. Based on the results obtained, a study was performed which allow us to show that: 1) autophagy is induced after nutlin-3 treatment in EBV (+) latency III cells; 2) these cells strongly expressed beclin-1 and present a constitutively high level of autophagy; 3) autophagy is involved in the resistance of apoptosis observed in these cells. Furthermore, our results demonstrate that Bcl-2 also contributes to the resistance of EBV (+) latency III cells and that treatment with ABT-737, a Bcl-2 inhibitor, restores their susceptibility to nutlin-3 treatment. We thus assessed the efficiency of this compound in vivo, in monotherapy or associated with conventional treatments (Cyclophosphamide for BL and Rituximab for PTLD). Results obtained during these pre-clinical studies show that: 1) ABT-737 reduces tumor growth and increase the overall survival of mice xenografted with a lymphoblastoïd cell line (LCL, used as a model for PTLD studies) but has no effects on mice xenografed with BL cell lines; 2) the association ABT-737/Cyclophosphamide reduces tumor growth during treatment but doesn’t improve the overall survival of mice xenografed with BL cell lines; 3) the association ABT-737/Rituximab is very efficient and induces 70% of complete remission in mice xenografted with LCL.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 97-109.

INTRODUÇÃO: As alterações da mucosa do estômago excluso após Derivação Gástrica em Y-de-Roux (DGYR) para tratamento da obesidade mórbida não são bem conhecidas. Atualmente, pouco se sabe a respeito das consequências da cirurgia, especialmente, considerando que tal técnica necessita de vigilância para possíveis alterações de mucosa. Adicionalmente, é possível que o refluxo duodenal biliopancreático para dentro do estômago excluso, sem tamponamento pela ingestão de alimentos, pode, após décadas, danificar a mucosa gástrica e provavelmente aumentar o risco de câncer gástrico. OBJETIVO: Analisar as alterações da mucosa do estômago excluso através de: índice de proliferação celular (Ki-67), apoptose (caspase 3 e Bcl-2), função hormonal (gastrina) e infiltrado inflamatório (CD3 e CD8). MÉTODOS: Enteroscopia de duplo balão foi realizada em 35 pacientes submetidos à DGYR com mais de 36 meses de cirurgia. Foram realizadas múltiplas biópsias no coto gástrico funcional e na mucosa do estômago excluso. Biópsias gástricas de 32 pacientes obesos não operados foram utilizadas como grupo controle. Biópsias endoscópicas foram seccionadas a partir de blocos de tecidos fixados em formalina e embebidos em parafina. Amostras de 4 m de espessura foram examinadas por imuno-histoquímica pelo método de estreptavidina-biotinaperoxidase. RESULTADOS: Os dois grupos foram comparados por idade, gênero, presença de gastrite, metaplasia intestinal e de Helicobacter pylori. O número médio de células de gastrina positivas foi de 55,5 (desvio padrão (DP) = 11,7) no grupo controle e 29,6 (DP = 7,9), nos casos, p= 0,0003. Índice de proliferação (Ki-67) nos casos (corpo=24,7%, antro=24,9%) foi significativamente maior em comparação com os controles (corpo=15% e antro=17,7%), p = 0,002 e 0,01 ,respectivamente. Imunoexpressão de caspase 3 foi maior nos controles em comparação ao estômago excluso (31 x 46%), p = 0,02. Não houve diferença estatística entre as expressões de CD3 , CD8 , e Bcl- 2 nos controles e nos casos. Não houve associação entre os resultados imuno-histoquímicos e a presença de Helicobacter pylori ou alterações histológicas. CONCLUSÕES: Proliferação celular está aumentada e a apoptose está diminuída na mucosa do estômago excluso em comparação com os controles obesos não operados. Alterações na renovação celular e nas secreções hormonais nestas condições podem ser relevantes em seguimento a longo prazo / INTRODUCTION: Mucosal alterations in the excluded stomach mucosal after Roux-en-Y gastric bypass for morbid obesity have not been clearly clarified. Currently, little is known regarding the long-term consequences of the surgical specially considering that the technique hinders surveillance for possible mucosal alterations. Indeed, it is possible that duodenal reflux of bile and pancreatic secretions without any buffering offered by food intake may, after decades, damage the gastric epithelium and lead to an increase gastric cancer risk. OBJECTIVE: This study aims to analyze the mucosal alterations (proliferative status (Ki-67), apoptosis (caspase 3 and Bcl-2), inflammatory response (CD3 and CD8) and for hormonal function (gastrin)) in the excluded stomach. METHODS: Double-balloon enteroscopy was performed in 35 patients who underwent Roux-en-Y gastric bypass longer than 36 months. Multiple biopsies of the proximal pouch and the excluded gastric mucosa were collected. Gastric biopsies from 32 non-operated obese patients were utilized as controls. Endoscopic biopsies were cut from tissue blocks fixed in formalin and embedded in paraffin. Sections 4 m thick were examined for immunoexpression using the streptavidin-biotin-peroxidase method. RESULTS: The two groups were compared for age, gender, gastritis, intestinal metaplasia, and presence of Helicobacter pylori. The mean number of positive gastrin cells was 55.5 (standart deviation (SD) = 11.7) in the control group and 29.6 (SD=7.9) in the cases, p=0.0003. Ki-67 proliferative index in cases (body=24.7%, antrum=24.9%) was significantly higher compared to controls (body=15.0% and antrum=17.7%), p=0.002 and 0.01, respectively. Caspase 3 immunoexpression was higher in the controls compared to the excluded stomach (46 vs. 31%), p=0.02. There was no statistical difference between CD3, CD8, and Bcl-2 immunoexpressions among the controls and cases. CONCLUSIONS: Cell proliferation is increased and apoptosis is downregulated in the excluded gastric mucosa compared to the non-operated obese controls. Alterations in cell turnover and in hormonal secretions in these conditions may be of important in long-term follow-up