Ovarian cancer is the fifth leading cause of cancer death among women in United States and has an alarming 1.4% (1 in 71) lifetime risk. The lack of overt symptoms and the absence of a reliable screening test to detect ovarian cancer result in over 70% of women being diagnosed after the disease has spread beyond the ovary resulting in a poor prognosis. A key characteristic of ovarian cancer is the ability of tumor cells to evade apoptosis, or programmed cell death contributing to the limitless replicative potential, which is a hallmark of all carcinogenesis. There is conclusive evidence that levels of bcl-2 are elevated in ovarian cancer patients' indication that this protein is an ovarian cancer biomarker. The overall goal of this thesis is to functionalize a substrate for specific, sensitive and cost-effective bcl-2 capture. This surface will ultimately be incorporated into an acoustic wave-based diagnostic device for worldwide point-of-care (POC) ovarian cancer detection. This research looks to assess the capture of this analyte protein on a series of bioconjugated surfaces. For the research to be diagnostically applicable, certain factors reveal themselves as more important than others. Since the surface-bound capture antibody must recognize the bcl-2 protein, it is vital to ensure upright orientation of this specific antibody with high affinity for the analyte. Furthermore once integrated with a nanosensor, the surface will sense a change in the mass on the surface, which requires that the surface is highly resistant to non-specific binding. Bioconjugation techniques were employed to initiate self-assembled monolayers (SAM) of silanes, immobilize antibodies (via amine-crosslinking or direct adsorption of protein A/G) and disperse polyethylene glycol (PEG) reagents to reduce non-specific binding on the glass substrates. 3-aminopropyltrimethoxysilane (3-APTMS) and chlorodimethyloctylsilane (ODMS) were deposited on the surface to create initial hydrophilic and hydrophobic properties on which molecular self-assembly could occur. Testing a variety of assemblies with and without the presence of silanes, amine-crosslinking and PEGylation reagents, the substrate displaying the highest efficacy of bcl-2 capture was revealed. These various surfaces were assessed through contact angle and a novel sandwich enzyme linked immunosorbent assay (ELISA) for sensitivity and specificity of bcl-2 standard capture. The consistently low background and facile assembly of the ODMS based substrate with direct adsorption of protein A/G and the PEGylation reagent, Pluronic, was deemed the best functionalized surface for non-specific recruitment of the bcl-2 protein. The substrate also consistently displayed low signal-to-noise ratio which was of extreme importance in this research to guarantee the prevention of false-positive results when detecting nascent carcinogenic behavior. Elucidation of this substrate assembly is the first step towards the long term objective of this thesis, which is to construct a cost-effective early ovarian cancer detection device which can be implemented at the point-of-care to those who need it the most. This is ultimately expected to dramatically improve health outcomes for females worldwide.

Notre équipe étudie les mécanismes de résistance à l’apoptose induite par divers agents dans les cellules de lymphomes B infectées ou non par le virus d’Epstein-Barr (EBV). EBV est un virus oncogénique de la famille des gamma-herpès virus qui est associé notamment au lymphome de Burkitt (LB) et aux syndromes lymphoprolifératifs post-transplantation (PTLD). Des résultats précédents ont montré que l’utilisation de la nutline-3, une molécule capable de se fixer sur MDM2, active p53 dans ces cellules tumorales. Cependant cette activation de p53 provoque l’apoptose des cellules B EBV(-) alors que les cellules B EBV(+) en latence III (exprimant toutes les protéines virales dites « de latence ») sont beaucoup plus résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans cette résistance afin de mettre en place des stratégies thérapeutiques pour la contourner. La première partie de ma thèse a été consacrée à l’étude du rôle de l’autophagie dans la résistance des cellules EBV(+) en latence III à l’apoptose. L’autophagie est un processus de dégradation des protéines qui joue un rôle physiologique complexe impliqué à la fois dans la survie et dans la mort cellulaire. Les travaux effectués ont montré que: 1) l’autophagie est induite en réponse au traitement par la nutline dans les cellules EBV(+) en latence III ; 2) ces cellules expriment fortement la Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie participe à la résistance de ces cellules à l’apoptose. La seconde partie de ma thèse a été consacrée au développement de nouvelles molécules ciblant les protéines anti-apoptotiques de la famille de Bcl-2. En effet, outre Bcl-2 qui est surexprimé dans les cellules EBV(+), les cellules de LB et les PTLD surexpriment aussi Mcl-1, une autre protéine anti-apoptotique. Or il a été montré que cette protéine était fréquemment à l’origine de résistance à des inhibiteurs déjà développés (et en essais cliniques) contre Bcl-2. Le développement de molécules ciblant Mcl-1 s’avère donc utile pour les contrer. Pour cela une collaboration avec une équipe de chimiste (dirigée par Fanny Roussi à l’Institut de Chimie des Substances Naturelles à Gif-sur-Yvette) a été mise en place. Nous avons identifié et étudié les mécanismes d’action de plusieurs molécules inhibitrices potentielles de Mcl-1 et/ou Bcl-xL capables d’induire l’apoptose dans nos deux modèles de lymphomes. / Our team investigates the mechanisms of resistance to apoptosis induced in various B-cell lymphomas including some infected by the Epstein-Barr virus (EBV). EBV is an oncogenic member in the gamma-herpesvirus family. Among other pathologies, it is associated with Burkitt’s lymphoma (BL) and post-transplant lymphoproliferative disorders (PTLD). Previously, our laboratory has found that in these tumor cells, the binding of nutlin-3 to MDM2 results in the activation of p53. However, although p53 activation leads to apoptosis in EBV(-) cells, EBV(+) latency III cells which express all viral « latency » proteins are much more resistant. During this PhD project, I studied the mechanisms involved in this resistance and made attempts to define new therapeutic strategies that would bypass them. First, the role played by autophagy was investigated. This catabolic process which degrades proteins and organelles is physiologically complex as it is involved in both cell survival and cell death. Our work has demonstrated that: 1) autophagy was induced in nutlin-3 treated EBV(+) latency III cells; 2) Beclin-1 was strongly expressed in these cells whose autophagy was constitutively activated; 3) autophagy was involved in the resistance to apoptosis observed in these cells. Second, I turned my efforts to the identification of new molecules targeting anti-apoptotic members of the Bcl-2 family. Like Bcl-2, the antiapoptotic protein Mcl-1 is heavily expressed in LB and PTLD cell lines but in this case, independently of their EBV status and this is a frequent cause for the observed resistance to Bcl-2 inhibitors that are currently tested in clinical trials. Molecules targeting Mcl-1 could thus prove promising to circumvent this resistance. In a collaboration with a Chemistry team supervised by Fanny Roussi at the Institut de Chimie des Substances Naturelles in Gif-sur-Yvette, we have identified the mechanisms of action of potential inhibitors of Mcl-1 and/or Bcl-xL, another anti-apoptotic molecules which induce apoptosis in our two lymphoma models.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Avaliou-se a expressÃo das proteÃnas bax, bcl-2 e P27 no carcinoma espinocelular invasivo da cavidade bucal atravÃs da tÃcnica da imunohistoquÃmica para conhecer-se o perfil apoptÃtico destes tumores. Buscou-se detectar os fatores epidemiolÃgicos e o comportamento clÃnico dos pacientes acometidos por esta neoplasia. Procurou-se empregar parÃmetros estatÃsticos diferentes do odds ratio para comparaÃÃes posteriores. Analisando-se quarenta e oito pacientes da Santa Casa da MisericÃrdia de Fortaleza, Cearà foi possÃvel conhecer os fatores sexo, escolaridade, renda familiar, tabagismo (forma e tempo de consumo,tempo de abandono, quantidade consumida), etilismo(tipo de bebida, tempo de consumo, quantidade consumida, freqÃÃncia). Com a anÃlise do laudo histopatolÃgico da peÃa cirÃrgica soube-se das informaÃÃes referentes ao tumor em si (localizaÃÃo, gradaÃÃo histolÃgica, tamanho, linfonodos envolvidos). Obteve-se fragmentos da peÃas cirÃrgicas e foram confeccionadas lÃminas para a verificaÃÃo da invasividade atravÃs da coloraÃÃo em hematoxilina e eosina. Em seguida foi realizada a reaÃÃo de imunohistoquÃmica foi pelo mÃtodo da estrepto-avidina-biotina-peroxidase objetivando-se avaliar a expressÃo das proteÃnas supracitadas. As lÃminas com marcaÃÃo positiva foram submetidas a contagem de no mÃnimo mil cÃlulas e este resultado foi empregado nos mÃtodos LI (Labelling Index) e HS(HScore). A avaliaÃÃo dos resultados foi atravÃs de mÃtodos descritivos, sendo os testes realizados o qui-quadrado e o teste exato de Fisher com nÃvel de significÃncia a dez por cento.Os resultados apontaram uma mÃdia de idade de cinqÃenta e sete anos e o maior nÃmero de indivÃduos do sexo masculino, analfabetos, com renda familiar de um salÃrio-mÃnimo na sua maioria. A forma de consumo de tabaco, em maior nÃmero encontrada, foi o cigarro industrializado com vinte anos de consumo. A ingestÃo de bebidas destiladas superou a de fermentadas com mÃdia de vinte anos. O soalho bucal foi a localizaÃÃo com maior nÃmero de casos, com uma mÃdia de tamanho de trÃs e meio centÃmetros, o estadiamento patolÃgico predominante foi o pT2, com gradaÃÃo histolÃgica moderadamente diferenciada em maior quantidade. Dentre os cruzamentos realizados, houve correlaÃÃo estatÃstica entre o tamanho de tumores com as faixas etÃrias(P=0,084 para α=10%); tamanho dos tumores com envolvimento de linfonodos(P=0,085 para α=10%); sexo dos pacientes com envolvimento dos linfonodos (P=0,03 para α=10%). Os resultados das reaÃÃes de imunohistoquÃmica mostraram um maior percentual de casos positivos para bax (77,1%) seguido de P27(45,9%) e bcl-2 (16,6%). As mÃdias dos LI encontradas apontaram bax com 67, 766; seguida de bcl-2 com 10,804; e P27 com 7,989. Cruzando-se os H-scores dos marcadores entre si encontrou-se correlaÃÃo positiva entre bax e P27 (0,245 na correlaÃÃo de Pearson). Os parÃmetros estatÃsticos avaliados foram: mÃdia e seu erro padrÃo; mediana; moda; desvio padrÃo; curtose e seu erro padrÃo; mÃnimo; mÃximo e quartis. Os resultados mostram uma tendÃncia a apoptose nos carcinomas espinocelulares bucais / The evaluation of the expression of bax, bcl-2 and p27 proteins at invasive squamous cell carcinoma of oral cavity was done using the immunohistochemistry technique to know about apoptotic profile of these neoplasms. The epidemiological factors and the clinical behavior of the patients were detected, too. Statistical parameters different from odds ratio was employed for future comparisons. Forty-eight patients from Santa Casa da MisericÃrdia de Fortaleza, CearÃ, Brazil was analyzed and with this analysis was possible to know the prognostic factors: sex of patients, instruction grade, familiar gains, tobacco consumption (form of consumption, duration of consumption, period of forsaking and quantity consumed), alcohol consumption (nature of drinking, duration of consumption, period of forsaking, quantity consumed, periodicity). In a posterior moment, with the histopathological report from the surgical specimen was possible to know about localization of neoplasm, size of neoplasm, histological grade, nodes involvement and invasiveness. Pieces of the neoplasm were achieved from surgical specimen and glass slides were done to analyze the invasiveness by hematoxilin-eosin coloration. After the immunohistochemistry reaction by streptoavidin biotin technique was done to evaluate the expression of proteins above. The glass slides with positive reaction were submitted under a counting and, at least, one thousand cells were counted. The results from counting were submitted under the LI(Labelling Index) and HS(H Score) methods. The evaluation of the results was made using descriptive methods and the statistical tests were qui-square and Fisherâs exact test with 10% significance. The results showed the mean age was 57 years old, more males than females, analphabets and one minimum wage the mean familiar gain. The main form of tobacco consumption was industrialized cigarette with 20 years of consumption. The swallow of distillers drinking was bigger than fermentation ones with 20 years of drinking at mean. The floor of the mouth was the anatomic site with more number of cases and the mean size of neoplasm was 1.4 inches. The preponderant pathological staging detected was pT2 and the preponderant histological grade was moderately differentiated. Among the cross tabs realized, there were statistical correlation between size of tumors and age (P=0.084; α=10%), between size of tumors and nodes involvement (P=0,085; α=10%), between sex of patients and nodes involvement (P=0.03; α=10%). The results from immunohistochemical reactions were more positive to bax (77.1% of positive cases), P27(45.9% of positive cases) and bcl-2 (16.6% of positive cases). The mean of LI showed bax at first position (67.766); second bcl-2 (10.804) and P27(7.989). The cross tabs among HS showed statistical positive correlation between bax and P27 (0.245 at Pearsonâs correlation). The statistical parameters were: mean and its standard error, median, mode, standard deviation, kurtosis and its standard error, minimum, maximum and percentiles. The conclusions showed apoptosis propensity at oral squamous cell carcinoma