O câncer de colo do útero é o terceiro tipo de câncer mais frequente em mulheres no mundo, e a infecção persistente pelo papilomavirus humano (HPV) oncogênico é condição necessária, mas não suficiente para seu desenvolvimento. As oncoproteínas virais E6 e E7 interferem direta ou indiretamente na ação de várias proteínas celulares. Entretanto, as variantes proteicas, resultantes de polimorfismos genéticos, podem apresentar comportamento distinto mediante a infecção pelo HPV. O objetivo deste estudo foi avaliar possíveis associações entre polimorfismos nos genes TP53 (p53 PIN3, p53 72C>G) e p21 (p21 31C>A) e o desenvolvimento de neoplasias cervicais, considerando os níveis de expressão das proteínas p53, p21, p16 e ciclina D1, e fatores de risco clássicos para o câncer cervical. Foram selecionadas 466 mulheres residentes no Rio de Janeiro, 281 com diagnóstico histopatológico de neoplasia cervical de baixo (LSIL) e alto grau (HSIL) e câncer (grupo de casos) e 185 sem história atual ou pregressa de alteração citológica do colo uterino (grupo controle). A técnica de PCR-RFLP (reação em cadeia da polimerase - polimorfismo de comprimento de fragmento de restrição), foi empregada na análise dos polimorfismos p53 72C>G e p21 31C>A, usando as enzimas de restrição BstUI e BsmaI, respectivamente. A avaliação do polimorfismo p53 PIN3 (duplicação de 16 pb) foi feita por meio da análise eletroforética direta dos produtos de PCR. A expressão das proteínas p53, p21, p16, ciclina D1 e Ki-67 e a pesquisa de anticorpos anti-HPV 16 e HPV pool foram avaliadas por imunohistoquímica (Tissue Microarray - TMA) em 196 biópsias do grupo de casos. O grupo controle se mostrou em equilíbrio de Hardy-Weinberg em relação aos três polimorfismos avaliados. As distribuições genotípicas e alélicas relativas a p53 PIN3 e p53 72C>G nos grupos controles e de casos não apresentaram diferenças significativas, embora o genótipo p53 72CC tenha aumentado o risco atribuído ao uso de contraceptivos das pacientes apresentarem lesões mais severas (OR=4,33; IC 95%=1,19-15,83). O genótipo p21 31CA(Ser/Arg) conferiu proteção ao desenvolvimento de HSIL ou câncer (OR=0,61, IC 95%=0,39-0,97), e modificou o efeito de fatores de risco associados à severidade das lesões. A interação multiplicativa de alelos mostrou que a combinação p53 PIN3A1, p53 72C(Pro) e p21 31C(Ser), representou risco (OR=1,67, IC95%=1,03-2,72) e a combinação p53 PIN3A1, p53 72C(Pro) e p21 31A(Arg) conferiu efeito protetor (OR=0,26, IC95%=0,08-0,78) para o desenvolvimento de HSIL e câncer cervical. Observou-se correlação positiva da expressão de p16 e p21 e negativa da ciclina D1 com o grau da lesão. A distribuição epitelial de p16, Ki-67, p21 e p53 se mostrou associada à severidade da lesão. Os polimorfismos analisados não apresentaram associação com a expressão dos biomarcadores ou positividade para HPV. Nossos resultados sugerem a importância do polimorfismo p21 31C>A para o desenvolvimento das neoplasias cervicais e ausência de correlação dos polimorfismos p53 PIN3 e p53 72C>G com a carcinogênese cervical, embora alguns genótipos tenham se comportado como modificadores de risco. Nossos resultados de TMA corroboram o potencial de uso de biomarcadores do ciclo celular para diferenciar as lesões precursoras do câncer cervical. / Cervical cancer is the third most common female cancer worldwide, and persistent infection by the Human Papillomavirus (HPV) is a necessary but not sufficient condition to cause it. The viral oncoproteins E6 and E7 interfere directly or indirectly with the action of various cellular proteins. However, the protein variants, resulting from genetic polymorphisms, may act differently when encountering HPV infection. The aim of this study was to evaluate possible associations between polymorphisms in the TP53 (p53 PIN3, p53 72C>G) and p21 (p21 31C>A) genes, and the development of cervical neoplasia, considering the expression levels of p53, p21, p16 and cyclin D1 proteins, together with classic risk factors for cervical cancer. A total of 466 women resident in Rio de Janeiro were selected, being 281 with histopathological diagnosis of low (LSIL) or high grade (HSIL) cervical neoplasia or cancer (test group), and 185 with no current or previous history of alteration of cervical cytology (control group). The PCR-RFLP technique (polymerase chain reaction restriction fragment length polymorphism) was used to analyze the p53 72C>G and p21 31C>A polymorphisms, using BstUI and BsmaI restriction enzymes, respectively. Genotyping of the p53 PIN3 (duplication of 16 pb) polymorphism was performed by direct electrophoretic analysis of the PCR products. The expression of p53, p21, p16, cyclin D1 and Ki-67 proteins and the study of anti-HPV 16 and anti-HPV pool positivities were evaluated by immunohistochemisty (Tissue Microarray - TMA) in 196 biopsies of cases. The control group obeyed the Hardy-Weinberg principle in relation to the three polymorphisms analysed. The genotypic and allelic frequencies regarding p53 PIN3 and p53 72C>G in the control and test groups were not significantly different, although the p53 72CC genotype has increased the risk of more severe lesions attributed to the use of contraceptives (OR=4.33; IC 95%=1.19-15.83). The p21 31CA(Ser/Arg) genotype showed to protect against the development of HSIL or cancer (OR=0,61, IC 95%=0,39-0,97), and modified the effect of risk factors associated to the lesion severity. The multiplicative interaction of alleles showed that the combination p53 PIN3A1, p53 72C(Pro) and p21 31C(Ser) represented risk (OR=1,67, IC95%=1,03-2,72) and the combination p53 PIN3A1, p53 72C(Pro) and p21 31A(Arg) conferred protection (OR=0,26, IC95%=0,08-0,78) against the development of HSIL and cervical cancer. It was observed positive and negative correlations of, respectively, p16 and p21, and cyclin D1 expression with the cervical lesion grade. The epithelial distribution of p16, Ki-67, p21 and p53 was associated with the lesion severity. The polymorphisms analyzed showed neither association with the expression of the biomarkers nor positivity for HPV. Our results suggest the importance of polymorphism p21 31C>A in the development of cervical neoplasia and the lack of correlation between the polymorphisms p53 PIN3 and p53 72C>G with cervical carcinogenesis, although some genotypes acted as risk modifiers. Our TMA results corroborated the potential use of cell cycle biomarkers as an adjunctive tool to differentiate cervical precursor lesions.

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.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Die zelluläre Antwort auf einen spezifischen Stimulus wird nicht nur durch den Stimulus selbst, sondern auch von dem Zustand der Zelle bestimmt. Um ein tieferes Verständnis für die Variabilität in einer Zellpopulation zu gewinnen, ist es notwendig, die verschiedenen zellulären Antworten mit definierten zellulären Zuständen zu verbinden. In dieser Arbeit wurde ein System etabliert, welches es ermöglicht, die zelluläre Antwort auf DNA-Schäden und den Einfluss unterschiedlicher zellulärer Zustände zu studieren sowie die zu Grunde liegenden molekularen Mechanismen zu identifizieren. Im Zuge dessen wurde eine auf CRISPR/Cas9 basierende Methode entwickelt, mit der Fluoreszenzreporter für endogene Signalproteine in nicht transformierten Brustepithelzellen (MCF10A) generiert wurden. Anhand dieses Reportersystems konnte durch time-lapse Mikroskopie die Dynamik des Tumorsuppressors p53 und eines seiner Zielgene, des Zellzyklusinhibitors p21, verfolgt werden. Dabei wurde deutlich, dass die p21 Antwort der einzelnen Zellen auf DNA-Schäden sehr heterogen ausfällt. Über eine Form-basierte Gruppierungsmethode wurden vier verschiedene Subpopulationen mit charakteristischen p21 Dynamiken identifiziert. Um den Einfluss der Zellzyklusphase zu untersuchen, wurde die Zellteilung vor Bestrahlung analysiert und so Rückschlüsse auf die initiale Zellzyklusphase gezogen. 24h nach Bestrahlung wurde ein EdU labeling durchgeführt und der Zellzyklus mittels semi-supervised Klassifizierung bestimmt. Durch Einführen einer Mutation in der Bindedomäne von p21 wurde gezeigt, dass proliferating cellular nuclear antigen (PCNA) für die Heterogenität der p21 Antwort verantwortlich ist. Alles in allem bietet mein Projekt eine Pipeline, um auf Einzelzellebene zu erforschen, wie zelluläre Antworten durch den Zellzyklus beeinflusst werden. Dieser Ansatz könnte zukünftig Anwendung in der Erforschung von Medikamentenresistenz finden, zumal zelluläre Heterogenität in der Tumortherapie zu fractional killing führt. / The cellular response to a given stimulus is not only governed by the stimulus itself, but also depends on the state of the cells. However, it remains obscure how cellular states influence cell fate decisions. In this thesis, I established a framework to study how the cellular response to DNA damage is affected by varying cell states and to identify the underlying molecular mechanisms. To this end, I generated fluorescent reporters using CRISPR/Cas9 in non-transformed breast epithelial cells (MCF10A) and measured the dynamics of the tumor suppressor p53 and one of its target genes, the cell cycle inhibitor p21 using time-lapse microscopy. I found DNA damage induced highly diverse p21 dynamics in individual cells. A shape-based clustering identified four subpopulations of characteristic p21 dynamics. To examine the source of variability, I analyzed initial cell cycle states by monitoring cell division prior to damage, and determined final cellular state by EdU labelling and a semi-supervised classification 24h post damage. The results suggested that p21 dynamics depend on cell cycle phases and determine cell cycle progression. Furthermore, proliferating cellular nuclear antigen (PCNA)--a cell cycle dependent factor-- was shown to determine p21 heterogeneity using a mutant p21 deficient in interaction with PCNA. Overall, my project provides a pipeline to study at the single cell level how cellular response is affected by cellular states. Considering that cellular heterogeneity leads to fractional killing in tumor therapies, this approach also suggests future application on studying drug-resistance in cancer therapy.

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.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Made available in DSpace on 2014-12-17T15:32:31Z (GMT). No. of bitstreams: 1 RosileneCS.pdf: 376311 bytes, checksum: 8a0459347381c3b197002ddd72f02225 (MD5) Previous issue date: 2005-11-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Oral squamous cell carcinoma (OSCC) is the most common malignancy in oral cavity and human papillomavirus (HPV) may have an important role in its development. The aim of this experiment was to investigate the HPV DNA and viral types in 90 cases of OSCC. Moreover, a comparative analysis between the cases of OSSC with and without HPV DNA was performed by using cell cycle markers p21 and pRb in order to detect a possible correlation of these proteins and HPV infection. DNA was extracted from paraffin embedded tissue and amplified by PCR (polymerase chain reaction) with primers PCO3+ e PCO4+ for a fragment of human &#946;-globin gene. After this procedure, PCR for HPV DNA detection was realized using a pair of generic primers GP5+ e GP6+. Immunohistochemical study was performed by streptoavidin-biotin technique and antibodies against p21 and pRb proteins were employed. Eighty-eight cases were positive for human &#946;-globin gene and HPV DNA was found in 26 (29.5%) of then. It could not be detected significant correlation between HPV and age, sex and anatomical sites of the lesion. The most prevalent viral type was HPV 18 (80.8%). Regarding the immunohistochemical analysis, it was detected significant association between HPV presence and pRb immunoexpression (p=0,044), nevertheless, the same was not observed in relation to p21 protein (p =0,416). It can be concluded that the low detection of HPV DNA in OSCC by the present experiment suggests a possible role of the virus in the development and progression in just a subset of this disease / O carcinoma epiderm?ide oral ? a neoplasia maligna mais freq?ente da cavidade oral e o papilomav?rus humano (HPV) parece ter um relevante papel na indu??o desta les?o. Neste trabalho investigou-se o DNA do HPV e tipos virais em 90 casos de carcinoma epiderm?ide oral (CEO). Realizou-se tamb?m uma an?lise comparativa entre os grupos de CEO com DNA do HPV e sem o DNA do v?rus, empregando-se os marcadores do ciclo celular p21 e pRb, a fim de estabelecer poss?vel correla??o entre a express?o imuno-histoqu?mica dessas prote?nas e a infec??o pelo HPV. O DNA foi extra?do de tecido emblocado em parafina e amplificado por PCR (rea??o em cadeia da polimerase) com um par de primers designados PCO3+ e PCO4+ para um fragmento do gene da &#946;-globina humana. Posteriormente, realizou-se PCR para detec??o do DNA de HPV utilizando-se um par de primers gen?ricos designados GP5+ e GP6+. A tipagem viral foi realizada pela hibridiza??o dot blot. No m?todo imuno-histoqu?mico utilizou-se a t?cnica da streptavidina-biotina com um painel de anticorpos monoclonais para as prote?nas p21 e pRb. Dos 88 casos positivos para o gene da &#946;-globina humana, em 26 (29,5%) foi detectado o DNA do HPV. N?o houve associa??o significativa entre o HPV e as vari?veis idade e sexo dos pacientes e localiza??o anat?mica da les?o. O tipo viral prevalente foi o HPV 18 (80,8%). Quanto ? an?lise imuno-histoqu?mica, foi observada associa??o estatisticamente significativa entre a presen?a do HPV e a express?o imunohistoqu?mica de pRb (p=0,044), entretanto, n?o houve qualquer diferen?a estatisticamente significativa entre a express?o da prote?na p21 e a presen?a do v?rus (p =0,416). P?de-se concluir que o baixo percentual de detec??o do DNA do HPV no carcinoma epiderm?ide oral no presente trabalho, sugere uma poss?vel participa??o do HPV no desenvolvimento e progress?o de apenas um subgrupo dessas les?es