L' oncoprotéine E6 - qui joue un rôle crucial dans le processus d'oncogenèse induit par les papillomavirus a longtemps résisté à toute analyse. Depuis 1995 l'équipe Oncoprotéines a concentré ses efforts sur cette problématique. Ce qui a permis la résolution par RMN de la structure du domaine C-terminal de E6 en 2006. C'est dans ce cadre que j'ai commencé ce Doctorat en 2008, avec objectif de continuer la quête de données structurales sur E6 tout en acquérant des informations sur ses modes d'interaction avec ses cibles cellulaires. Les travaux de cette thèse ont permis l'obtention de la structure cristallographique de E6 (HPV16) en complexe avec un peptide de E6AP, en utilisant une approche originale capable de produire des protéines E6 stables et solubles. Cette structure constitue la première information structurale publiée sur des protéines E6 entières, attendue depuis plus de 20 ans par la communauté scientifique. J'ai effectué également durant cette thèse une analyse du système d'interaction de la protéine E6 basée sur une large étude d'interaction entre les protéines E6 (7 types) et 93 peptides porteurs de motif LxxLL.

Mathematical models provide unique insights to real-world problems. Within the context of infectious diseases, models are used to explore the dynamics of infections and control mechanisms. Human papillomavirus (HPV) globally infects about 630 million people, many of these infections develop into cancers and genital warts. Vaccines are available to protect against the most prevalent and devastating strains of HPV. The introduction of this vaccine as part of a national immunization program in Canada is a complex decision for policy-makers in which mathematical models can play a key role. We use the current recommendations provided by the World Health Organization to explore the integral role mathematical models have in the decision to incorporate the HPV vaccine within a national immunization program. We then provide a review of the literature discussing the role of mathematical models in the decision to include a vaccine in a national immunization program within the context of the HPV vaccine. Next, we evaluate the current standing of mathematical models used within the context of HPV immunization, to highlight the types of models used, underlying assumptions and general recommendations made about these immunization programs. Then, we create and analyze a model to explore the possibility of bettering the current HPV vaccine strategy in Canada. We focus on the effects of the grade of vaccination and the number of doses required to eradicate the targeted strains of HPV.

Problématique : Bien que le tabac et l’alcool soient les facteurs causaux principaux des cancers épidermoïdes de l’oropharynx, le virus du papillome humain (VPH) serait responsable de l’augmentation récente de l’incidence de ces cancers, particulièrement chez les patients jeunes et/ou non-fumeurs. La prévalence du VPH à haut risque, essentiellement de type 16, est passée de 20% à plus de 60% au cours des vingt dernières années. Certaines études indiquent que les cancers VPH-positifs ont un meilleur pronostic que les VPH- négatifs, mais des données prospectives à cet égard sont rares dans la littérature, surtout pour les études de phase III avec stratification basée sur les risques. Hypothèses et objectifs : Il est présumé que la présence du VPH est un facteur de bon pronostic. L’étude vise à documenter la prévalence du VPH dans les cancers de l’oropharynx, et à établir son impact sur le pronostic, chez des patients traités avec un schéma thérapeutique incluant la chimio-radiothérapie. Méthodologie : Les tumeurs proviennent de cas traités au CHUM pour des cancers épidermoïdes de la sphère ORL à un stade localement avancé (III, IVA et IVB). Elles sont conservées dans une banque tumorale, et les données cliniques sur l’efficacité du traitement et les effets secondaires, recueillies prospectivement. La présence du VPH est établie par biologie moléculaire déterminant la présence du génome VPH et son génotype. Résultats: 255 spécimens ont été soumis au test de génotypage Linear Array HPV. Après amplification par PCR, de l’ADN viral a été détecté dans 175 (68.6%) échantillons tumoraux ; le VPH de type 16 était impliqué dans 133 cas (52.25 %). Conclusion: Une proportion grandissante de cancers ORL est liée au VPH. Notre étude confirme que la présence du VPH est fortement associée à une amélioration du pronostic chez les patients atteints de cancers ORL traités par chimio-radiothérapie, et devrait être un facteur de stratification dans les essais cliniques comprenant des cas de cancers ORL. / Background: HPV is recognised as a good prognostic factor in head and neck (H&N) cancer. However, most of the data is derived from randomised trials with different treatment options or small heterogeneous cohorts. This trial aims to determine the prevalence and prognostic impact of HPV on overall survival (OS), disease-free survival (DFS), local regional control (LRC) and treatment toxicity, in patients with locally advanced SCCHN treated with concomitant platinum-based chemoradiation therapy (CRT) and followed prospectively. Methods: Prospective data on efficacy and toxicity was available for 560 patients treated with CRT. Of these, 270 fixed and paraffin embedded specimens were collected. DNA was extracted from specimens and HPV detection was performed as previously described (Coutlée, J Clin Microbiol, 2006). Analysis was performed using Kaplan-Meier survival curves, Fisher's test for categorical data and log-rank statistics for failure times. Results: Median follow-up was 4.7 years. DNA extraction was successful in 255 cases. HPV prevalence was 68.6%, and 53.3% for HPV-16 specifically. For HPV+ and HPV- respectively, median LRC were 8.9 and 2.2 years (log-rank p = 0.0002), median DFS were 8.9 and 2.1 years (log-rank p=0.0014) and median OS were 8.9 and 3.1 years (log-rank p=0.0002). Survival was statistically significantly different based on HPV genotype, stage, treatment period and chemotherapy regimen. COX adjusted analysis for T, N, age, and treatment remained significant (HR 0.45, p=0.004). Subgroup analysis for genotype, TNM, primary site and chemotherapy regimen will be presented at the meeting. Conclusions: An increasing proportion of oropharyngeal cancer is linked to HPV. This large study with confirms that HPV status is strongly associated with improved prognosis among H&N cancer patients receiving CRT, and should be a stratification factor for clinical trials including H&N cases. Toxicity of CRT is not modified for the HPV population.

This dissertation thesis touches very current and among public and experts often discussed topic. It concerns particularly matter of papillomaviral infections and vaccination against them. The research proved vaccination-coverage level of respondents and also their knowledge level. The vaccination-coverage level of girls is very high (almost 80%). A lot of girls also used possibility of full vaccination in the age of 13. This dissertation thesis should serve as a source and summary information about HPV viruses, diseases caused by them, about ways of transmission and epidemiological measures. Furthermore it also informs about cervical cancer, risk factors, diagnostical possibilities, therapy and at last but not lest about prevention of cervical carcinoma.

O câncer de colo de útero (CCU) é um dos mais danosos que afetam as mulheres, especialmente em países em desenvolvimento, onde os programas de prevenção (baseados no exame de Papanicolaou) não se mostram efetivos. Vacinas contra o HPV foram disponibilizadas em 2007 como prevenção primária do CCU. Apresentam boa eficácia e segurança, porém um elevado custo para sua incorporação. O objetivo desta dissertação é avaliar a incidência de câncer de colo de útero no estado de Roraima, seu impacto econômico, e o perfil sócio-econômico das portadoras de CCU tratada no SUS, além de realizar análises de custo-efetividade e custo-utilidade da inclusão da vacinação para o HPV aos programas preventivos vigentes. A dissertação é composta por 2 ensaios cujo tema unificador é a análise econômica de estratégias de combate ao CCU. No primeiro ensaio, todos os exames histopatológicos e citológicos emitidos em Roraima em 2009 foram revisados, juntamente com os prontuários médicos de todas as pacientes portadoras de CCU. Todos os procedimentos terapêuticos e diagnósticos realizados em 2009 em portadoras de CCU foram registrados. Prospectivamente foram entrevistadas as pacientes portadoras de CCU abordando tópicos sócio-econômicos. Foram registrados 90 casos de CCU em Roraima em 2009. A incidência correspondeu a 46,2 casos novos por 100.000 mulheres, a maior do Brasil. As portadoras do CCU demonstram perfil econômico desfavorável, exclusão social, e baixo conhecimento e adesão precária aos programas preventivos de saúde feminina. O impacto anual do CCU para gestão em saúde de Roraima é superior a R$8 mil por caso. Este ensaio ressalta a inequidade de acesso aos programas preventivos em Roraima, excluindo da cobertura de rastreio o subgrupo que mais se beneficiaria, e gerando custos elevados. O segundo ensaio realiza avaliações econômicas da incorporação da vacina para o HPV para o estado de Roraima. Foi desenvolvido um modelo de coorte de Markov como instrumento analítico de simulação da história natural do HPV até a progressão para CCU, levando em consideração os programas preventivos. As probabilidades de transição foram baseadas preferencialmente em dados empíricos locais e nacionais. Após calibração satisfatória e considerando valores de caso-base, a adição a vacinação para HPV reduziria em 35% a incidência de CCU, em cenário de realização de 3 testes de Papanicolaou ao longo da vida. A razão incremental de custo-efetividade foi de R$ 2.556 para cada ano de vida ajustado pela qualidade (AVAQ) poupado. As análises de sensibilidade confirmam a robustez deste resultado. A vacinação possui perfil favorável do ponto de vista de custo-utilidade, e sua inclusão no calendário vacinal resultaria em redução substancial da incidência e mortalidade por CCU em Roraima. / Invasive cervical cancer (ICC) is one of the most damaging that affect women, especially in developing countries, where preventive programs (based on the Pap smear) haven’t achieved effectiveness. HPV vaccines have recently been proposed as primary prevention of ICC. They show good efficacy and safety, but a high cost for its acquisition. Objectives: The objective of this dissertation is to evaluate the incidence of ICC in Roraima, its economic impact, and the socioeconomic profile of ICC carriers assisted in the public health system, and to perform a costeffectiveness and cost-utility analysis of adding HPV vaccination to current preventive programs. In the first essay, all histopathologic and cytologic reports issued in Roraima in 2009 were reviewed, along with the medical records of all patients diagnosed with ICC. All therapeutic and diagnostic procedures performed in 2009 in women with ICC cervix carcinoma were recorded. These patients were argued prospectively, addressing socio-economic topics. We registered 90 cases of cervical cancer in Roraima in 2009. The incidence corresponded to 46.2 new cases per 100,000 women. Carriers of the ICC showed unfavorable economic profile, social exclusion, deficient knowledge on ICC prevention and low adherence to the screening preventive programs. The annual impact of the ICC in Roraima is over R$ 8,000 per case. This essay highlights the inequity of access to preventive programs in Roraima, that excludes the subgroup of the population who would mostly benefit from screening, resulting in elevated costs. The second essay conducted economic evaluations of the incorporation of HPV vaccine to preventive programs in Roraima. A Markov cohort model was developed as an analytic tool to simulate the natural history of HPV and its progress to ICC, considering the current preventive programs. Transition probabilities were based mainly on empirical data of local and national studies. After satisfactory calibration values and considering the base case, the addition of HPV vaccination would reduce by 35% the incidence of ICC, in a scenario of three Pap tests throughout life. The incremental ratio of cost-effectiveness was R$ 2,556 for each year of quality-adjusted life (QALY) saved. The sensitivity analysis confirms the robustness of this result. Vaccination has a favorable profile in terms of cost-utility, and its inclusion in the immunization schedule would result in substantial reduction in incidence and mortality of ICC in Roraima.

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Cervical cancer represents an enormous health, psychological and social stress for every woman. The most important risk factor in the development of cervical carcinoma, which the second most common malignant cancer in women, is infection with a high-risk strain of human papillomavirus - a very frequent sexually transmitted disease. More than 100 types of HPV are acknowledged to exist, with HPV 16 and 18 being classified as high-risk types in particular. Worldwide, 500,000 new cases of cervical cancer are diagnosed every year. In the Czech Republic, there are 1,000 new cases of cervical cancer each year, out of which up to 400 women die. It is estimated that there will be up to 1,000,000 new cases of cervical cancer by 2050 unless the prevention is improved. Every woman is at risk of developing cervical cancer. HPV is sexually transmitted, however not only by sexual intercourse but also by skin-to-skin-contact with infected areas. Other risk factors in the development of the disease are: first sexual intercourse at early age, the number of sexual partners, smoking, other sexually transmitted diseases and a long term use of hormonal contraception. Use of condoms, which protects against sexually transmitted diseases, reduces the transmission of HPV by up to 70%. Having regular gynaecological check-ups with Pap smears is crucial for cervical cancer screening, as the screening suggests the presence of cytological abnormalities and pre-cancer. However, it cannot detect all types of premalignant changes and early stages of the carcinoma. Two vaccines have recently been developed, effective against the most frequent oncogenic strains of HPV (16 and 18), which currently cause about 70% of cervical cancer cases. Active immunisation against human papillomavirus is the first vaccination against carcinoma. Together with screening, it represents the best prevention method against cervical carcinoma. Based on the research of technical literature, the first part of the dissertation gives an overall view of the issue of cervical carcinoma. The second part of the dissertation deals with the research, eliciting the knowledge and attitude of women from Southern Bohemian towns in the field of cervical carcinoma prevention in the period of December 2008 - March 2009 and comparing it to technical literature.

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Introdução: o carcinoma epidermoide invasor (CE) de vulva é uma doença rara, que corresponde a cerca de 3-5% dos tumores malignos do trato genital feminino e a 90% de todas as neoplasias malignas primárias da vulva. Existem duas vias para o desenvolvimento de neoplasias intraepiteliais (NIV) e CE vulvar: uma via não relacionada ao papilomavírus humano (HPV) e outra relacionada ao HPV, com características clínicas, patológicas e epidemiológicas distintas. Objetivos: estudar as duas vias da carcinogênese vulvar, realizando correlação da expressão imunohistoquímica do p53 com a histologia. Métodos: foi realizado estudo do tipo casocontrole com 76 casos. Esses foram reclassificados conforme a terminologia da Sociedade Internacional para o Estudo das Doenças Vulvares (ISSVD, 2004), tendo sido realizada imuno-histoquímica para p53 e revisão de dados clínicos. Resultados: foram identificados 26 casos normais, 15 casos da via associada ao HPV (12 de NIV usual; 3 de CE condilomatoso) e 13 da via não associada ao HPV (5 de NIV diferenciada; 8 de CE queratinizante). A expressão do p53 nas vias carcinogênicas apresentou diferenças significativas: na via não associada ao HPV o p53 apresentou maior percentagem de células coradas (>25%, p<0,001), padrão basal com extensão ao terço médio para as NIV diferenciadas e difuso ou infiltrativo para os CE (p<0,001). A via carcinogênica associada ao HPV apresentou marcação de p53 menos extensa (até 10% das células, p<0,001), com padrão basal para as NIV usuais, sendo negativo para p53nos CE condilomatosos (p<0,001). Encontramos diferenças entre as idades (p<0,05), sendo que as pacientes da via não associada ao HPV apresentaram média de 66 anos e as da via associada, média de 44 anos. Conclusão: existe um padrão característico, baseado na histologia e expressão do p53, que separa as lesões vulvares em duas vias carcinogênicas distintas. O uso rotineiro de imuno-histoquímica para o p53 simultânea ao diagnóstico histológico em todos os casos de NIV e CE vulvar poderá auxiliar na definição da via carcinogênica, permitindo um melhor acompanhamento clínico das pacientes. / Introduction: The squamous cell carcinoma of the vulva is a rare disease, which accounts for about 3-5 % of malignant tumors of the female genital tract and 90 % of all primary neoplasms of the vulva. There are two pathways for the development of intraepithelial neoplasia (VIN) and vulvar squamous cell carcinoma: a pathway unrelated to human papillomavirus (HPV) and other HPV-related, with significant differences in clinical, pathological and epidemiological aspects. Objectives: To study the two pathways of vulvar carcinogenesis, correlating the results of p53 with histology. Methods: A retrospective case-control with 76 cases. These were reclassified according to the terminology of the International Society for the Study of Vulvar Diseases (ISSVD, 2004). Was performed immunohistochemistry for p53 and review of clinical data. Results: We identified 26 normal cases, 15 cases of HPVrelated pathway (12 usual VIN; 3 warty squamous carcinoma) and 13 not related with HPV (5 differentiated VIN; 8 keratinizing squamous cell carcinoma). The p53 showed significant differences: in cases related to HPV p53 showed a higher percentage of stained cells (> 25 %, p<0.001 ), basal pattern extending to the middle third to the differentiated VIN and diffuse or infiltrative for keratinizing carcinoma (p< 0.001). The p53 in HPV-related cases was less extensive (up to 10 % of the cells, p<0.001), with a basal pattern for usual VIN and negative pattern for warty carcinoma (p< 0.001). We found significant differences between age groups, with the group of patients with HPV-related average of 44 years and the patients in the group unrelated to HPV average of 66 years. Conclusion: There is a characteristic pattern, based on the results of histology and p53, which separates the vulvar lesions in two distinct carcinogenic pathways. We propose the routine use of p53 simultaneously to histological diagnosis in all cases of VIN and vulvar squamous cell carcinoma, as this would assist in defining the carcinogenic pathway allowing better monitoring of the patient.

The diploma work concerns the actual problem of papilomavirus infection and the vaccination against its originators. The gravity lies in the human-organism attack and the further proceeding changes in the human cells and mucous membranes. In many cases this leads to the pre-cancer states or even worse, to the cervix carcinoma. The work is divided into two parts, first theory, and second practice. The aim was to evaluate the vaccination against papilomviruses at the female-students of the University firstly, secondly to map the topic knowledge, and thirdly to monitor if the female-students visit their gynaecologist on regular basis. The theory concerns all the epidemiology, virology, clinical and diagnosis aspects of the papilomavirus infections, including the vaccination as a prevention possibility. In practice, there were run two anonymous questionnaire searches, first aimed on the female-students, second on the male-students only. The questions were focused on the level of the topic knowledge and the level of the both sex students vaccination, including the reasons of their non-interest in the possible vaccination, next the frequency of the girls? screenings in the gynaecologist practices. The research proved certain level of the respondents? topic knowledge and compared the existing differences among faculties. Another research conclusion is that the vaccination of the female-respondents is not very high, only 23,75% has already been vaccinated against the virus. Many young ladies underestimate the vaccination, and have more or less serious reasons why not to have them vaccinated. More than half of the respondents don?t do so because of the vaccine high price. This work should serve the women and young ladies, but also the young men as a source of the needed information. This means to improve the virus knowledge itself, its means of transmission, the diagnosis process or the treatment possibilities; overall the ways of primary prevention, with the ways of infection expanding prevention.