High-risk mucosal human papillomaviruses (HPVs) are major aetiological agents for the development of cervical cancer. Thus, the current goal of cervical cancer treatment is to develop vaccines against HPV s. Such vaccines would either prevent cervical cancer by eliminating HPV infection or be useful for treating established lesions by the destruction of cells displaying HPV proteins. The aim of this thesis was to characterise immune responses to the E5 protein of HPV -16, one of several antigens with possible use in vaccination. To determine whether immune responses to HPV -16 E5 existed and whether they could be correlated with disease severity or with the presence of HPV -16 DNA, both cell mediated (Chapter Two) and humoral (Chapter Three) immunity was investigated in women with and without cervical disease. Cellular responses in a minority of women were inversely correlated with disease severity. However, E5 specific antibodies were negatively correlated with the absence of HPV -16 DNA. Thus, although some immune responses were evident, these were generally limited to a small number of subjects and were not associated with the detection of HPV-16 E5 mRNA or DNA sequence variants. Due to the immune responses in women, E5 was further investigated to determine if the absence of HPV -16 E5 specific immune responses was due to the poor antigenicity of HPV -16. Mice were immunised with synthetic peptides corresponding to full length HPV -16 E5 (Chapter Four). As with the human data, cellular responses and weak antibody responses were detected in mice. Some mice also exhibited cytotoxic T -lymphocyte responses and when E5/major histocompatibility class I (MHC-I) interactions were investigated, a number of peptides showed a high percentage of binding. The E5/MHC-I interactions were further investigated (Chapter Five). The surface expression of MHC-I on cells containing HPV-16 or -18 DNA was found to be lower than on HPV DNA negative cell lines even after stimulation with interferon-gamma. Stimulation with E5 synthetic peptides increased expression of cell surface MHC-I molecules on cell lines negative for HPV DNA. Furthermore, the presence of the E5 gene reduced the expression of the ovalbumin gene in normal human keratinocytes. In conclusion, the data contained within this thesis indicate that HPV-16 E5 CMI is inversely correlated with disease status. It is possible to induce cell mediated responses to HPV -16 E5 and low-titre antibody responses. The presence of HPV16 E5 DNA may impair normal cellular function.

This thesis examined the evidence, policy decision-making, and implementation of HPV vaccination in Canada as a case study to explore normalization versus individualized decision making in public health programs. Mixed methods were used: a systematic review, content analyses and policy document analysis. Overall, the scientific evidence supported an effect of vaccination against HPV infection and precancerous cervical lesions, but evidence regarding cervical cancer incidence or mortality is lacking. Scientific and medical communities appeared optimistic about the vaccine, but cautious about its readiness for routine implementation. Policy decision-making was initially cautious, but shifted towards active program implementation, possibly related to the availability of federal funding. The educational materials and media coverage both sent clearly normalizing messages about HPV vaccination. The discussion suggests that HPV vaccination might be more suited to an individualized than population approach, but many factors coincided to promote its implementation, in Canada, within a traditional public health model.

Human papillomaviruses (HPV) are among the most common sexually transmitted infections and are responsible for 5% of all human cancers. HPV type 16 is the most prevalent of the high-risk HPVs (a subgroup of HPVs with potential to cause cancer), accounting for ~55% of HPV-associated cancers. HPV16 is a nonenveloped virus, composed of the major capsid protein L1, the minor capsid protein L2, and a circular double-stranded DNA genome (vDNA) condensed with human histones. HPV initially infects undifferentiated basal keratinocytes and viral replication is dependent on epithelial differentiation. Like many other DNA viruses, HPV must deliver its vDNA to the host cell nucleus to successfully replicate. Initial binding of HPV16 to host cells is through L1 interactions with cell surface heparan sulfate receptors. Shortly after virus binding, L2 is believed to undergo furin cleavage-dependent conformational changes, resulting in spanning of the protein across the local membrane and exposure of the central and C-terminal regions of L2 (which was lumenal and and inaccessible before furin cleavage) to the host cell cytosol. L2 is critical for transport of the L2/vDNA from endosomes to the trans-Golgi network (TGN). We hypothesize that furin-dependent early L2 spanning, through the direct binding and recruitment of cytosolic sorting factors, may contribute to viral endocytosis and subcellular retrograde trafficking (trafficking from endosomes to Golgi) of vDNA. We have developed a Tac receptor (CD25 or IL2 receptor, a transmembrane cell surface protein) chimera system to study L2-dependent endocytosis and trafficking. In this system the Tac ecto- and transmembrane domains are fused to the ~400 amino acid portion of L2 that is likely cytosolic upon L2 spanning. Through transient expression of Tac-L2 chimera we use anti-Tac ectodomain antibodies to label and track cell surface populations by immunofluorescence and confocal microscopy. We have also adopted this system to study endocytosis through a cell surface biotinylation approach. Both approaches suggest that L2 may enhance endocytosis and preliminary evidence suggests that the Tac-L2 chimera may recruit the cytosolic retromer complex (the host cytosolic factors help protein retrograde trafficking) to preferentially traffic to the TGN. Retromer-dependent trafficking of cargo from early endosomes to the TGN is known to involve certain members of the sorting nexin family, specifically the SNX-BAR proteins. We performed a small siRNA screen and identify SNX6 and SNX32 (aka SNX6b) as SNX-BAR proteins that may be specifically involved in retrograde trafficking of HPV16 L2/vDNA during infection. Future work will focus on the mechanisms through which L2 and SNX6 influence HPV16 entry and trafficking.

O câncer de cervical é causado por uma infecção persistente por algum dos tipos oncogênicos de Papilomavírus Humano (HPV) e continua a ser um problema de saúde pública, especialmente nos países em desenvolvimento. Por Peptide Phage Display, foram selecionadas sequências de peptídeos com afinidade de ligação a linhagens celulares ou a tumores associados ao HPV. Dentre elas, foi possível identificar sequências contidas em moléculas importantes para a progressão de tumores, como a -manosidase e triptase, enzimas que desempenham um papel importante na progressão tumoral. Para caracterizar o efeito da inibição de -manosidase II na terapia de tumores cervicais em camundongos, utilizamos a droga Swainsonina (SW), previamente descrita como inibidor desta enzima. Nós testamos o efeito desta droga tratando animais inoculados com células tumorais que expressam os oncogenes E6 e E7 de HPV16. Observamos que os animais tratados com Swainsonina apresentaram crescimento tumoral significativamente mais rápido do que os animais controle. Investigando os mecanismos por trás desse efeito, descobrimos que embora SW module parcialmente os macrófagos associados aos tumores, o tratamento induz o acúmulo de células com fenótipo mieloderivado supressor no baço dos animais, potencializando o efeito tolerogênico dos tumores sobre o sistema imune. Sendo assim, sugerimos cautela no uso deste fármaco para a terapia de pacientes com tumores HPV&#43. Outro braço do trabalho foi avaliar o papel da triptase, que é produzida por mastócitos, no infiltrado inflamatório. Para isto padronizamos um modelo de co-cultura de esferóides tumorais da linhagem TC-1 com a linhagem de mastócitos murinos PT18. Através deste modelo pudemos observar que a linhagem tumoral consegue induzir a desgranulação dos mastócitos independentemente de anticorpos. Além disso, quando em co-cultura, a linhagem tumoral parece estar aumentando a meia-vida dos mastócitos e estimulando a proliferação destes. Em experimentos in vivo observamos que tumores induzidos com as células PT18 e TC-1 cresceram mais rapidamente do que tumores induzidos apenas com TC-1. Através da imunofenotipagem dos tumores ficou evidenciado um aumento de células CD31+ e no infiltrado inflamatório total de tumores induzidos com o co-cultivo. Justificando o fato destes crescerem mais rapidamente, sugerindo que os mastócitos podem ter efeitos tanto proliferativos quanto no processo de angiogênese tumoral. / Cervical cancer is caused by a persistent infection by some of the oncogenic types of Human Papillomavirus (HPV) and continues to be a public health problem, especially in developing countries. By Peptide Phage Display peptide sequences were selected with binding affinity to cell lines or to HPV-associated tumors. Among them, it was possible to identify sequences contained in molecules important for the progression of tumors, such as -mannosidase and tryptase, enzymes that play an important role in tumor progression. To characterize the effect of -mannosidase II inhibition on cervical tumor therapy in mice, we used the drug Swainsonina (SW), previously described as an inhibitor of this enzyme. We tested the effect of this drug by treating animals inoculated with tumor cells expressing HPV16 E6 and E7 oncogenes. We observed that Swainsonine treated animals had significantly faster tumor growth than control animals. Investigating the mechanisms behind this effect, we found that although SW partially modulates tumor-associated macrophages, the treatment induces the accumulation of cells with suppressive myeloderivative phenotype in the animals spleens, enhancing the tolerogenic effect of tumors on the immune system. Therefore, we suggest caution in the use of this drug for the therapy of patients with HPV&#43 tumors. Another arm of the study was to evaluate the role of tryptase, which is produced by mast cells, in the inflammatory infiltrate. For this we standardized a co-culture model of tumor spheroids of the TC-1 lineage with the murine mast cell line PT18. Through this model we could observe that the tumoral lineage can induce mast cell degranulation independently of antibodies. In addition, when co-cultured, the tumoral lineage appears to be increasing the half-life of mast cells and stimulating the proliferation of these. In in vivo experiments we observed that tumors induced with PT18 and TC-1 cells grew faster than tumors induced only with TC-1. Tumor immunophenotyping revealed an increase in CD31&#43 cells and in the total inflammatory infiltrate of tumors induced with co-culture. Justifying the fact that they grow faster, suggesting that mast cells can have both proliferative effects and the process of tumor angiogenesis.

Introdução: A infecção pelo Papilomavirus humano (HPV) enquadra-se como um problema de saúde pública, sendo considerada a infecção sexualmente transmissível (IST) mais comum. Estima-se, mundialmente, que aproximadamente 600 milhões de pessoas possuam o vírus do HPV e que em torno de 75 a 80% das pessoas adquiram- no em algum momento da vida. Objetivo: Analisar os aspectos sociodemográficos e clínicos em mulheres com infecção genital pelo HPV associando-os com o tipo de lesão genital e a sorologia reagente ou não reagente para o HIV e avaliar o seguimento clínico de mulheres com lesão intraepitelial de alto grau (HSIL) causada pelo HPV, que foram submetidas à cirurgia de alta frequência (CAF). Metodologia: Estudo transversal, retrospectivo, com abordagem quantitativa, realizado no Serviço de Moléstias Infecciosas em Ginecologia e Obstetrícia (SEMIGO) do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP- USP). Foram estudadas 1027 mulheres com diagnóstico de infecção genital pelo HPV, nas suas expressões lesão intraepitelial de baixo grau (LSIL), HSIL, condilomatoses (vulvar, vaginal, cervical e perianal), ou por identificação de sequências de DNA- HPV por meio de técnicas de biologia molecular. Foram elaborados dois formulários estruturados especificamente para este estudo, um referente às variáveis sociodemográficas e clínicas e outro ao seguimento clínico, ambos validados quanto à forma e conteúdo por três especialistas em infecção por HPV. A coleta de dados foi realizada por meio de consulta ao banco de dados eletrônico do local de estudo, elaborado com o software File Maker Pro 11. A população foi caracterizada por meio de estatística descritiva e os dados foram processados e analisados pelo software Statistical Package for the Social Sciences (SPSS) versão 16.0 for Windows. Para verificar a associação, realizou-se o teste qui-quadrado e análise de regressão logística com cálculo de odds ratio e com intervalo de confiança de 95%. Resultados: A faixa etária predominante foi entre 20 e 29 anos 387 (37,7%), 662 (64,4%) mulheres eram de etnia branca, 597 (58,1%) eram casadas ou viviam como casadas, 438 (42,6%) possuíam de cinco a oito anos de estudo e 529 (51,5%) exerciam atividade não remunerada. Quanto ao tipo de lesão, 237 (23,0%) mulheres possuíam LSIL, 391 (38,1%) HSIL, 349 (34,0%) condiloma e 50 (4,9%) diagnóstico de HPV por meio técnicas de biologia molecular. Observou-se maior probabilidade de soropositividade para o HIV em mulheres não brancas (p<0,01; OR=1,990; IC=1,392-2,843), que estudaram de 0 a 4 anos (OR=4,384; IC=1,706-11,266), viúvas (p<0,01; OR=4,223; IC=1,869-9,542), que fumavam (p<0,01; OR=2,389; IC=1,660-3,437), que tiveram mais de 10 parceiros sexuais (p<0,01, OR=3,487, IC=2,170-5,602) e que se prostituíam (p=0,0039, OR=3,699, IC=1,434-9,540). Em relação ao seguimento clínico de mulheres com HSIL submetidas à CAF, o comparecimento aos retornos pré-estabelecidos diminuiu com o passar do tempo. Conclusão: Faz-se necessário assegurar o acesso das mulheres a um exame preventivo de qualidade, através de programas de rastreamento eficientes, bem como garantir seu seguimento clínico. / Introduction: Human papillomavirus (HPV) infection is considered a public health issue, at it is the most common sexually transmitted infection (STI). Globally, it is estimated that 600,000,000 people carry HPV, and that around 75 to 80% of people will be infected in their lifetime. Objective: To analyze the sociodemographic and clinical aspects of women with genital HPV infection, associate those aspects with the type of genital lesion and a positive or negative test result for HIV, evaluate the clinical follow-up of women with high-grade squamous intraepithelial lesions (HSIL) caused by HPV, who had been submitted to loop electrosurgical excision procedure (LEEP). Methodology: This cross-sectional, retrospective study was performed, using a quantitative approach, at SEMIGO (Portuguese acronyms standing for Gynecology and Obstetrics Infectious Disease Clinic) of the Ribeirão Preto Faculty of Medicine University Hospital (University of São Paulo). The study subjects were 1027 women diagnosed with genital HPV infection, as low-grade squamous epithelial lesions (LSIL), HSIL, condylomatosis (vulvar, vaginal, cervical and perianal), or by identifying DNA-HPV sequences using molecular biology techniques. Two structured questionnaires were designed specifically for this study; one referring to the sociodemographic and clinical variables, and the other to the clinical follow-up, both validated regarding form and content by three HPV infection experts. Data collection was performed through a survey on the electronic database of the study location, using File Maker Pro 11. The population was characterized by descriptive statistics and the data were processed and analyzed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 16.0. The chi-square test and logistic regression analysis were used to verify the association, calculating the odds ration and considering a confidence interval of 95%. Results: Most women (387 or 37.7%) were between 20 and 29 years old, 662 (64.4%) were white, 597 (58.1%) were married or lived in common-law, 438 (42.6%) had one to five years of education, and 529 (51.5%) were unemployed. Regarding the type of lesion, 237 (23.0%) women had LSIL, 391 (38.1%) HSIL, 349 (34.0%) condyloma, and 50 (4.9%) were diagnosed for HPV by molecular biology techniques. It was observed that women were more likely to be infected by HIV if they were not white (p<0.01; OR=1.990; CI=1.392-2.843), had 0 to 4 years of education (OR=4.384; CI=1.706-11.266), were widowed (p<0.01; OR=4.223; CI=1.869-9.542), smoked (p<0.01; OR=2.389; CI=1.660-3.437), had over 10 sexual partners (p<0.01, OR=3.487, CI=2.170-5.602) and were prostitutes (p=0.0039, OR=3.699, CI=1.434-9.540). In terms of the clinical follow-up of women with HSIL submitted to LEEP, their attendance to appointments reduced over time. Conclusion: It is necessary to guarantee women accessibility to high-quality preventive exams through effective screening programs, in addition to ensuring their clinical follow-up.

Thesis advisor: Junona Moroianu / The E7 protein of low risk HPV11 has been shown to interact with multiple proteins, including pRb, in both the cytoplasm and the nucleus. High risk HPV16 E7 and low risk HPV11 E7 share a novel nuclear import pathway independent of karyopherins but dependent on the GTPase Ran (Angeline, et al., 2003; Knapp, et al., 2009; Piccioli, et al., 2010). We continued to analyze the nucleocytoplasmic transport of HPV11 E7 in vivo through transfection assays in HeLa cells with EGFP-HPV11 E7 wild type and mutant fusion constructs. We found that nuclear localization of HPV11 E7 is mediated by a nuclear localization signal located in the C-terminal domain which contains a unique zinc-binding domain. Mutations of cysteine residues that interfered with zinc-binding clearly disrupted the nuclear localization of the EGFP-11cE7 and EGFP-11E7 mutants. These data suggest that the integrity of the zinc-binding domain is essential for the nuclear localization of HPV11 E7. In addition, we discovered that HPV11 E7 has a leucine-rich C-terminal nuclear export signal (NES) (76IRQLQDLLL84) mediating the nuclear export of HPV11 E7 in a CRM1-dependent manner. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology Honors Program. / Discipline: Biology.

A infecção genital pelo Papilomavírus Humano (HPV) é considerada a doença sexualmente transmissível mais freqüente em todo o mundo, representando importante problema de saúde pública devido à sua alta prevalência e transmissibilidade. Estima-se que cerca de 75% da população sexualmente ativa entre em contato com um ou mais tipos de HPV durante sua vida, com prevalência mais elevada entre mulheres jovens. Estudos epidemiológicos têm demonstrado que a infecção pelo vírus da imunodeficiência humana (HIV) está associada a elevadas prevalências da infecção pelo HPV. A literatura acerca da infecção pelo HPV em gestantes é escassa e controversa. O objetivo do trabalho foi identificar a prevalência da infecção pelo HPV em gestantes e identificar a possível influência da infecção pelo HIV-1 nesta prevalência. Foi realizada amostragem de pacientes do Ambulatório de Pré-natal do Setor de Moléstias Infecto-contagiosas e do Pré-natal de Baixo Risco do Departamento de Ginecologia e Obstetrícia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo. Todas as pacientes foram informadas sobre o estudo e assinaram o termo de consentimento livre e esclarecido. Foram coletados lavados cérvico-vaginais, que foram submetidos à extração do DNA utilizando a técnica de salting out. Realizou-se a detecção do HPV nas amostras de DNA através da técnica de reação em cadeia de polimerase (PCR), e as amostras positivas para o HPV foram testadas para os tipos 6, 11, 16 e 18. Foram arroladas ao todo 97 pacientes, sendo 44 portadoras do HIV e 53 sem esta infecção. Do total de pacientes avaliadas, 66 foram positivas para o HPV. A prevalência para a infecção pelo HPV foi de 79,5% e 58,5% nas pacientes portadoras ou não do HIV, respectivamente. A infecção pelo HIV aumentou o risco de ser portadora do HPV, principalmente do tipo oncogênico. Contagem de linfócitos T CD4+ abaixo de 200 células/mm3 e carga viral do HIV maior que 10000 cópias aumentaram o risco de infecção pelo HPV. Este estudo mostrou haver maior prevalência da infecção pelo HPV em grávidas portadoras do HIV, permitindo inferir que a infecção por esse retrovírus seja um fator de risco significativo para o aumento da infecção pelo HPV em gestantes. / Genital infection with human papillomavirus (HPV) is considered to be the most frequent sexually transmitted disease around the world, representing an important public health problem due to its high prevalence and transmissibility. It is estimated that 75% of the sexually active population gets in contact with one or more HPV types during their lives, with higher prevalence among younger women. Epidemiologic studies have demonstrated that human immunodeficiency virus (HIV) infection is associated with a high prevalence of HPV infection. The literature about HPV infection during pregnancy is scarce and controversial. The aim of this study was to estimate the prevalence of HPV infection in pregnant women and identify the possible influence of HIV-1 infection on this prevalence. Patients were selected from the Prenatal Outpatient Clinic of the Infectious Diseases Sector and from the Low-risk Prenatal Outpatient Clinic of the Obstetrics and Gynecology Department of the University Hospital, Medical School of Ribeirão Preto, São Paulo University. All patients were informed about the study and signed an informed consent term. Cervical-vaginal washes were collected and submitted to DNA extraction by the salting-out technique. HPV was detected in the DNA samples by the polymerase chain reaction (PCR) technique and the HPV-positive samples were tested for types 6, 11, 16 and 18. Ninety-seven patients were included in the study, 44 of them being HIV-positive and 53 HIV-negative. Of the patients evaluated, 66 were positive for HPV. The prevalence of HPV infection was 79.5% and 58.5% in HIV-positive and -negative women, respectively. HIV infection increased the risk of harboring HPV, mainly oncogenic types. A CD4+ T-cell count below 200 cells/mm3 and HIV viral load above 10000 copies/mL increased the risk of HPV infection. This study showed a higher prevalence of HPV infection in HIV-positive pregnant women, suggesting that this retrovirus infection is a significant risk factor for the increase of HPV infection in pregnant women.

Introdução: A infecção pelo Papilomavirus humano (HPV) enquadra-se como um problema de saúde pública, sendo considerada a infecção sexualmente transmissível (IST) mais comum. Estima-se, mundialmente, que aproximadamente 600 milhões de pessoas possuam o vírus do HPV e que em torno de 75 a 80% das pessoas adquiram- no em algum momento da vida. Objetivo: Analisar os aspectos sociodemográficos e clínicos em mulheres com infecção genital pelo HPV associando-os com o tipo de lesão genital e a sorologia reagente ou não reagente para o HIV e avaliar o seguimento clínico de mulheres com lesão intraepitelial de alto grau (HSIL) causada pelo HPV, que foram submetidas à cirurgia de alta frequência (CAF). Metodologia: Estudo transversal, retrospectivo, com abordagem quantitativa, realizado no Serviço de Moléstias Infecciosas em Ginecologia e Obstetrícia (SEMIGO) do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP- USP). Foram estudadas 1027 mulheres com diagnóstico de infecção genital pelo HPV, nas suas expressões lesão intraepitelial de baixo grau (LSIL), HSIL, condilomatoses (vulvar, vaginal, cervical e perianal), ou por identificação de sequências de DNA- HPV por meio de técnicas de biologia molecular. Foram elaborados dois formulários estruturados especificamente para este estudo, um referente às variáveis sociodemográficas e clínicas e outro ao seguimento clínico, ambos validados quanto à forma e conteúdo por três especialistas em infecção por HPV. A coleta de dados foi realizada por meio de consulta ao banco de dados eletrônico do local de estudo, elaborado com o software File Maker Pro 11. A população foi caracterizada por meio de estatística descritiva e os dados foram processados e analisados pelo software Statistical Package for the Social Sciences (SPSS) versão 16.0 for Windows. Para verificar a associação, realizou-se o teste qui-quadrado e análise de regressão logística com cálculo de odds ratio e com intervalo de confiança de 95%. Resultados: A faixa etária predominante foi entre 20 e 29 anos 387 (37,7%), 662 (64,4%) mulheres eram de etnia branca, 597 (58,1%) eram casadas ou viviam como casadas, 438 (42,6%) possuíam de cinco a oito anos de estudo e 529 (51,5%) exerciam atividade não remunerada. Quanto ao tipo de lesão, 237 (23,0%) mulheres possuíam LSIL, 391 (38,1%) HSIL, 349 (34,0%) condiloma e 50 (4,9%) diagnóstico de HPV por meio técnicas de biologia molecular. Observou-se maior probabilidade de soropositividade para o HIV em mulheres não brancas (p<0,01; OR=1,990; IC=1,392-2,843), que estudaram de 0 a 4 anos (OR=4,384; IC=1,706-11,266), viúvas (p<0,01; OR=4,223; IC=1,869-9,542), que fumavam (p<0,01; OR=2,389; IC=1,660-3,437), que tiveram mais de 10 parceiros sexuais (p<0,01, OR=3,487, IC=2,170-5,602) e que se prostituíam (p=0,0039, OR=3,699, IC=1,434-9,540). Em relação ao seguimento clínico de mulheres com HSIL submetidas à CAF, o comparecimento aos retornos pré-estabelecidos diminuiu com o passar do tempo. Conclusão: Faz-se necessário assegurar o acesso das mulheres a um exame preventivo de qualidade, através de programas de rastreamento eficientes, bem como garantir seu seguimento clínico. / Introduction: Human papillomavirus (HPV) infection is considered a public health issue, at it is the most common sexually transmitted infection (STI). Globally, it is estimated that 600,000,000 people carry HPV, and that around 75 to 80% of people will be infected in their lifetime. Objective: To analyze the sociodemographic and clinical aspects of women with genital HPV infection, associate those aspects with the type of genital lesion and a positive or negative test result for HIV, evaluate the clinical follow-up of women with high-grade squamous intraepithelial lesions (HSIL) caused by HPV, who had been submitted to loop electrosurgical excision procedure (LEEP). Methodology: This cross-sectional, retrospective study was performed, using a quantitative approach, at SEMIGO (Portuguese acronyms standing for Gynecology and Obstetrics Infectious Disease Clinic) of the Ribeirão Preto Faculty of Medicine University Hospital (University of São Paulo). The study subjects were 1027 women diagnosed with genital HPV infection, as low-grade squamous epithelial lesions (LSIL), HSIL, condylomatosis (vulvar, vaginal, cervical and perianal), or by identifying DNA-HPV sequences using molecular biology techniques. Two structured questionnaires were designed specifically for this study; one referring to the sociodemographic and clinical variables, and the other to the clinical follow-up, both validated regarding form and content by three HPV infection experts. Data collection was performed through a survey on the electronic database of the study location, using File Maker Pro 11. The population was characterized by descriptive statistics and the data were processed and analyzed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 16.0. The chi-square test and logistic regression analysis were used to verify the association, calculating the odds ration and considering a confidence interval of 95%. Results: Most women (387 or 37.7%) were between 20 and 29 years old, 662 (64.4%) were white, 597 (58.1%) were married or lived in common-law, 438 (42.6%) had one to five years of education, and 529 (51.5%) were unemployed. Regarding the type of lesion, 237 (23.0%) women had LSIL, 391 (38.1%) HSIL, 349 (34.0%) condyloma, and 50 (4.9%) were diagnosed for HPV by molecular biology techniques. It was observed that women were more likely to be infected by HIV if they were not white (p<0.01; OR=1.990; CI=1.392-2.843), had 0 to 4 years of education (OR=4.384; CI=1.706-11.266), were widowed (p<0.01; OR=4.223; CI=1.869-9.542), smoked (p<0.01; OR=2.389; CI=1.660-3.437), had over 10 sexual partners (p<0.01, OR=3.487, CI=2.170-5.602) and were prostitutes (p=0.0039, OR=3.699, CI=1.434-9.540). In terms of the clinical follow-up of women with HSIL submitted to LEEP, their attendance to appointments reduced over time. Conclusion: It is necessary to guarantee women accessibility to high-quality preventive exams through effective screening programs, in addition to ensuring their clinical follow-up.

Philosophiae Doctor - PhD / Purpose: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. Design: Retrospective observational case series with laboratory investigations. Methods: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records.

Philosophiae Doctor - PhD / PURPOSE: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. DESIGN: Retrospective observational case series with laboratory investigations. METHODS: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records.