O câncer de colo do útero é a segunda principal causa de morte em mulheres por câncer, sendo causado principalmente pela infecção persistente por HPV. A principal forma atual de prevenção desse câncer é a realização de exames citológicos periódicos e a vacinação profilática disponibilizada recentemente pelo Ministério da Saúde. Entretanto, tais ações visam a prevenção da infecção por HPV ou a detecção de lesões, pois não há um tratamento específico contra infecções e lesões já estabelecidas. Dentre as proteínas expressas por HPV, L2 está presente no capsídeo viral e é bem conservada entre diversos tipos de HPV, enquanto E6 é uma proteína oncogênica capaz de induzir a transformação maligna das células. Este estudo visa o desenvolvimento de uma vacina profilática e terapêutica capaz de proteger contra a infecção por diversos tipos virais, assim como combater as células já modificadas por qualquer tipo de HPV. Assim, foi construído um vetor vacinal contendo peptídeos selecionados das proteínas L2 e E6 de HPV16 testadas em modelo murino, para avaliar sua eficiência como vacina de DNA ministrada antes ou após desafio com células tumorais. Os ensaios em modelo animal demonstraram que a vacina foi capaz de induzir a produção de anticorpos específicos anti-L2 e anti-E6, assim como induzir a produção de citocinas TNF e impedir o desenvolvimento tumoral de células HPV-positivas. Portanto, foi possível concluir que o vetor construído foi capaz de induzir uma resposta imune humoral e celular em camundongos, capaz de prevenir contra o HPV, assim como tratar os cânceres a ele associados. / Cervical cancer is the second most frequent cause of death in women due to cancer, mainly caused by persistent HPV infection. The primary prevention method of this cancer is through periodic cytological exams and prophylactic vaccination which has been recently made available by the Ministry of Health. However, such actions aim towards prevention of HPV infection or the detection of lesions, since there is no specific treatment against pre-existing infections and injuries. Among the proteins expressed by HPV, L2 is present in the viral capsid and is well conserved among several types of HPV, whereas E6 is an oncogenic protein, capable of inducing malignant mutations in cells. This study aims towards the development of a prophylactic and therapeutic vaccine, capable of protecting against infections caused by several viral types, as well as combating cells which have already been modified by any type of HPV. Thus, a vaccine vector, containing peptides selected from HPV16s L2 and E6 proteins, tested in the murine model, was constructed in order to evaluate its efficiency as a DNA vaccine, to be administered either before or after challenging with tumour cells. The animal model assays demonstrated that the vaccine was able to induce the production of anti-L2 and anti-E6 specific antibodies, as well as TNF cytokines, and to prevent tumour development in HPV-positive cells. Therefore, it was possible to conclude that the designed vector was indeed able to induce humoral and cellular immune responses in mice, capable of preventing against HPV, as well as of treating cancers associated with it.

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. High risk HPV types, including HPV 16, can cause cervical carcinomas upon infecting squamous basal epithelial cells. The HPV E2 protein is a multifunctional protein that regulates viral DNA replication and expression of a large number of cellular and viral genes, including the E6 and E7 viral oncogenes. Previous research in the Moroianu lab has identified a novel alpha-helical nuclear localization signal (NLS) in the C-terminal domain of HPV 16 E2 protein (75). Here, we focused on continuing the dissection of the HPV 16 E2 NLS and on identification of the nuclear import mechanism used by this protein. We identified several residues in the C-terminal domain of HPV 16 E2 (327KHK329) and within the NLS (K299, C300) that enhance the function of the NLS. Additionally, we determined that dimerization of the C-terminal domain plays an important role in the nuclear import of HPV 16 E2 as a mutation that disrupted it led to a significant decrease in the nuclear localization of the protein. We discovered that importin 11 karyopherin is a nuclear import receptor for HPV 16 E2. Our data suggest a nuclear import mechanism for HPV 16 E2 whereby UbcM2/UBE2E3 E2-type ubiquitin-conjugating enzyme acts as an adapter to bind HPV 16 E2 to importin 11 karyopherin for its nuclear import. This is a previously undescribed nuclear import mechanism which may have implications for the control of HPV 16 E2 functions. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Infecções pelos Papilomavirus Humanos (HPVs) estão relacionadas com uma série de malignidades nas regiões genitais, anais e recentemente a uma parcela dos tumores na região da cabeça e pescoço. Enquanto muito já fora explorado a respeito dessas infecções em mulheres, os fatores de risco virais e epidemiológicos para persistência da infecção e desenvolvimento de lesão em homens ainda estão sendo estabelecidos. Dessa forma, em meados de 2005, começou o Estudo HIM (HPV in men), um estudo multicêntrico envolvendo 4200 homens que tem como principal objetivo um melhor entendimento da história natural das infecções por esses vírus nessa população. Sabe-se que um importante evento no processo neoplásico em lesões do colo do útero é a integração do DNA viral no genoma da célula hospedeira. Embora o mecanismo de integração ainda não esteja claro, parece haver uma relação da progressão da doença com o aumento da proporção de lesões com genomas na forma integrada. Diante do exposto, o objetivo desse trabalho foi explorar as metodologias baseadas em PCR (convencional e em tempo real) e de sequenciamento de última geração (NGS) para verificar integração do DNA viral nas células de esfregaços genitais, do canal anal e cavidade oral de homens assintomáticos participantes do Estudo HIM. Além disso, teve como objetivo secundário verificar a carga viral nos diferentes sítios anatômicos. Foram inicialmente incluídas 125 amostras das quais 56 puderam ser incluídas nas análises de integração pela metodologia baseada em PCR e 90 amostras foram selecionadas para os ensaios de carga viral. Dentre as amostras testadas pela metodologia de PCR para integração, 11 foram desafiadas pela metodologia baseada em sequenciamento. Entre as 11 amostras sequenciadas, encontramos concordância entre os resultados em apenas 3 e isso pode ser atribuído à sensibilidade diferencial das metodologias diante de baixas cargas virais. Entre as amostras testadas pela metodologia baseada em PCR pudemos verificar que 30,4% das amostras apresentavam-se na forma puramente epissomal, 10,7% na forma puramente integrada e 58,9% das amostras apresentam as duas formas virais (classificadas como mistas). A amostra que se confirmou integrada pela metodologia de sequenciamento apresentou integração em 4 pontos do DNA humano com a ruptura no gene viral E2, conforme amplamente relatado na literatura. Grande variação na carga viral foi encontrada entre as amostras (0,00008 a 4200 cópias/célula), e isso se mostrou diretamente relacionado com as limitações das metodologias para verificar integração. Embora outras relações não puderam ser verificadas em nosso estudo pelo limitado número de amostras analisadas até o momento, estes dados ampliados poderão responder se a carga viral e integração seriam um fator de risco para a persistência da infecção ou o desenvolvimento de lesão em homens. / Human Papillomavirus (HPV) infections are associated with a range of malignancies in the genital and anal regions and recently, with an appreciable number of head and neck tumors. While much has been explored about these infections in women, viral and epidemiological risk factors for persistent infection and lesions development in men are still being established. Thus, in mid-2005, a multicenter study entitled HIM study (HPV in men) - involving 4200 men - which aims at a better understanding of the natural history of infection by these viruses in this population, was started. It is known that a key event in the transformation process in cervical lesions is the integration of viral DNA into the host cell genome. Although the integration mechanism is not yet clear, there seems to be a relationship of the disease progression with an increase of the proportion of lesions with integrated viral genomes. In view of the above, the aim of this study was to explore the methodologies based on PCR (conventional and real-time) and next-generation sequencing (NGS) to verify integration of viral DNA into cells from genital swabs, anal canal and oral cavity of asymptomatic men participating in the HIM study. Furthermore, the secondary objective was to verify the viral load in cells from different anatomical sites. Initially, 125 samples were selected of which 56 could be included in integration tests with the methodology based on PCR and 90 samples were selected for viral load determination. Of the samples tested by PCR method for integration, 11 were subjected DNA sequencing. We found correlation between the results in only 3 of the 11 samples and this can be attributed to the methodologies\' differential sensitivity to low viral loads. Among the samples tested by PCR-based methodology, we observed that 30.4% of the samples harbored purely episomal viral DNA, 10.7% were integrated and 58.9% of the samples showed the two viral forms (classified as mixed). The sample which was confirmed as integrated by sequencing presented integration into 4 human DNA points with the breakpoint within the E2 viral gene, as widely reported in literature. Large variation in viral load was observed among the samples (0.00008 to 4200 copies / cell), and this was shown to be directly related to the limitations of the methods used to verify integration. Evaluation of a larger number of samples may contribute to define the role of HPV DNA integration as a risk factor for the persistence of HPV infection or development of lesions in men.

INTRODUÇÃO: A lesão intraepitelial cervical de alto grau causada pelo HPV, precursora do câncer cervical, é facilmente diagnosticada e seu tratamento pode ser realizado de maneira ambulatorial, sem muitas complicações. Mesmo assim, o seguimento das pacientes tratadas deve ser feito de maneira criteriosa e sistemática para que a recorrência ou persistência da doença não passe despercebida. Conseguir identificar o grupo de pacientes com maior probabilidade de recorrência ou persistência da doença facilitaria sobremaneira esse seguimento, diminuindo o ônus econômico e psicológico que o seguimento generalizado produz. O objetivo deste trabalho é identificar marcadores que possam indicar o grupo de pacientes com maior possibilidade de recorrência da lesão intraepitelial cervical de alto grau. MÉTODOS: Neste estudo 114 mulheres com diagnóstico de lesão intraepitelial cervical de alto grau foram submetidas à conização cervical por cirurgia de alta frequência (CAF) no Setor de Patologia do Trato Genital Inferior (PTGI) da Divisão da Clínica Ginecológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) no período entre março de 2006 e maio de 2009. O seguimento foi realizado a cada seis meses durante o período de 24 meses. No seguimento foi coletada a citologia cervical, captura híbrida para HPV e realizada a colposcopia. Foram avaliados os testes de captura híbrida para HPV coletadas durante o seguimento, imuno-histoquímicos para oncoproteína viral E6 e proteína p16 na peça cirúrgica como possíveis marcadores de recorrência. RESULTADOS: A avaliação anatomopatológica da peça cirúrgica diagnosticou 85 (74,6%) casos de lesão intraepitelial cervical de alto grau e 29 (25,4%) casos de lesão intraepitelial de baixo grau. Nessas peças, 45 (39,5%) apresentaram expressão positiva para oncoproteína E6 e 69 (60,5%) apresentaram expressão negativa para E6; 74 (64,9%) apresentaram expressão positiva para p16 e 40 (35,1%) apresentaram expressão negativa para p16. A oncoproteína E6 não se associou com a apresentação mais grave da doença. Já a proteína p16 esteve positiva em 68 (80%) casos diagnosticados como lesão intraepitelial de alto grau e negativa em 23 (79,3%) casos com diagnóstico anatomopatológico de lesão intraepitelial de baixo grau ou cervicite crônica. A CH coletada no primeiro retorno apresentou sensibilidade de 83,3%, especificidade de 87,8%, VPP de 50% e VPN de 97,3%. Comparando a CH coletada no primeiro retorno com a citologia coletada após os 24 meses de seguimento, a CH apresentou sensibilidade de 75%, especificidade de 83,1%, VPP de 20% e VPN de 98,3%. CONCLUSÕES: A expressão das oncoproteína E6 e proteína p16 na peça cirúrgica não demonstraram ter valor para predizer recorrência no seguimento de pacientes tratadas por lesão intraepitelial cervical de alto grau, mesmo a p16 estando associada à presença de lesões mais graves. A CH pode ser usada como um teste preditivo de recorrência durante o seguimento de pacientes tratadas por lesão intraepitelial de alto grau. O VPN do teste é bastante alto e seu resultado negativo, seis meses após o tratamento indica uma taxa baixíssima de recorrência da doença / INTRODUCTION: The high grade cervical intraepithelial lesion caused by HPV, a pre-malignant condition, is easily diagnosed and its treatment can be done in outpatients without many complications. Nevertheless the patients follow-up must be done in a very systematic way to avoid any recurrence or persistence of the disease. To be able to identify the group of patients with higher rate of recurrence or persistence of the disease would make this follow-up much easier and decrease the economic and psychological burden of stressed outcome. The goal of our study is to identify markers that could indicate the group of patients more likely to recur. METHODS: In this study, 114 women diagnosed with high grade cervical intraepithelial lesion were treated with LLETZ at the Discipline of Gynecology, Faculty of Medicine, São Paulo University from March 2006 and May 2009. The follow-up visits after the treatment included Pap smear, HPV DNA test and colposcopy and occurred every 6 months for 24 months. The markers evaluated were the HPV DNA test collected during the follow-up and immunohistochemical tests performed on the surgical specimen: E6 oncoprotein and protein p16. RESULTS: We found 85 cases (74,6%) of HSIL and 29 cases (25,4%) of LSIL in the surgical specimen obtained with the LLETZ. The E6 oncoprotein was expressed in 45 (39,5%) and not expressed in 69 (60,5%) of the specimen; 74 (64,9%) expressed p16 and 40 (35,1%) didnt express p16. The E6 oncoprotein was not associated with severe presentation of the disease. The protein p16 was positive in 68 (80%) cases of diagnosed HSIL and negative in 23 (79,3%) cases with diagnosed LSIL or chronic cervicitis. The HPV DNA test collected at the first follow-up consult at 6 months presented a sensitivity of 83,3% specificity of 87,8%, positive predictive value (PPV) of 50% and negative predictive value (NPV) of 97,3%. Comparing the HPV DNA test collected at the first follow-up visit and the cervical cytology collected at the fourth and last follow-up visit at 24 months, the HPV DNA test presented a sensitivity of 75%, specificity of 83,1%, PPV of 20% and NPV of 98,3%. CONCLUSIONS: The E6 oncoprotein and protein p16 expression on the surgical specimen were not able to predict recurrence of the disease during the follow-up of the patients. The HPV DNA test can be used as a marker of the recurrence on the follow-up of patients treated for HSIL with LLETZ. The HPV DNA test negative result at the 6 month follow-up visit represents an extremely low recurrence rate

The development of a vaccine against some strains of the human papillomavirus (HPV) has led to many interesting public health questions [1]. We address some of these questions in the following work. We develop a compartmental mathematical model and examine the effect of waning immunity, vaccinating individuals prior to their becoming sexually active and the current government policy of vaccinating only females [2]. We calculate parameters based on data. We consider both time-dependent and age dependent ODE models and an age- and time-dependent PDE model and compare the results. We find the “effective” R0 value, Re0, for the time-dependent models. We introduce optimal control to both the time-dependent and age-dependent ODE models to assess the most cost-effective method for introducing the vaccine into a population. We find that the duration of protection offered by the vaccine can influence whether it is possible to eradicate infection from the population. We find the critical proportion to vaccinate to eradicate the disease. We see that introducing male vaccination would lead to a greater proportion of individuals to be vaccinated if the disease is to be eradicated. The PDE model shows that the proportion of females vaccinated has a large impact on the proportion of females infected. We show that it is cost-effective to vaccinate males and females. Our results support current government policy for age of vaccination [2]. We conclude that potential waning immunity will impact the success of the vaccine. We broadly support government policy for vaccination but recommend including male vaccination to most cost-effectively eradicate the disease.

Introduction: Cervical cancer is the fourth most common malignancy diagnosed in women worldwide. It results from cellular transformation by the high-risk human papillomavirus (HPV) oncogenes E6 and E7, which accounts for more than 99% of diagnosed cases. HPV links its life cycle to epithelial proliferation and differentiation, which requires the cells to remain active in cell cycle. p63 modulates epithelial development as well as proliferation, differentiation and DNA damage response (DDR), which makes it an important target for HPV oncoproteins to allow viral replication and survival in infected cells. Methods: In this study, small interfering RNAs targeting E7 oncoprotein and p63 in the HPV16 positive cervical cancer cell line CaSki were used. Western blotting, proliferation assays, apoptosis assays and cell cycle analysis were applied to examine the effects of E7 and p63 depletion on cell fate. Overexpression of different types of HPV-E7 was performed in the N/Tert-1 keratinocyte cell line to study the effect of E7 overexpression on p63 level. Results: E7 drives the expression of p63 at both transcript and protein levels in cervical cancer cell lines. Downregulation of E7 is accompanied by a remarkable inhibition of cell proliferation and cell cycle arrest in the G0/G1 phase. Depletion of E7 is associated with a significant reduction in p63 expression which is not due to impaired proliferation or induced differentiation. Downregulation of p63 is associated with delayed DDR in cervical cancer cells following treatment with ionising radiation. High-risk HPV E7s are more potent in inducing p63 upregulation and increasing the proliferation rates in keratinocytes. Conclusion: This work for the first time demonstrated that E7 modulates the expression of p63, which regulates DNA damage repair pathways, that promotes efficient and rapid repair of the DNA damage following ionising radiation treatment in cervical cancer cells. Tumour recurrence due to resistance to radiotherapy is common, mostly due to promoted DNA repair ability of cancer cells to reduce radiation-induced toxicity and increase cell survival in response to ionising radiation. These findings might be the key to the development of radioresistance in cervical cancer. The HPV E7-p63 axis may be a novel therapeutic target to enhance radio-sensitivity in HPV-transformed tumours.

Orientador: João Manuel Grisi Candeias / Resumo: Foram estudadas 112 gestantes atendidas no Ambulatório de Infecções Genitais (AIG) do Hospital das Clínicas, UNESP, Botucatu, no período de março de 2001 a julho de 2005. Todas as gestantes apresentavam lesões genitais sugestivas, pela patologia, de doença HPV induzida. Para essas lesões foi avaliado o local da coleta das mesmas e a presença do vírus HPV e da bactéria Chlamydia trachomatis. Nessas pacientes também se analisou a idade, o estado civil, o uso de anticoncepcional, infecções genitais e a paridade. Ambos os agentes causam problemas na gestação, tais como baixo peso aos nascer e parto prematuro. Casuística e Métodos: As pesquisas tanto de HPV como de Chlamydia t0rachomatis foram realizadas através de PCR utilizando-se, respectivamente, os primers GP5+ e GP6+ e PCT1 e PCT2. Os HPV's foram tipados por sequenciamento. Resultados: A positividade de HPV foi de 79,31%. Com relação aos tipos os mais encontrados foram o 6 e o 18. Para a Chlamydia a positividade foi de 15,8%. Conclusão: Os dados encontrados em nosso estudo são compatíveis com os encontrados na literatura em todo o mundo, demonstrando que, mesmo em países desenvolvidos, a taxa de mulheres contaminadas com HPV é muito alta. Com relação à Chlamydia, os dados também são compatíveis com os da literatura mostrando associação clara entre a presença dos dois agentes e problemas gestacionais. / Abstract: Not available. / Mestre

<p><strong>Purpose:</strong> To assess awareness and attitudes regarding HPV, HPV-vaccines, use of condom and participation in pap-smear screening among high-school students in Uppsala County. The intention was also to investigate if there were any differences between students at theoretical programmes and vocational programmes.</p><p><strong>Methods:</strong> 608 students from seven high-schools in Uppsala County answered a questionnaire covering demographics, awareness and attitudes regarding HPV, HPV-vaccine, use of condoms and pap-smear tests.</p><p><strong>Results:</strong> A majority of the students had never heard of HPV (86 %, n=521), HPV-vaccine (94 %, n=537) or the link between HPV and cervical cancer (88 %, n=563). Most respondents had a positive attitude towards HPV-vaccine (84 %, n=508), but the biggest obstacle was the high cost (37 %, n=227). The students believed that it was less likely that they would use a condom with a new partner if vaccinated (mean=78, SD=26, p<0.001), or if they or their partner used contraceptive pills (mean=62, SD=32, p<0.001) compared to how likely it was that they would use a condom in general with a new partner. The girls rated the probability that they would participate in a pap-smear screening as relatively low if vaccinated (mean=59 SD=27). Students at theoretical programmes had better knowledge about HPV and HPV-vaccines. They were also more positive to the use of condoms and participation in pap-smear screening. Furthermore, more students at theoretical programmes (11%, n=46) than at vocational programmes (9%, n=16) planned to be vaccinated (<em>p</em>=0.048).</p><p><strong>Conclusion:</strong> The awareness regarding HPV and HPV-vaccine was low among high school students in Uppsala County, especially among students at vocational programmes. More information is required to increase the awareness and motivation to use condoms and participate in pap-smear screening.</p>

Human papillomavirus (HPV) is widespread in the population and an important concern for public health. HPV-associated benign and cancerous disease is vaccine preventable yet vaccine uptake has been suboptimal. Adolescents are the primary target for vaccination yet their perspective has been inadequately examined. Ontario provides population-based preventive HPV vaccination to adolescent girls yet in the program’s first 2 years only approximately half of eligible girls received it. Effective strategies to improve vaccine uptake are needed. This thesis proposes a theory and ethics-based model to guide health promotion planning for HPV vaccination. Adopting an adolescent perspective, the model is applied and comprises: 1) a systematic review to identify barriers and facilitators to HPV vaccination from the viewpoint of young females; 2) GIS uses for communicating geospatial health information regarding vaccination; and 3) a roadmap for the future including recommendations for guiding principles, research, intervention development, and health policy.

Human Papilloma virus (HPV) can lead to cervical cancer, a common form of cancer in Sweden and globally. A vaccine has been developed for the most carcinogenic strains of HPV. In Sweden it is planned that the vaccine be included in the public national vaccination program for children. The aim of this study was to illustrate attitudes towards future sexual habits and preventive cervical cancer screening among young women and parents of girls vaccinated against HPV. This study is a part of a larger project where interviews with young women aged 17 to 26 years and parents of girls aged 13 to 17 years compose the foundation. In this study the interviews were analysed with qualitative latent content analysis, all together 21 interviews. The main result was that in both groups it was generally thought that neither future sexual habits nor participation in cervical cancer screening would be considerably affected. The young women however had deficient knowledge about what they were vaccinated against and the purpose of cervical cancer screening. The parents had better understanding. Accurate information about HPV, the HPV vaccine and cervical cancer screening in connection with vaccination is important for the outcome of future sexual habits and participation in cervical cancer screening. / Humant papillomvirus (HPV) kan leda till cervixcancer som är en vanlig cancersort både i Sverige och globalt. Ett vaccin har utvecklats mot de mest onkogena HPV-typerna. I Sverige ska HPV-vaccinet ingå i det allmänna nationella vaccinationsprogrammet för barn. Syftet med studien var att belysa inställningen till framtida sexualvanor och gynekologiska cellprovskontroller (GCK) bland unga kvinnor och föräldrar till flickor som HPV-vaccinerats. Denna studie är en del av ett större projekt där intervjuer med unga kvinnor 17-26 år samt med föräldrar till flickor 13-17 år utgör grunden. I denna studie analyserades intervjuerna med kvalitativ latent innehållsanalys, sammanlagt 21 intervjuer. Det huvudsakliga resultatet som framkom var att majoriteten av de unga kvinnorna och föräldrarna till flickorna inte trodde att framtida sexualvanor skulle förändras samt att deltagande i framtida GCK inte skulle påverkas nämnvärt. Det framkom dock att de unga kvinnorna hade bristfälliga kunskaper om vad de var vaccinerade emot och vad GCK var. Föräldrarna till flickorna hade större kunskap om vad vaccinering och GCK innebar än de unga kvinnorna. Vikten av rätt sorts information om HPV, HPV-vaccin och GCK i samband med vaccination framkom som ett betydande resultat för utfallet för de framtida sexualvanorna och deltagandet i GCK.