Thesis (M.P.H.)--Georgia State University, 2007. / Title from file title page. Russ Toal, committee chair; Michael Eriksen, Cristen J. Suhr, committee members. Electronic text (76 p. : ill., col. map) : digital, PDF file. Description based on contents viewed Feb. 25, 2008. Includes bibliographical references (p. 66-72).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (M.Sc.). / Written for the Dept. of Epidemiology, Biostatistics and Occupational Health. Title from title page of PDF (viewed 2008/12/05). Includes bibliographical references.

Thesis (M.P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Masters Abstracts International, Volume: 46-05, page: 2666. Adviser: Christine Markham. Includes bibliographical references.

Zugl.: Tübingen, Univ., Diss., 2007

Papillomaviruses (PV) are epithelium specific DNA viruses that can cause health problems ranging from harmless warts to invasive cancer. Papillomavirus induced tumours most often arise in the cervix where human papillomavirus (HPV) infections were shown to cause 99.7 % of all malignancies. This study aims to map binding sites of the multifunctional host protein CCCTC binding factor (CTCF) to the papillomavirus genome, validate them and determine the function of CTCF in the papillomavirus life cycle. Computer predictions of CTCF binding sites in the sequence of 8 different PV revealed a CTCF binding pattern including a conserved high-affinity binding site around nucleotide 3000 in high risk HPV and around nucleotide 5400 in low risk HPV. This binding pattern was experimentally confirmed using electrophoretic mobility shift assays (EMSA). The binding site around nucleotide 3000 in HPV18 was mutated and human foreskin keratinocytes (HFK) were transfected with mutant and wild type HPV18 to analyse the effect of the mutation on viral gene expression and life cycle. Western blotting of methylcellulose differentiated HFK revealed earlier expression of E2 and decreased expression of E1^E4 in the mutant compared to the wild type. Immunostaining of organotypic raft cultures grown from the mutant maintaining cells showed a significant increase in proliferating cells compared to the HFK maintaining the wild type. This was accompanied by pseudo-differentiation of keratinocytes since the cells of the granular layer of the raft expressed the terminal differentiation marker loricrin but maintained the morphology of undifferentiated cells. Thus CTCF was shown to have a major impact on the HPV life cycle and it may play a role in HPV induced carcinogenesis. Furthermore a function of CTCF in long term maintenance of the viral episome was revealed as cells maintaining the CTCF mutant were shown to lose episomes more quickly compared to wild type maintaining cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Papillomaviruses (PVs) are non-enveloped DNA viruses that are the primary etiological agents of cervical and oropharyngeal cancers. Vaccines for H(human)PV have proven to be effective prophylactic treatments; however, there is no treatment available for those currently infected. To develop new therapies, we require a clear understanding of viral pathogenesis and regulation. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.

A new opportunity to reduce cervical cancer deaths as well as other HPV associated diseases arises from recently developed prophylactic vaccines. A large body of scientific literature concludes that the vaccines provide 100% protection against the oncogenic (high-risk) HPV types 16 and 18, which are responsible for about 70% of all cervical cancers in women. The vaccines also protect against infections with HPV 6 and 11, the cause of about 90% of genital warts (condylomataacuminata) in both males and females. South Africa is faced with uncertainties about how to implement a HPV vaccine immunisation programme aimed at the improvement of health in relation to the burden of disease caused by cervical cancer and related HPV-implicated diseases whilst struggling to provide the most basic of healthcare services in the midst of a HIV pandemic and a diminished base of key healthcare professionals. In such a context and relative to other priorities and the comparative benefits of different interventions, should, and if so why should South Africa invest in a HPV vaccine immunisation programme; likewise, if not, why not? In this research, report I will provide the main ethical and legal issues related to the implementation of a National HPV vaccine immunisation intervention programme. The research method used is a literature review of some ethical & legal issues in HPV vaccine immunisation analysing findings by way of critical thinking and moral reasoning. The outcomes of my research report suggest that South Africa is bound ethically and legally to provide HPV vaccine immunisation as an intervention to reduce the scourge of HPV infections, especially cervical cancer-based on the outcomes, I make recommendations concerning policy changes at the National level. These changes include interventions targeting the youth with an HPV vaccine immunisation programme included as one type of intervention.

Le virus du papillome humain (VPH) est l'agent étiologique du cancer du col de l'utérus, d'un certain nombre d'autres cancers anogénitaux et d'un sous-groupe de cancers de la tête et du cou. Les VPH à haut-risque sont nécessaires au développement de ces cancers, mais les cellules infectées doivent acquérir d'autres mutations pour devenir cancéreuses. Dans une cellule saine, l'apparition d'altérations génétiques est contrôlée par des voies de signalisation sophistiquées qui détectent, signalent et réparent les dommages à l'ADN. Parmi ces dommages, les cassures double brin de l'ADN (DSBs) sont particulièrement nocives pour la survie cellulaire, car si elles ne sont pas réparées, elles peuvent conduire à l'apparition de mutations et dans des cas extrêmes à la mort cellulaire. Notre objectif principal était de comprendre les mécanismes menant à la dérégulation des voies de réparation des DSBs dans les cellules VPH-positives. Notre hypothèse est que l'interférence du VPH avec certains facteurs perturbe la réparation des DSBs et donc la stabilité du génome de la cellule hôte, ce qui peut conduire au développement de cancers. Nos observations ainsi que celles d'autres groupes ont permis de montrer que les cellules cancéreuses VPH-positives accumulent de manière excessive de foyers nucléaires 53BP1 (53BP1-NBs, 53BP1 nuclear bodies), suggérant que les cellules sont soumises à des niveaux élevés de stress réplicatif et que le virus interfère avec la réparation des cassures d'ADN en phase S/G2. De plus, nous avons démontré que RNF168, une ubiquitine E3-ligase impliquée dans la réparation des DSBs, est essentielle à l'amplification du génome viral dans les kératinocytes en différenciation et que la protéine est ciblée directement par l'oncoprotéine E7 des VPHs à haut risque. Cette interaction a lieu via un domaine de 40 acides aminés conservé et jusqu'alors non caractérisé de RNF168, le E7BD (E7-binding domain). L'interaction avec E7 affecte la fonction de RNF168 au DSBs, révélant un mécanisme par lequel une protéine virale contribue directement à l'acquisition d'instabilité génomique. Comme la liaison de E7 au E7BD entrave la fonction de RNF168 aux DSBs, nous pensons que ce domaine joue un rôle clé dans la régulation des fonctions de RNF168. Afin de déterminer la fonction du E7BD, nous avons optimisé une méthode de « marquage de proximité » basée sur l'utilisation de la protéine TurboID, afin de déterminer l'interactome de RNF168 dépendant de ce domaine. La biotine ligase TurboID qui agit dans des intervalles de temps très courts nous a permis de visualiser les interactions lors du recrutement des facteurs de réparation aux dommages, un processus extrêmement rapide. Ainsi nous avons pu valider l'utilisation de TurboID pour établir l'interactome des protéines impliquées dans la réparation des DSBs telles que RNF168. De plus, nous avons identifié plusieurs nouveaux partenaires de l'E3-ligase qui sont perdus lorsque le E7BD est absent. Par la suite, l'impact de certains de ces interacteurs sur les fonctions de RNF168 a été évalué par une approche d'ARN interférence. Nous avons ainsi pu identifier un nouvel interacteur de RNF168, l'hexokinase HK1, qui régule les quantités de RNF168 disponible dans la cellule. Finalement, nous avons décidé de tirer profit de cette approche et développé un système permettant la détection de protéines faiblement exprimées ou de locus d'ADN spécifiques à l'aide de la protéine TurboID. Nous avons démontré que l'utilisation de TurboID-RNF168 permet de visualiser le recrutement de RNF168 à différents types de dommages sans affecter la dynamique de réparation des DSBs. De plus, l'utilisation de TurboID-dCas9 nous a permis de visualiser en immunofluorescence des loci répétés tel que les télomères, mais également l'ADN viral intégré dans le génome de cellules cancéreuses VPH-positives. Ainsi ce projet a contribué à l'amélioration des connaissances sur les processus de maintien de la stabilité génomique dans les cellules infectées par le VPH et nous pensons que les découvertes présentées dans ce travail contribuent à la compréhension de la régulation de protéines cellulaires comme RNF168. / The human papillomavirus (HPV) is the etiological agent of cervical cancer, a subset of anogenital cancers as well as head and neck cancers. High-risk HPV is necessary for the development of cancers, but infected cells must acquire additional mutations to become cancerous. In a normal cell, the acquisition of genetic alterations is controlled by numerous signaling pathways that detect, signal, and repair DNA damage. Among those, DNA double-strand breaks (DSBs) are particularly dangerous. If left unrepaired, they can lead to the acquisition of mutations and ultimately to cell death. Our main objective was to understand the mechanisms leading to the deregulation of DSBs repair pathways in HPV-positive cells. We hypothesize that HPV components can interfere with DSBs repair and therefore, with the stability of the host cell genome, which leads to cancer development. Our observations and those of other groups have shown that HPV-positive cancer cells present an abnormal accumulation of 53BP1-NBs, suggesting that the cells are subjected to high levels of replicative stress and that the virus interferes with the repair in the S / G2 phase of the cell cycle. In addition, we have demonstrated that RNF168, a ubiquitin E3-ligase involved in the repair of DSBs, is essential for the amplification of the viral genome in differentiating keratinocytes and that the protein is directly targeted by the oncoprotein E7. This interaction occurs via a conserved and uncharacterized 40 amino acid domain of RNF168, the E7BD (E7-binding domain). The interaction with E7 affects the function of RNF168 at DSBs, revealing a mechanism by which a viral protein directly contributes to the acquisition of genomic instability. Furthermore, the binding of E7 to the E7BD hinders the function of RNF168 at DSBs, suggesting that this domain plays a key role in the regulation of RNF168 functions. To determine the role of the E7BD, we optimized a method of proximity labeling using the TurboID protein to determine the RNF168 E7BD-dependent interactome. The TurboID biotin ligase biotinylated proximal proteins in very short time intervals allowed us to visualize the interactions during the recruitment of DNA damage repair factors. We were thus able to validate the use of TurboID to establish the interactome of proteins involved in DSBs repair such as RNF168. In addition, we have identified several new partners of the E3-ligase that are lost in the absence of the E7BD. Subsequently, the impacts of some of these interactors on the functions of RNF168 were evaluated by an RNA interference approach. This enabled us to identify the hexokinase HK1 as a new partner and regulator of RNF168. Finally, we decided to take advantage of this approach and developed a system for the detection of weakly expressed proteins or specific DNA loci using the TurboID protein. We demonstrated that TurboID-RNF168 allows the visualization of the protein recruitment to different types of DNA damage without affecting the dynamics of DSBs repair. In addition, we used TurboID-dCas9 to visualize repeated loci such as telomeres, and viral DNA integrated into the genome of HPV positive cancer cells by immunofluorescence. In conclusion, the results present in this thesis will contribute to our knowledge of genomic stability maintenance in HPV infected cells and our understanding of the regulation of cellular proteins such as RNF168.

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.