MSc (Microbiology) / Department of Microbiology / BACKGROUND: Human papillomavirus (HPV) is a common sexually transmitted virus known to be a causative agent of cervical cancer (CC), one of the most frequent cancers in women worldwide. HPV is a double stranded DNA virus of approximately 7,900 bp; belonging to Papillomaviridae family. To date, about 202 low risk (LR) and high risk (HR) HPV genotypes have been identified. However, available vaccines against HPV infection are designed based on the most common known genotypes. Therefore, it is critical to understand the scope and diversity of HPV genotypes in all geographical locations which can help to inform the design and development of future vaccines. OBJECTIVE: The objective of this study was to describe the burden and diversity of HPV genotypes in a cohort of women in routine care in northern South Africa. METHODS: Eighty seven women consented to participate in the study and each provided a specimen for analysis. With the help of qualified health care practitioners, Aptima Cervical Specimen Collection and Transport Kit (Hologic, San Diego, CA) was used to collect cervical specimens from each study participant following the manufacturer’s procedure. Total DNA was purified from the cervical pellet using QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified DNA was then subjected to a single round conventional PCR in a reaction volume of 100 μl to amplify HPV L1 gene comprising of approximately 450 bp. A portion of each PCR amplicon from each participant was denatured, hybridized and genotyped using the Linear Array HPV genotyping Test Kit (Roche Molecular Systems, Inc. Branchburg, NJ USA). The kit is designed to detect 37 HPV genotypes (genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108). To detect the HPV genotypes, the Linear Array (LA) reference guide was used for results interpretation following the manufacturer’s instructions. The other portion of each of the amplicons was subjected to next generation sequencing (NGS) using the Illumina MiniSeq platform. Using the Nextera XT DNA Library preparation kit, an initial input of 1ng genomic DNA was tagmented, cleaned up, normalized and pooled. The pooled library was then denatured with 0.1 N NaOH and diluted into a final volume of 500 μl at 1.8 pM then sequenced using the Local Run Manager option following the manufacturer’s instructions. The generated sequence data was downloaded into fastaQ format and analysed using Genious 11.0.5 software. RESULTS: Of the 87 participants, the overall proportion of women harbouring HPV DNA by linear array (LA) PCR was 23% (n=20). Of the 20, 16 (80%) were living with HIV. However, this difference was not significant (p=0.077). Genotyping data generated by Roche LA method was successful for all the 20 positive amplicons. In this study, 27 (73%) of the 37 HPV genotypes incorporated in the Roche Linear Array method were detected. The detected genotypes include: types 84, 83, 81, 73, 72, 71, 70, 69, 68, 66, 62, 61, 59, 54, 53, 52, 51, 45, 42, 39, 35, 26, 18, 16, 6, IS39 and CP6108. Most women (15/20;75%) harboured multiple infections compared to single infection. In terms of genotypes distribution, the most frequent genotypes detected LR HPV types in increasing order of frequency included HPV type 61 and 83 (12%), 62 (36%) and 81 (43%). On the other hand, HPV type 66, 53, 52, 51, 18 and 16 were the most common genotypes detected HR HPV types. In contrast, although genotyping data was successfully generated from 15 of 20 women (75%), NGS technology was seen to be more sensitive compared to Roche LA method. Nearly all the detected genotypes identified by the commercial kit were detected by NGS. In addition, NGS detected 10 namely: HPV types 11, 31, 33, 40, 55, 56, 58, 64, 67, and 82 that were not detected by the LA yet incorporated in the kit. Moreover, it was observed that NGS identified additional 6 HPV types including HPV types 2, 27, 30, 35, 85 and 102 not incorporated in the Roche LA kit. A similar distribution of HPV multiple infections was observed in the study population, however, high frequency of 93% (14 of 15) was detected by NGS. The proportion of women harbouring one or more of the 22 LR HPV types was 100% (n=15).The most frequent LR genotypes in increasing order of frequency was HPV type 62 and 70 (27%), 6 (40%) and 11 (47%). HPV types 40, 42, 54, 72, 64, and 81 were the least detected genotypes with n=1 (7%) each. Furthermore, the common combination observed among the participants was type 6 and 11. In contrast, the most frequent detected genotypes in the study population by NGS under the HR HPV types in increasing order of frequency include type 35 (21%), 39, 56 and 82 (29%), 68 (36%) and 51 (50%). In addition, HPV types 26, 31, 45, 53, 56, 58 and 66 were the least detected genotypes n=1 (7%) in the study population. HPV 39 and 68 were observed as the common combination detected under HR HPV types. Following genotyping by LA and NGS, the demographic and clinical data of all the 20 positive subjects by PCR were subjected to statistical analysis to determine the association between HPV positive DNA status and associated risk factors. Smoking status (p=0.000), age at first sexual intercourse (p=0.011), vaccination status (p=0.000), gender of sexual partner (p=0.000), highest level of education (p=0.004), marital status (p=0.008) and number of sexual partners (p=0.000) were found to be having a positive statistical association. CONCLUSION: Amplification of targeted HPV DNA from cervical specimens demonstrated the presence of HPV infection in the study cohort, with a proportion of 23%. The findings illustrate that there is a diversity of HPV genotypes prevalent in the study population as shown by Roche LA and NGS methods. However, the NGS method was observed to be more sensitive than Roche LA in detecting HPV genotypes. Furthermore, NGS identified 6 additional HPV types not incorporated in the Roche LA. Thus, there are genotypes that may be present in the study population that the Roche commercial kit may fail to detect. Therefore, is it imperative to use both genotyping methods to confirm HPV genotypes. / NRF

The aim of the study was to gain an in-depth understanding of the girl children’s parents perceptions regarding the papillomavirus vaccine roll-out programme at schools in Tshwane District, Gauteng Province. The researcher used a qualitative exploratory research design to address the research objective of the study as the qualitative method enables the researcher to explore and describe the study phenomenon. Data were collected from 12 parents of girl children who received the papillomavirus vaccine at one of the schools in Soshanguve township, which is one of the biggest townships in the Tshwane District. Data was analysed manually using content analysis. The following four superordinate themes emerged from data analysis: a) Communication of the programme, b) Motivation for allowing their children to be immunized. c) Response to immunisation, d) Suggested ways of enhancing the programme. These themes were discussed in relation to existing literature. Recommendations were made based on the findings to enhance the papillomavirus vaccination programme and for future research. / Health Studies / M.A. (Nursing Science)

Les virus du papillome humain (VPH) sont de petits virus à ADN double brin infectant les épithéliums de la peau et des muqueuses. La réplication nécessaire au maintien de leur génome dans les cellules infectées dépend des protéines virales E1 et E2. Au cours de la réplication, E1 est recrutée à l’origine de réplication par E2 afin d’être assemblée en doubles hexamères capables de dérouler l’ADN. E1 contient un domaine C-terminal responsable de l’activité ATPase/hélicase, un domaine central de liaison à l’origine et une région N-terminale régulant la réplication in vivo. Cette région contient des signaux de localisation et d’export nucléaire qui modulent le transport intracellulaire de E1. Chez le virus du papillome bovin (VPB), il a été proposé que ce transport est régulé par la sumoylation de E1. Finalement, la région N-terminale de E1 contient un motif de liaison aux cyclines permettant son interaction avec la cycline E/A-Cdk2. La phosphorylation de E1 par cette dernière régule différemment l’export nucléaire des protéines E1 du VPB et du VPH. Dans la première partie de cette étude, nous avons démontré que bien que la protéine E1 des VPH interagit avec Ubc9, l’enzyme de conjugaison de la voie de sumoylation, cette voie n’est pas requise pour son accumulation au noyau. Dans la seconde partie, nous avons déterminé que l’accumulation nucléaire de E1 est plutôt régulée pas sa phosphorylation. En fait, nous avons démontré que l’export nucléaire de E1 est inhibé par la phosphorylation de sérines conservées de la région N-terminale de E1 par Cdk2. Puis, nous avons établi que l’export nucléaire de E1 n’est pas nécessaire à l’amplification du génome dans les kératinocytes différenciés mais qu’il est requis pour le maintien du génome dans les kératinocytes non différenciés. En particulier, nous avons découvert que l’accumulation nucléaire de E1 inhibe la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase S et que cet effet anti-prolifératif est contrecarrée par l’export de E1 au cytoplasme. Dans la troisième partie de cette étude, nous avons démontré que l’arrêt cellulaire induit par E1 dépend de sa liaison à l’ADN et à l’ATP, et qu’il est accompagné par l’activation de la voie de réponse aux dommages à l’ADN dépendante de ATM (Ataxia Telangiectasia Mutated). Ces deux événements semblent toutefois distincts puisque la formation d’un complexe E1-E2 réduit l’activation de la voie de réponse aux dommages par E1 sans toutefois prévenir l’arrêt de cycle cellulaire. Finalement, nous avons démontré que la réplication transitoire de l’ADN viral peut avoir lieu dans des cellules arrêtées en phase S, indépendamment de l’activation de la voie de réponse aux dommages à l’ADN et de la kinase ATM. Globalement, nos résultats démontrent que l’export nucléaire de E1 est régulé par sa phosphorylation et non par sa sumoylation. Ils démontrent également que l’export nucléaire de E1 est essentiel au maintien du génome dans les kératinocytes, possiblement parce qu’il prévient l’inhibition de la prolifération cellulaire et l’activation de la voie de réponse aux dommages à l’ADN en limitant l’accumulation de E1 au noyau. / Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin and the mucosa. HPV rely on two viral proteins, E1 and E2, to replicate and maintain their genome in the nucleus of infected cells. During replication, the E1 helicase is recruited to the origin of replication by E2 and is assembled into a double-hexamer that unwinds DNA ahead of the replication fork. E1 is comprised of a C-terminal enzymatic domain with ATPase/helicase activity, a central origin-binding domain and a N-terminal regulatory region that is required for viral DNA replication in vivo. The latter region of E1 contains a nuclear localization signal and a nuclear export signal that regulate its shuttling between the nucleus and cytoplasm. For bovine papillomavirus (BPV) E1, this shuttling was suggested to be controlled by the sumoylation of E1. In addition to the NES and NLS, the N-terminal region of E1 contains a conserved cyclin-binding motif that is required for the interaction of E1 with cyclin E/A-Cdk2. Cdk2 phosphorylation of E1 has been reported to control the nuclear export of E1 from BPV and HPV, albeit differently. In the first part of this study, we showed that although HPV E1 interacts with Ubc9, the conjugating enzyme of the sumoylation pathway, this pathway is not required for its accumulation in the nucleus. In the second part, we found that the nuclear accumulation of E1 is, instead, regulated by phosphorylation. Specifically, we found that Cdk2-dependent phosphorylation of conserved serines in the E1 N-terminal region inhibits the nuclear export of HPV E1. Furthermore, we reported that nuclear export is not essential to amplify the viral genome in differentiating keratinocytes but that it is required for its long-term maintenance in undifferentiated keratinocytes. Importantly, we found that the nuclear accumulation of E1 induces a S-phase arrest that is detrimental to cellular proliferation and that this anti-proliferative effect can be counteracted by the export of E1 from the nucleus to the cytoplasm. In the last part of this study, we showed that this arrest is dependent on the DNA- and ATP-binding activities of E1. Furthermore, we found that the cell cycle arrest induced by E1 is accompanied by the activation of a DNA damage response (DDR) dependent on the ATM (Ataxia Telangiectasia Mutated) pathway. However, these two events seem to be distinct since complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Importantly, we demonstrated that transient viral DNA replication still occurs in S-phase arrested cells, independently of the induction of a DDR and of the ATM kinase. Collectively, these data indicate that nuclear export of E1 is regulated by phosphorylation and not by sumoylation. They also revealed that nuclear export of E1 is essential for maintenance of the viral episome in keratinocytes, at least in part to limit its nuclear accumulation and prevent its detrimental effect on cellular proliferation and induction of a DDR.

Les papillomavirus sont de petits virus à ADN double brin qui infectent les cellules de l’épithélium de la peau et des muqueuses d’une variété de vertébrés causant des lésions bénignes telles des verrues. Certains de ces virus sont également associés au développement de lésions malignes, notamment le cancer du col utérin. La protéine régulatrice E2 des papillomavirus est impliquée dans diverses fonctions contribuant à l’établissement de l’infection par ces virus. Entre autre, E2 régule la transcription des gènes viraux, participe à l’initiation de la réplication de l’ADN viral en s’associant à l’hélicase virale E1 et est responsable du maintien et de la ségrégation de l’épisome viral au cours de la division cellulaire. Toutes ces activités sont attribuables à la capacité de E2 à s’associer au génome viral et à interagir avec des protéines virales et cellulaires. De plus, ces fonctions sont elles-mêmes régulées par des modifications post-traductionnelles de la protéine E2. Plusieurs études ont été réalisées afin de découvrir les mécanismes de régulation des fonctions de E2 mais le rôle exact des différents domaines de E2 dans ces contrôles reste à être défini. En premier lieu, nous nous sommes intéressés à l’interaction entre E2 et Brd4(L) qui avait été définie comme étant essentielle à la ségrégation de l’épisome. Plusieurs caractéristiques associées à la protéine Brd4(L) telles que sa capacité à lier les lysines acétylées des histones, son interaction avec le complexe Mediator et sa participation à l’activation de la transcription en formant un complexe avec pTEFb, nous ont permis d’émettre l’hypothèse que l’interaction E2-Brd4(L) est nécessaire à l’activité transcriptionnelle de E2. Nous avons démontré que la protéine Brd4(L) interagit avec le domaine de transactivation de E2 de divers types de papillomavirus. De plus, cette interaction implique les résidus de E2 essentiels à son activité transcriptionnelle. Ainsi, ces résultats proposent que l’association E2-Brd4(L) serve à la régulation de la transcription des gènes viraux. Dans un second temps, nos recherches se sont concentrées sur l’existence d’une interface de dimérisation au sein du domaine de transactivation de E2 et de son implication dans les activités transcriptionnelles et réplicatives de la protéine. Nos études ont aussi mis en évidence que l’intégrité de la structure de ce domaine contribue au bon fonctionnement de la réplication du génome viral. Cette découverte suggère que la dimérisation de E2 peut réguler l’initiation de la réplication et propose l’existence d’un niveau de régulation additionnel impliquant l’état de la structure quaternaire de la protéine E2 et une modulation de l’interaction entre E1 et E2 à cette étape du cycle viral. Finalement, l’étude de l’instabilité de la protéine E2 nous a permis de définir une région importante dans le domaine flexible de la protéine, nécessaire à sa dégradation par le protéasome. De plus, la présence de résidus conservés localisés dans ce domaine, sont associés à la dégradation et portent la signature d’un signal de localisation nucléaire de type PY-NLS, suggérant que la stabilité de la protéine E2 est régulée par sa localisation au sein de la cellule. Ces études démontrent l’existence de nouvelles stratégies de régulation des activités transcriptionnelle et réplicative de la protéine E2 des papillomavirus. La compréhension de ces mécanismes nous permet de mieux cerner les étapes favorisant l’établissement et la progression du cycle viral et d’identifier de nouvelles cibles thérapeutiques contre les infections aux papillomavirus. / Papillomaviridae is a family of small double-stranded DNA viruses known as papillomaviruses (PV) which infect skin and mucosal epithelial cells where they cause benign lesions such as warts. A subset of these viruses is associated with the development of malignant lesions and is the causal agent of cervical cancer. Papillomavirus E2 regulatory protein is involved in several functions leading to the establishment of the viral infection. These activities include the regulation of viral genes transcription, it participation to the initiation of viral DNA replication by recruiting the viral helicase E1, and to the maintenance and segregation of the viral episome during cellular division. All these functions are associated to the ability of E2 to bind specifically the viral genome, to interact with viral and cellular proteins and to acquire post-translational modifications. The first article of this thesis led to the identification of Brd4(L) as the major protein associated to E2 protein of different papillomavirus types. This interaction involves the amino acids associate to the transcription function of E2. The protein Brd4(L) was identified originally as a factor that maintains epigenetic memory by it interaction with acetylated histones during mitosis. This association with the chromatin, it interaction with Mediator complex and it participation to the cellular transcription by recruiting pTEFb complex allowed us to propose that the interaction between Brd4 and E2 is essential to the regulation of viral gene transcription. The second part of this work based on previous characterization of the transactivation domain dimerization interface; investigate the role of this surface in the transcriptional and replicative activities of E2. Our studies demonstrated that the integrity of the TAD dimerization interface may contribute to the DNA replication activity of E2. This discovery suggests that the dimerization interface may regulate the viral DNA replication by the redox state of the E2 protein. A fine characterization of this interface may provide new aspect of the interaction between E1 and E2 in the context of viral cycle. Finally, the third section of this thesis define a region of E2 protein associated to it degradation by the proteasome. This study also demonstrates that the stability of E2 is related to its cellular localization and suggests that the highly conserved residues found in this region may represent a PY-NLS nuclear localization signal signature. This thesis shows the existence of different approaches to regulate the transcriptional and the replicative activities as well as the stability of the papillomavirus E2 protein to favor the establishment and the progression of viral cycle.

Infecção persistente por tipos oncogênicos de papilomavírus humano (HPV) é considerada como o principal fator de risco para desenvolvimento de carcinoma invasivo do colo uterino (CCU) e de lesões intraepiteliais cervicais (SIL). Fatores genéticos do hospedeiro, como o polimorfismos de genes HLA (human leukocyte antigen), também têm sido implicados na suscetibilidade a estas patologias e à infecção por (HPV), como observado em diversos estudos caso-controle. Neste estudo investigou-se em uma coorte de mulheres (Ludwig-McGill cohort) se a variabilidade dos genes HLA-DRB1 e -DQB1 influenciam na história natural das infecções por HPV e no risco de SIL. A tipificação de DRB1 e DQB1 foi realizada em 620 amostras provenientes de um estudo epidemiológico prospectivo. A positividade para HPV foi testada em amostras da mesma paciente coletadas a cada 4 meses, obtidos durante o primeiro ano de seguimento, enquanto os resultados de citologia perfazem os 2 primeiros anos de seguimento. Infecções persistentes de curta ou longa duração foram definidas como 2 e 3 resultados consecutivos positivos para o mesmo tipo de HPV, respectivamente. As associações foram estimadas através de razões de chance e intervalos de confiança de 95%, ajustadas para potenciais fatores de confusão. Os resultados obtidos indicam que a prevalência da infecção por HPV e o risco de persistência variam dependendo do haplótipo HLA. O haplótipo DRB1*0301-DQB1*0201 mostrou-se protetor contra a infecção por HPV, e DRB1*1102-DQB1 *0301 contra infecções persistentes. Já os haplótipos DRB1*1601-DQB1*0502 e DRB1 *0807-DQB1*0402 foram fatores de risco para infecções persistentes por HPV. Não foi observada uma forte concordância entre risco de infecção por HPV e risco de SIL associados a determinado HLA, em parte porque o número de pacientes com SIL foi um fator limitante neste estudo. Um risco aumentado de SIL, independente da infecção por HPV, foi associado com DRB1*0301 e DR12. Portadoras de DR4 e DQB1*0601 tiveram uma maior probabilidade de desenvolver SIL e HSIL, respectivamente. Uma associação negativa entre o alelo DQB1*0301 e HSIL foi verificada. Análise do dimorfismo na posição 86 da cadeia β de HLA-DR mostrou que valina nesta posição tem um efeito protetor para prevalência e persistência de infecção por HPV, e maior risco de SIL no grupo com infecções transitórias por HPV. Em outra análise, investigamos a distribuição de grupos alélicos de DRB1 em uma série independente de amostras provenientes de pacientes com CCU. Observamos um risco diminuído de desenvolvimento de CCU associado a DR3. Por outro lado, DR4 e DR8/12 mostraram-se fatores de risco para o CCU nesta população. Estes resultados sugerem que o polimorfismo de HLA desempenha um papel na história natural das infecções por HPV, SIL e CCU. Também analisamos respostas linfoproliferativas em pacientes com CCU, contra peptídeos derivados de E6 e E7 de HPV16. As respostas positivas foram mais freqüentes contra peptídeos de E6 do que E7. Não observamos resposta contra um peptídeo ou região em particular. Parte desta diversidade nas respostas linfoproliferativas pode ser relacionada com o polimorfismo de genes HLA e seu papel na seleção de epítopos. / Persistent infection with oncogenic human papillomavirus (HPV) is the major risk factor for the development of malignant lesions in the uterine cervix. Host factors have also been implicated in the pathogenesis of these diseases. Associations between human leukocyte antigen (HLA) polymorphisms and cervical cancer, precursor lesions or HPV infections have been reported by case-control studies in several populations. This study investigated through cohort analysis if human leukocyte antigen (HLA)-DRB1 and DQB1 variability is related to human papillomavirus (HPV) infection and squamous intraepithelial lesions (SIL) prevalence and persistence. HLA-DRB1 and DQB1 genes were typed in 620 samples from the Ludwig-McGill cohort. HPV positivity was tested in specimens collected every 4 months during the first year of follow-up. Persistent and long-term infections were defined as at least 2 or 3 consecutive positive results for the same HPV type, respectively. Analysis of SIL included data obtained during the two first years of follow-up. The magnitudes of associations were estimated by unconditional logistic regression analysis adjusted for potential confounders. Certain HLA alleles and haplotypes were associated with HPV either HPV prevalence or persistence. The DRB1*0301-DQB1*0201 haplotype was associated with a lower risk for HPV infection and DRB1*1102-DQB1*0301 for HPV persistence. DRB1*1601-DQB1*0502 and DRB1*0807-DQB1*0402 were associated with a increased risk for persistent HPV infection. It was not observed a strong concordance between the associations verified for HPV prevalence/persistence and SIL, possibly due to the limited number of SIL specimens. A higher risk for SIL, independent of HPV infection, was observed for DRB1*0301 and DR12. DR4 and DQB1*0601 carriers showed a higher frequency of SIL and HSIL, respectively. A negative association between DQB1*0301 and HSIL was verified. Valine at position 86 of the DRβ chain was associated with reduced risks of HPV positivity and persistence, as compared to glycine carriers. However, valine carriers had a higher risk of SIL if transiently infected by HPV. We also analyzed an independent sample of patients with invasive cervical, and a protective effect was observed for DR3. On the other hand, DR4 and DR8/12 were associated with a higher risk for cervical cancer in this population. Our results suggest that HLA class II polymorphisms and pocket 1 profile are involved in clearance and maintenance of HPV infection and the risk of SIL and CCU, consistent with the hypothesis that genetic background is important in the natural history of HPV infections and associated lesions. We also analyzed lymphoproliferative responses against HPV16 E6 and E7 peptides, in patients with invasive cervical cancer. Lymphoproliferative responses were more frequent for E6 peptides than for E7 peptides. The responses were not restricted to a particular peptide, which is expected based on HLA variability observed among patients.

Foi estudado o perfil nosológico da população ribeirinha do Baixo Rio Machado em Rondônia, área de Amazônia Ocidental no Brasil. Neste estudo, escolhemos abordar o perfil das seguintes doenças: malária, hepatite viral B e C, parasitose intestinal e papilomavírus humano. Estas doenças foram priorizadas devido à importância clínica e epidemiológica e também escassez de estudos referentes nas populações ribeirinhas amazônicas. Para isto, foi realizado um estudo descritivo no Baixo Rio Machado que se localiza a 250 km de Porto Velho (capital do Estado de Rondônia) pela margem direita do Rio Madeira, que é um afluente volumoso do Rio Amazonas, no Brasil. Nesta área, vivem aproximadamente 806 pessoas distribuídas em 55 comunidades ribeirinhas isoladas. Em Rondônia, encontramos locais, como o Rio Machado, que abrigam a presença de portadores assintomáticos do Plasmodium, fato que pode contribuir para a persistência desta doença na região. Para caracterizar o perfil da malária ribeirinha, focalizando aspectos da infecção assintomática e sintomática, foram realizados hemoscopia e Reação em Cadeia da Polimerase (PCR) com amplificação do DNA ribossomal do parasita para Plasmodium vivax e Plasmodium falciparum para o diagnóstico da malária. Foi considerado como assintomático o paciente com hemoscopia positiva ou PCR positivo que permaneceu sem sintomas durante pelo menos 60 dias. Foi realizado acompanhamento longitudinal de 70 dias após o primeiro corte transversal (nC1=585 pessoas) para observação dos pacientes assintomáticos diagnosticados por hemoscopia, onde 25 pacientes (4,25%) permaneceram assintomáticos durante todo esse seguimento. Após, foi realizado mais dois cortes transversais a cada 6 meses (nC2=583 pessoas, nC3=607 pessoas), com tratamento dos indivíduos assintomáticos diagnosticados por PCR no corte transversal anterior ou hemoscopia atual e também dos casos sintomáticos. Foi estudado, na população que permaneceu na área durante toda a pesquisa (n=379), o perfil e o impacto do tratamento da infecção assintomática. Houve diminuição da infecção por P. falciparum de seis vezes e aumento da prevalência de malária por P. vivax de aproximadamente três vezes. O diagnóstico por PCR foi de 2-5 vezes mais eficiente do que por hemoscopia e a prevalência de infecção assintomática foi de 16 a 22% nos cortes transversais. Assim, foi observada a importância do tratamento dos indivíduos assintomáticos para o controle da malária no local, sendo que o incremento de malária por Plasmodium vivax deve ter ocorrido devido a recaídas da doença. Com o estudo entomológico da área, foi verificado que o vetor Anopheles possui atividade hematofágica moderada e principalmente em região peridomiciliar, sendo o A. darlingi a principal espécie da região. Existem poucos dados sobre a prevalência de hepatites na população ribeirinha de Rondônia, o que dificulta a vigilância epidemiológica nestas regiões amazônicas. Foi realizado um estudo de prevalência de hepatite B e C na área, com 123 pacientes pertencentes a 5 comunidades ribeirinhas, incluindo todas as pessoas que estavam presentes na área no momento do corte transversal, de todas as faixas etárias. Foi realizada sorologia para os marcadores de hepatite B: HbsAg, Anti-Hbc (total) e Anti-Hbs e sorologia para hepatite C com o marcador anti-HCV. Foram encontrados 12 (14,7%) pacientes com hepatite B aguda, 29 (38,7%) pacientes apresentaram imunidade vacinal para hepatite B e 7 (9,3%) pacientes apresentaram hepatite C, sendo que um (1,3%) paciente tinha co-infecção para hepatite B e C. Notou-se que esta população ribeirinha está exposta às hepatites virais, sendo necessário intensificar a vigilância epidemiológica na área, assim como a cobertura vacinal e também fornecer cuidados preventivos, curativos e paliativos em relação a estas doenças. A infecção pelo papilomavírus humano (HPV) é altamente prevalente, sendo detectada em aproximadamente 10% a 20% da população sexualmente ativa entre 15 e 49 anos de idade. A introdução de testes mais acurados para a detecção do DNA do HPV em investigações epidemiológicas confirmou a importância do HPV, principalmente dos tipos de alto risco, como o principal fator de risco para o desenvolvimento de neoplasia intra-epitelial cervical e câncer do colo uterino. Não foram encontrados estudos sobre a existência do HPV em populações ribeirinhas amazônicas na literatura médica. Foi realizado, então, um estudo transversal para levantar a prevalência de HPV nesta população, com 84 participantes em idade sexualmente ativa presentes na área. Após coleta de consentimento informado e questionário clínico-epidemiológico, foi realizado exame para isolar o HPV. A tipagem do HPV foi realizada com amplificação dos DNAs por PCR empregando iniciadores genéricos seguida de hibridização em pontos, capaz de identificar mais de 40 tipos diferentes de HPVs. Foram encontradas 18 pacientes contaminadas pelo HPV, perfazendo 21,4% da amostra. Os tipos de HPV encontrados foram: 53, 58, 31, 56, 16, 83, 55, 66, 45, 51, 40, 42, 6, 68. Os tipos de HPV mais freqüentes foram 51 (23%), 58 (19%), 53 (7,7%), 83 (7,7%), sendo HPV 16 encontrado em 3,8% das pacientes HPV positivas e a prevalência do HPV de alto risco oncogênco foi de 13,1%. Desta forma, foi encontrado alta prevalência de HPV na população ribeirinha amazônica estudada, evidenciando a necessidade de vigilância epidemiológica para câncer de colo uterino na região. Por fim, foi estabelecido a prevalência de parasitoses intestinais na população, abordando a correlação das enteroparasitoses com malária assintomática e anemia, realizando um inquérito coproparasitológico na área incluindo pacientes de todas as faixas etárias, que estivessem dispostos a participar do estudo. A análise das amostras foi realizada pelo método da Sedimentação Espontânea (método de Lutz ou Hoffmann, Pons & Janer). Entre os 268 exames de fezes realizados, a prevalência de parasitose intestinal encontrada na região em estudo foi de 86,6%. Entre os helmintos, Ascaris lumbricoides (47%), Ancilostomideos (37,3%), Trichuris trichiura (3,4%), Capillaria hepatica (2,3%), foram os parasitas mais encontrados. Entre os protozoários foram encontrados: Entamoeba coli (21,4%), Entamoeba histolytica (12%), Giardia intestinalis (15,4%), Endolimax nana (10,1%) e Iodamoeba butschlii (7,5%). A alta prevalência de parasitose intestinal encontrada na pesquisa foi concordante com outros dados desenvolvidos na Região Amazônica. Com este estudo, a região da Amazônia Ocidental insere-se no cenário geográfico mundial de distribuição da Capillaria hepatica, particularmente, a região do Baixo Rio Machado. / Was studied the nosological profile of the riverside population of the Baixo Rio Machado in Rondônia, Western Amazon area of Brazil. In this study, was chosed the profile of following diseases: malaria, hepatitis B and C, intestinal parasite and human papillomavirus. These diseases have been prioritized due to clinical and epidemiological importance and scarcity of studies in the riverside Amazon population. For this, was conducted a descriptive study in the Baixo Rio Machado which is located 250 km from Porto Velho (capital of Rondônia State) in the right bank of the Rio Madeira which is a large tributary of the Amazon River in Brazil. In this area, live about 806 people distributed in 55 riversides communities isolated. In Rondônia, we find places such as the Rio Machado, which shelter to the presence of asymptomatic carriers of Plasmodium, which may contribute to the persistence of this disease in the region. To characterize the profile of malaria riverside, focusing on aspects of symptomatic and asymptomatic infections, were performed haemoscopia and Polymerase Chain Reaction (PCR) with amplification of ribosomal DNA for the parasite Plasmodium vivax and Plasmodium falciparum to the diagnosis of malaria. Was considered as asymptomatic patient with positive haemoscopia or positive or PCR that remained without symptoms for at least 60 days. Longitudinal follow-up was conducted for 70 days after the first cross section (nC1 = 585 people) for observation of asymptomatic patients diagnosed by haemoscopia where 25 patients (4.25%) remained asymptomatic throughout the follow-up. After was performed two cross cuts every 6 months (nC2 = 583 people, nC3 = 607 people), with treatment of asymptomatic individuals diagnosed by PCR in previous transverse cross-sections or haemoscopia a current and also symptomatic cases. We studied in the population that remained in the area throughout the study (no= 379), the profile and impact of treatment of asymptomatic infection. There was a decrease in infection by P. falciparum of six times and increased prevalence of malaria by P. vivax approximately three times. The diagnosis by PCR was 2-5 times more efficient than haemoscopia and the prevalence of asymptomatic infection was 16 to 22% in transverse cross-sections. Thus, was saw the importance of treatment of asymptomatic individuals for the control of malaria in the place, and the increase in malaria by Plasmodium vivax should have occurred due to relapse of the disease. With entomological study of the area, was saw that the vector Anopheles has moderate blood activity and especially in peridomiciliary region and the A. darlingi the main species in the region. There are few data on the prevalence of hepatitis in riverside population of Rondônia, which makes surveillance in Amazonian regions. Was conducted a study of prevalence of hepatitis B and C in the area, with 123 patients belonging to 5 communities, including all the people who were present in the area at the cross section of all ages. Serology was performed for markers of hepatitis B: HBsAg, anti-HBc (total) and anti-HBs and serology for hepatitis C with anti-HCV marker. Was found 12 (14.7%) patients with acute hepatitis B, 29(38.7%) patients demonstrated immunity to hepatitis B vaccine and 7(9.3%) patients had hepatitis C, with one (1.3%) patient had co-infection for hepatitis B and C. Realize that this riverside population is exposed to viral hepatitis, being necessary to intensify epidemiological surveillance in the area, as well as immunization coverage and provide preventive care, curative and palliative for these diseases. The infection by human papillomavirus (HPV) is highly prevalent, being detected in approximately 10% to 20% of the sexually active population between 15 and 49 years of age. The introduction of more accurate tests for the detection of HPV DNA in epidemiological investigations confirmed the importance of HPV, particularly types of high risk, as the main risk factor for the development of cervical intraepithelial neoplasia and cervical cancer. Was found no studies on the existence of HPV in riverside Amazonian populations in the medical literature. The transversal cross-section study was take up to raise the prevalence of HPV in this population, with 84 participants in sexually active age in the area. After collection of informed consent and clinical-epidemiological questionnaire, was conducted to isolate the HPV test. The typing of HPV was taking with the DNAs for PCR using generic primers followed by hybridization in points, able to identify over 40 different types of HPV. Was found 18 patients infected by HPV, comprising 21.4% of the sample. The HPV types found were: 53, 58, 31, 56, 16, 83, 55, 66, 45, 51, 40, 42, 6, 68. The most common HPV types were 51 (23%), 58 (19%), 53 (7.7%), 83 (7.7%) with HPV 16 found in 3.8% of HPV positive patients and prevalence the high risk HPV oncogenic was 13.1%. Thus, was find high prevalence of HPV in riverside Amazonian population studied, highlighting the need for surveillance for cancer of the cervix in the region. Finally, was established the prevalence of intestinal parasites in the population, deal the correlation of intestinal with asymptomatic malaria and anemia, taking the fecal examination conducting in the area including patients of all ages, who were willing to participate in the study. The analysis of samples was performed by the spontaneous sedimentation method (method of Lutz or Hoffmann, Pons & Janer). Among the 268 fecal examinations, the prevalence of intestinal parasite found in the region under study was 86.6%. Among helminths, Ascaris lumbricoides (47%), Ancilostomideos (37.3%), Trichuris trichiura (3.4%), Capillaria hepatica (2.3%) were found more parasites. Among the protozoa were: Entamoeba coli (21.4%), Entamoeba histolytica (12%), Giardia intestinalis (15.4%), Endolimax nana (10.1%) and Iodamoeba butschlii (7.5%).The high prevalence of intestinal parasite found in the survey was consistent with other data developed in the Amazon region. In this study, the region of the Western Amazon is part of the scenario of global geographic distribution of Capillaria hepatica, particularly the region of the Baixo Rio Machado.

Submitted by Verônica Esteves (vevenesteves@gmail.com) on 2017-09-26T13:12:36Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE-CAROLINE ALVES DE OLIVEIRA MARTINS.pdf: 1666399 bytes, checksum: dc894448ce671170ce04894a85e6038f (MD5) / Approved for entry into archive by Verônica Esteves (vevenesteves@gmail.com) on 2017-09-26T13:13:39Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE-CAROLINE ALVES DE OLIVEIRA MARTINS.pdf: 1666399 bytes, checksum: dc894448ce671170ce04894a85e6038f (MD5) / Made available in DSpace on 2017-09-26T13:13:39Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE-CAROLINE ALVES DE OLIVEIRA MARTINS.pdf: 1666399 bytes, checksum: dc894448ce671170ce04894a85e6038f (MD5) Previous issue date: 2017 / Hospital Geral de Bonsucesso / O objetivo do estudo foi analisar a relação entre os diversos fatores de risco para infecção pelo vírus do Papiloma Humano (HPV) e suas lesões, avaliar a prevalência de câncer e das lesões precursoras do trato genital inferior e da infecção por HPV em mulheres transplantadas, além dos tipos mais prevalentes de HPV. Com este fim, foi realizado um estudo transversal com uma amostra aleatória de 61 pacientes. Os resultados encontrados foram: 10 casos de lesão (16.4%), uma prevalência geral de infecção por HPV de 54.5%, e o HPV 16 como o tipo de HPV de alto risco mais encontrado, seguido pelo HPV 51/53/70. Observou-se também, através da realização de regressão logística múltipla, a associação, com significância estatística, entre o uso de hormônio e ocorrência de infecção por HPV de alto risco (p = 0.037). Não foi observada associação, com significância estatística, entre os diversos fatores e a ocorrência de lesões. A imunossupressão foi considerada fator determinante para ocorrência de lesões. O HPV 16 foi o tipo mais frequente observado nas pacientes com e sem lesão. A maior prevalência foi observada na faixa etária entre 31 e 54 anos. Desta forma, observa-se que a alta prevalência de infecção por HPV e de suas lesões precursoras, em relação à literatura, confirmam a importância do rastreio e seguimento diferenciados das pacientes transplantadas. / This study aimed to analyze the relationship between several risk factors for human papillomavirus (HPV) infection and its lesions and to assess the prevalence of lower genital tract precursor lesions, cancer, and HPV infection in female transplant recipients, besides the most prevalent HPV types. The methodology adopted was a crosssectional study with a random sample of 61 patients. The results found were: 10 cases (16.4%) of lesions, 54.5% of the overall prevalence of HPV infection, and that HPV 16 was the most common high-risk HPV type, followed by HPV 51/53/70. A multiple logistic regression was done, and hormone use presented a statistically significant association with high-risk HPV infection (p = 0.037). No statistically significant association was identified for the set of all factors with the lesions studied. Immunosuppression was considered the determining factor for the occurrence of lesions. HPV 16 was the most frequent type observed in patients with and without lesion. The highest prevalence was observed in the age group between 31 and 54 years. Therefore, it was observed the high prevalence of HPV infection and its precursor lesions confirmed the importance of differential screening and follow-up of transplanted patients.

O Papilomavírus humano (HPV) é provavelmente o agente mais prevalente das doenças sexualmente transmissíveis do trato genital.Este estudo foi realizado para comparar as prevalências de HPV nos 144 raspados penianos de homens HIV positivos e negativos.Utilizou PCR PGMY09/11 e hidridização em pontos. A prevalência de HPV nos indivíduos HIV positivo foi de 59% e no HIV negativo de 67%.A lesão aceto-branca pela peniscopia não demonstrou significativa positividade para HPV.Pacientes HIV positivo mostraram múltiplos tipos de HPV e os tipos oncogênicos (16/18) foram os de maior freqüência. Os HPV tipo 6/11 foram os mais freqüentes nos dois grupos. Observou-se maior prevalência de HPV nos HIV positivos com linfócitos T CD4 menor que 200 células/mm3. A carga viral plasmática do HIV não foi um fator de positividade para HPV / Genital tract human papillomaviruses (HPV) are probably the most prevalent sexually transmitted pathogens. This study is to compare HPV DNA prevalence in 144 penile smears, obtained from HIV positive and negative men. It was used PCR employing the PGMY09/11 generic HPV primers and dot blot hybridization. HPV prevalence was 59% in HIV positive men and 67% in HIV negative. Acetic white lesions by peniscopy did not show significant positive of HPV in neither HIV positive and negative groups. HIV positive men had more often multiple and oncogenic HPV types (16/18). HPV types 6/11 were more frequent in both groups. The HIV positive group with lower 200 T CD4 cell counts load reported more HPV prevalence. HIV load was not a positive factor for HPV

Our understanding of humoral immune response to human papillomavirus (HPV) infection has been mainly derived from studies in women. Very little is known about humoral immune response to HPV in men. There is also a growing interest in understanding the burden of HPV exposure in the subgroups of the male population, including men who have sex with women (MSW), men who have sex with men (MSM) and men who have sex with both men and women (MSMW). This dissertation was undertaken to understand and characterize humoral immune response, measured by detectable serum antibody IgG, to HPV 6, 11, 16 and 18 infection, to estimates seroprevalence of HPV 6, 11, 16 and 18, to determine the associations of sociodemographic and sexual behavioral factors with seroprevalence of individual HPV types, and to evaluate the role of serum antibodies in the subsequent acquisition of infection with the same HPV type, genetically related and un-related HPV types. Three studies that compose of this dissertation were conducted within the framework of two longitudinal studies of HPV infection in men: a single-site natural history study of male residents of Tucson, Arizona (the 1st study: N=285); and a multinational natural history study of healthy men residing in São Paulo, Brazil, Cuernavaca, Mexico, and Tampa, Florida (the 2nd study: N=1477; the 3rd study: N=2187). Men were recruited using similar eligibility criteria in both natural history studies and followed every 6 months for a maximum of 18 months in the single-site study and 48 months in the multi-national study. HPV DNA status was assessed using the PGMY09/11 L1 consensus primer system and the Linear Array HPV Genotyping Protocol. Testing of serum antibodies to HPV 6, 11, 16 and 18 was performed with virus-like particle-based ELISA assays. Data from our studies indicate that exposure to HPV 6, 11, 16 and 18, the four HPV types targeted in the currently license HPV vaccines, is common. Of 285 male residents of Tucson, Arizona, 28.8% of them were seropositive to HPV 16 and/or 18 at study entry. Similarly, approximately one third of 1477 participants of the multi-national male HPV natural history study were seropositive to at least one vaccine HPV type, with the percentage of 21.8% in U.S. site, 33.4% in Mexico site, and 49.1% in Brazil site. It is also noted that seroprevalence of individual vaccine HPV types is greatly elevated among men of different sexual practices. Seroprevalence of HPV 6, 11, 16 and/or 18 was twice as high among MSM and MSMW compared to MSW. Likewise, seroprevalence of individual HPV types was two fold or higher among MSW and MSMW. Our findings suggest that the predominant predictors of seropositivity to HPV 6, 11, 16 and 18 are age and same-sex sexual behaviors. Seroprevalence increased with age among young-to-middle-aged men with significant upward age trends observed for HPV 11, 16 and 18. MSM, compared to MSW, more likely to be seropositive to HPV 16 or 18. Similarly, men who practiced same-sex anal sex, compared to those who did not, were significantly more likely to be seropositive to HPV 6, 11, 16 and 18, respectively. Among 276 men free of HPV 16 at enrollment in Tucson, We did not detect statistically significant associations between the baseline serum antibodies to HPV 16 and/or 18 and subsequent risk of infection with homogeneous HPV types or related-HPV types. Of 2187 men residing in three countries who tested HPV 16 negative at enrollment, the risk of subsequent HPV 16 infection was not associated with enrollment HPV 16 serum antibodies status. Our data provide important estimates of population exposure to vaccine HPV types for future studies modeling potential vaccine impact and vaccine cost effectiveness in men. Our findings also support strategic vaccination of males as an effective preventive measure for HPV-related diseases and cancers in men and their sex partners, men and women alike.

Les virus du papillome humain (VPH) sont de petits virus à ADN double brin infectant les épithéliums de la peau et des muqueuses. La réplication nécessaire au maintien de leur génome dans les cellules infectées dépend des protéines virales E1 et E2. Au cours de la réplication, E1 est recrutée à l’origine de réplication par E2 afin d’être assemblée en doubles hexamères capables de dérouler l’ADN. E1 contient un domaine C-terminal responsable de l’activité ATPase/hélicase, un domaine central de liaison à l’origine et une région N-terminale régulant la réplication in vivo. Cette région contient des signaux de localisation et d’export nucléaire qui modulent le transport intracellulaire de E1. Chez le virus du papillome bovin (VPB), il a été proposé que ce transport est régulé par la sumoylation de E1. Finalement, la région N-terminale de E1 contient un motif de liaison aux cyclines permettant son interaction avec la cycline E/A-Cdk2. La phosphorylation de E1 par cette dernière régule différemment l’export nucléaire des protéines E1 du VPB et du VPH. Dans la première partie de cette étude, nous avons démontré que bien que la protéine E1 des VPH interagit avec Ubc9, l’enzyme de conjugaison de la voie de sumoylation, cette voie n’est pas requise pour son accumulation au noyau. Dans la seconde partie, nous avons déterminé que l’accumulation nucléaire de E1 est plutôt régulée pas sa phosphorylation. En fait, nous avons démontré que l’export nucléaire de E1 est inhibé par la phosphorylation de sérines conservées de la région N-terminale de E1 par Cdk2. Puis, nous avons établi que l’export nucléaire de E1 n’est pas nécessaire à l’amplification du génome dans les kératinocytes différenciés mais qu’il est requis pour le maintien du génome dans les kératinocytes non différenciés. En particulier, nous avons découvert que l’accumulation nucléaire de E1 inhibe la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase S et que cet effet anti-prolifératif est contrecarrée par l’export de E1 au cytoplasme. Dans la troisième partie de cette étude, nous avons démontré que l’arrêt cellulaire induit par E1 dépend de sa liaison à l’ADN et à l’ATP, et qu’il est accompagné par l’activation de la voie de réponse aux dommages à l’ADN dépendante de ATM (Ataxia Telangiectasia Mutated). Ces deux événements semblent toutefois distincts puisque la formation d’un complexe E1-E2 réduit l’activation de la voie de réponse aux dommages par E1 sans toutefois prévenir l’arrêt de cycle cellulaire. Finalement, nous avons démontré que la réplication transitoire de l’ADN viral peut avoir lieu dans des cellules arrêtées en phase S, indépendamment de l’activation de la voie de réponse aux dommages à l’ADN et de la kinase ATM. Globalement, nos résultats démontrent que l’export nucléaire de E1 est régulé par sa phosphorylation et non par sa sumoylation. Ils démontrent également que l’export nucléaire de E1 est essentiel au maintien du génome dans les kératinocytes, possiblement parce qu’il prévient l’inhibition de la prolifération cellulaire et l’activation de la voie de réponse aux dommages à l’ADN en limitant l’accumulation de E1 au noyau. / Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin and the mucosa. HPV rely on two viral proteins, E1 and E2, to replicate and maintain their genome in the nucleus of infected cells. During replication, the E1 helicase is recruited to the origin of replication by E2 and is assembled into a double-hexamer that unwinds DNA ahead of the replication fork. E1 is comprised of a C-terminal enzymatic domain with ATPase/helicase activity, a central origin-binding domain and a N-terminal regulatory region that is required for viral DNA replication in vivo. The latter region of E1 contains a nuclear localization signal and a nuclear export signal that regulate its shuttling between the nucleus and cytoplasm. For bovine papillomavirus (BPV) E1, this shuttling was suggested to be controlled by the sumoylation of E1. In addition to the NES and NLS, the N-terminal region of E1 contains a conserved cyclin-binding motif that is required for the interaction of E1 with cyclin E/A-Cdk2. Cdk2 phosphorylation of E1 has been reported to control the nuclear export of E1 from BPV and HPV, albeit differently. In the first part of this study, we showed that although HPV E1 interacts with Ubc9, the conjugating enzyme of the sumoylation pathway, this pathway is not required for its accumulation in the nucleus. In the second part, we found that the nuclear accumulation of E1 is, instead, regulated by phosphorylation. Specifically, we found that Cdk2-dependent phosphorylation of conserved serines in the E1 N-terminal region inhibits the nuclear export of HPV E1. Furthermore, we reported that nuclear export is not essential to amplify the viral genome in differentiating keratinocytes but that it is required for its long-term maintenance in undifferentiated keratinocytes. Importantly, we found that the nuclear accumulation of E1 induces a S-phase arrest that is detrimental to cellular proliferation and that this anti-proliferative effect can be counteracted by the export of E1 from the nucleus to the cytoplasm. In the last part of this study, we showed that this arrest is dependent on the DNA- and ATP-binding activities of E1. Furthermore, we found that the cell cycle arrest induced by E1 is accompanied by the activation of a DNA damage response (DDR) dependent on the ATM (Ataxia Telangiectasia Mutated) pathway. However, these two events seem to be distinct since complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Importantly, we demonstrated that transient viral DNA replication still occurs in S-phase arrested cells, independently of the induction of a DDR and of the ATM kinase. Collectively, these data indicate that nuclear export of E1 is regulated by phosphorylation and not by sumoylation. They also revealed that nuclear export of E1 is essential for maintenance of the viral episome in keratinocytes, at least in part to limit its nuclear accumulation and prevent its detrimental effect on cellular proliferation and induction of a DDR.