Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised in vitro and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an in vitro stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation. In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.

L'infection au papillomavirus humain (HPV) est connue pour être le principal facteur causal d'une série de maladies aussi bien bénignes (condylomatose ano-génitale, papillomatose lyringée, et autres) que malignes (cancer du col de l'utérus, certains cancers ORL, et autres). Deux vaccins prophylactiques (Gardasil® et Cervarix®) sont sur la marché depuis à peu près quatre ans pour prévenir cette infection. Le présent travail de thèse comportait trois objectifs principaux : i) étudier in-silico l'immunogénicité du vaccin Gardasil® ; ii) étudier in-silico l'histoire naturelle d'une infection à HPV et iii) évaluer in-silico le potentiel de l'hypothèse thérapeutique suivante : l'administration intramusculaire du vaccin Gardasil® chez des patients atteints d'une papillomatose laryngée induirait un effet bénéfique car l'arrivée des immunoglobulines au tissu affecté empêcherait l'HPV de compléter son cycle de vie et, par conséquent, la maladie de se propager. Les principales conclusions sont : i) pour qu'une papillomatose laryngée ne s'étende pas il faudrait, d'après nos simulations, que le taux d'IgGs sériques soit maintenu au-dessus de 200 mMU/mL ; ii) pour rester, sur une période de 10 ans, le plus longtemps possible au-dessus de ce seuil (d'effet thérapeutique), en administrant la quantité minimale de vaccin, il faudrait, d'après nos simulations, suivre le protocole suivant : l'immunisation de base (à 0, 2 et 6 mois), suivie de trois rappels successifs tous les six mois jusqu'au 24ème mois, suivis d'un rappel 18 mois plus tard ; iii) par ailleurs, il semble inutile (voire contreproductif), d'après nos simulations, de modifier le schéma traditionnel de base (0-2-6 mois)

<p>In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping.</p><p>P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools.</p><p>The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples.</p><p>To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension.</p><p>The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants.</p>

Anal cancer, like cervical cancer, is associated with Human Papillomavirus (HPV) infection. HIV+ patients have 38-60 fold increased risk of anal cancer compared to HIV- patients prompting many to suggest routine screening given the success of cervical Pap screening. Our goal is to describe our experience with routine anal Pap screening, determine which patients are most likely to have abnormal results, if anal disease on physical exam is predictive of cytology, and correlate cytology with histology findings. Charts of all patients with an anal Pap followed at the Hospital of Saint Raphael HIV Clinic were reviewed. Demographics, immune status, sexually transmitted disease history, cytology and histology data was extracted from medical charts. Patients with an anal Pap between November 1, 2002-November 30, 2004 were included. Those with an insufficient sample were excluded. Analysis was done using ÷2 for comparison of proportions and student t-test for continuous variables. Overall, 265/560 HIV+ patients had at least one anal Pap. Seventy-four of these 265 patients had an abnormal anal Pap. Mean age was 44 yrs, and 68% were men. Fifty-nine percent were African American, 34% White, and 17% Hispanic. Those with an abnormal Pap were more likely to be White (p=.03), and be gay or bisexual men (p=.02). They were also more likely to have lower CD4+ nadir (142 vs 223, p=.005) and CD4+ at time of anal Pap (353 vs 497, p<.001). Those with an abnormal anal Pap also had more anal disease (30% vs 9%, p<.001), history of warts (23% vs 12%, p=.02) and herpes (35% vs 22%, p=.02). Anal disease on physical exam had a sensitivity of 56% and specificity of 77% for abnormal cytology findings. On histology two patients had Anal Intraepithelial Neoplasia (AIN ) I, 2 AIN II, 3 AIN III, and 2 Squamous Cell Carcinoma In Situ. There was no correlation between cytology and histology. Routine anal cytology screening is a feasible tool to incorporate into an ethnically diverse HIV clinic for identifying precancerous anal lesions, a group which has been largely overlooked. Anal disease on physical exam is a poor predictor of abnormal cytology and there was no correlation between severity of disease on cytology and histology. However, further follow-up study is required to determine the impact on morbidity and mortality.

There is a paucity of research on the risk for sexually transmitted infections (STIs) and sexual behavior among general populations of men. Research with male populations predominantly has focused on those subgroups considered to be at high risk of disease transmission, such as gay and bisexual men, injection drug users, and adolescents/young adults. Considerably fewer studies have examined factors among men, in general, and heterosexual men, specifically. Therefore, I conducted analyses with a cross-national sample of adult, sexually active men in Brazil, Mexico, and the United States to investigate sexual behaviors and risk factors associated with the human papillomavirus (HPV) and other STIs. The research questions were: 1) How does sexual risk differ among men residing in Brazil, Mexico, and the US by age cohort?; 2) Do men's sexual behaviors change after being tested for HPV and other STIs?; and 3) Do men's sexual behaviors change after being informed of diagnosis with HPV and other STIs? These research questions were explored through a quantitative assessment of secondary data collected through a risk factor questionnaire administered using computer assisted self-interviewing. The study findings underscore the need for public health interventions to address STI risk and transmission among men across the lifespan. Additionally, this study revealed the potential of STI testing as an effective strategy to reduce sexual risk-taking among men. While this research identifies key issues of importance in improving men's sexual health, additional research is needed to provide an enhanced contextual understanding of socio-cultural, interpersonal, and community level factors that affect sexual behaviors and decision-making among men.

The Papanicolaou Smear is a screening test which detects premalignant lesions of the uterine cervix. By treating these lesions, cervical cancer can be evaded. In 1988, a cytological diagnosis which communicated a state of uncertainty in the atypicality of cervical cells was first created in the Bethesda Cytology Classification scheme. This diagnosis is now known as atypical squamous cells of undetermined significance (ASC-US) and still little is known about its natural history. / This paper analyzes the results of a longitudinal study incorporating repeated regular measurements of viral and cytological endpoints as well as lifestyle and behavioural aspects, to understand the natural history of an ASC-US Pap smear and identify determinants of ASC-US acquisition and clearance. / Overall, the median duration of ASC-US is short, and is dependent on the definition of clearance since most lesions regress to normal. The factors most predictive of ASC-US acquisition but not clearance relate to HPV infection.

In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping. P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools. The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples. To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension. The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants. / QC 20101028

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Resumo não disponível.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-27T18:18:34Z No. of bitstreams: 1 SÉRGIO MENEZES AMARO FILHO.pdf: 3118907 bytes, checksum: 5ace372e7cf9ac4518a96c77052798ac (MD5) / Made available in DSpace on 2013-09-27T18:18:34Z (GMT). No. of bitstreams: 1 SÉRGIO MENEZES AMARO FILHO.pdf: 3118907 bytes, checksum: 5ace372e7cf9ac4518a96c77052798ac (MD5) Previous issue date: 2012-03-30 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / O câncer cervical é o terceiro tipo de câncer mais comum entre mulheres no mundo. Dentre outros cofatores, a infecção pelo HPV de alto risco, tem sido bem documentada como fator necessário ao desenvolvimento desse tipo de câncer. A principal ação do HPV envolve a expressão massiva das oncoproteínas virais E6 e E7 que podem formar complexos específicos com proteínas supressoras de tumor, sendo capazes de alterar mecanismos do ciclo celular, modificando a expressão de proteínas celulares. Um dos principais avanços na medicina clínico patológica é o uso dessas proteínas como marcadores de forma a aumentar a acurácia do prognóstico e do próprio estadiamento do câncer cervical. Assim, a fim de reforçar a hipótese de que as proteínas associadas ao ciclo celular Ki-67, MCM-2, p53 e p16INK4a se encontram superexpressas no câncer cervical, foram analisadas em 87 amostras cervicais de pacientes com câncer invasivo (CCI) e 43 cérvix normais. Verificamos também se há uma expressão diferencial que pode ajudar a avaliação do estadiamento clínico da FIGO e quais tipos virais podem induzir a uma expressão diferencial dessas proteínas. Além disso, características sociodemográficas, comportamentais e clínicas das pacientes foram obtidas dos prontuários e analisadas. Para isso, a detecção de DNA de HPV foi realizada pela técnica da PCR e hibridização in situ. A expressão das proteínas foi observada por imunohistoquímica, seguida por quantificação manual e através do software ImagePro Plus. A análise estatística foi feita utilizando o software STATA/SE 10.1 aplicando os testes: Kruskall-Wallis, Student, Fisher e Qui-Quadrado. Nossos resultados mostraram forte associação (p<0,05) do CCI e dos estágios tumorais mais avançados (III e IV) com mulheres superiores a 55 anos, com mais de quatro gestações e sem escolaridade. A prevalência de DNA de HPV por PCR na população total foi de 73,4%. Nos grupos de CCI e controle foram, respectivamente, 94,3% e 29,3%. O tipo mais prevalente foi o HPV16 (70,8%), acompanhado pelo HPV33 (11,2%) e 35 (4,5%). Como esperado, foi observado aumento (p<0,05) na expressão de Ki-67, MCM-2, p53 e p16INK4a no CCI, quando comparados ao controle. A proteína p16INK4a com expressão difusa, citoplasmática e nuclear esteve associada ao câncer. Ki-67 apresentou forte expressão (>50%) conforme o agravamento da doença. Não foi observada associação entre a expressão de MCM-2, p53 e p16, e o estadiamento do tumor. Como conclusões, foram observadas maiores chances no desenvolvimento do CCI em mulheres com idades superiores a 55 anos, com mais de quatro gestações e sem escolaridades, estando esses fatores associados, também, à progressão tumoral. Apenas o marcador Ki-67 associouse ao estágio do CCI. Os tipos mais prevalentes encontrados, HPV16, 33, 35, 67 e 58, sugerem que novos estudos devam ser considerados para implementação de vacinação contra o HPV no Brasil. / Cervical cancer is the thrid most common cancer among women worldwide. Beside others cofactors, infection with high risk HPV has been well documented as a necessary cause for cancer development. The main action involves the expression of massive HPV E6 and E7 viral oncoproteins that can form specific complexes with tumor suppressor proteins that are able of changing mechanisms of the cell cycle, modifying the expression of cellular proteins. One of the major advances in medicine clinical pathology is the use of these proteins as markers in order to improve the accuracy of prognosis and the cervical cancer stages. Thus, in order to reinforce strengthen the hypothesis that the proteins involved in cell cycle Ki-67, MCM-2, p53 and p16INK4a are over expressed in the uterine cervix of patients with cervical cancer, we analyzed 87 samples from patients with invasive cervical cancer (ICC) that were compared with 43 normal cervices (controls). Was verified also whether there is a differential expression that can help to assess the FIGO staging for ICC and which HPV viral types can induce a differential expression of these proteins. Moreover sociodemographic, behavioral and clinical characteristics of patients were obtained from medical records and analyzed. Therefore, the detection of HPV DNA was performed by PCR and in situ hybridization. By means of immunohistochemistry the expression of Ki-67, MCM2, p53 and p16 were analyzed. Statistical analysis was performed using STATA / SE 1.10 by applying the Kruskal-Wallis test, T-Student, chi-square and Fisher. Our results showed a strong correlation (p<0.05) of the CCI and the later stages of the cancer in women over 55 years, more than four pregnancies and no school education. The overall HPV DNA prevalence was 73.4%. On the ICC group and control group the prevalence was, respectively, 94.3% and 29.3%. The most prevalent HPV types were HPV16 (70.8%), HPV33 (11.2%) followed by HPV 35 (4.5%). Women with HPV16 were associated with advanced age (50.8 years) compared to women with other types (58.2 years). As expected, was observed an increase in Ki-67, MCM-2, p53 and p16 expression in CCI (p<0.05), compared to control. The expression of p16 protein with diffuse cytoplasmic and nuclear staining was associated with cancer. The Ki-67 showed a strong expression (> 50%) as the later stage of the disease. There was no association between the expression of MCM-2, p53 and p16 and tumor staging. In conclusion, we observed higher risks of ICC development in older ages, multiple pregancies and no school education. Only the Ki-67 marker was associated with the stage of the ICC. The most prevalent HPV types found, HPV 16, 33, 35, 67 and 58, suggest that new studies should be evaluated and considered on implementation of HPV vaccination in Brazil.