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Desenvolvimento de um biossensor amperométrico de DNA para detecção do Papilomavírus humanoManoella Leite dos Santos, Cinthya 31 January 2008 (has links)
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Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer cervical tem sido uma patologia grave no Brasil e no mundo com cerca de 18.680
mulheres infectadas anualmente. O Papilomavírus Humano (HPV) tem sido biologicamente
associado ao desenvolvimento de lesões cervicais, devido a sua capacidade de infectar
células epiteliais, e progressão ao câncer do colo de útero. O diagnóstico tardio pode
inviabilizar o tratamento e levar a óbito muitas mulheres. Este diagnóstico poderia ser
antecipado pelo uso de biossensores de DNA, que são dispositivos analíticos que resultam
da integração entre uma sonda seqüência específica e um sinal transdutor. Entre outras
técnicas eletroquímicas, os biossensores amperométricos têm sido descritos como atrativos
devido à sua simplicidade, baixos custos de instrumentação, possibilidade de realização em
tempo real e geralmente alta sensibilidade. Neste trabalho, foi desenvolvido um
genossensor para detecção de seqüências do vírus HPV usando azul de metileno (AM)
como mediador químico. Os sinais biológicos captados foram transduzidos por Voltametria
de Pulso Diferencial (VPD). Os eletrodos de trabalho e referência foram produzidos através
de técnica de impressão sob uma superfície de polivinil álcool. Os mesmos foram
constituídos de carbono e Ag\AgCl, respectivamente. O eletrodo de trabalho foi modificado
com quitosana para a imobilização de pequenas seqüências de DNA para hibridização com
uma seqüência alvo. A seqüência alvo usada para hibridização foi o gene E6 do HPV18,
uma cadeia extensa com 500 pares de bases disponíveis para anelamento com seqüências
de 20 bases. Análises de bioinformática foram realizados para verificar possíveis regiões de
anelamento destas seqüências curtas ao genoma viral. Os resultados demonstraram um
aumento no sinal do AM quando a hibridização ocorreu, evidenciado pelo aumento dos
picos de corrente de VPD analisados graficamente. As diferenças entre os sinais emitidos a
partir de um eletrodo contendo reações de hibridização e outro isento das mesmas, foi
usada para detectar seqüências de DNA viral obtidas de amostras clínicas. Os resultados
indicaram ser o método passível de aplicações em diagnósticos clínicos, e a técnica de
impressão, um instrumento viável para a construção dos biossensores de DNA utilizando o
azul de metileno como indicador de sinais de hibridização
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The use of Human Papillomavirus promoters to target Cervical Cancer cellsLung, Mandy Siu Yu, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2008 (has links)
Human Papillomavirus (HPV) is one of the most common causes of sexually transmitted disease worldwide. Infections by high-risk HPVs, such as HPV-18, have been associated etiologically with cervical cancer. The successful development of HPV vaccines may be beneficial to the HPV-na??ve population, but women that have already been exposed to the virus are still at risk of developing HPV-associated malignancies. A need for a systemic cure for HPV-infection therefore still exists. Gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers; however, few studies have explored this possibility for cervical cancer. The aim of this project is to construct a gene expression vector that can specifically target HPV-infected cervical cancer cells, by making use of the activity and selectivity of the P105 promoter which is determined by transcription control elements within the HPV-18 long control region (LCR). The first part of this study involved the construction of LCR deletion plasmids, and examining the subsequent level of gene expression induced within different mammalian cell lines. The results suggest the LCR to be capable in achieving cervical cancer-specific gene expression. The 3′-end of the viral L1 gene upstream of the LCR appeared to have a repressive effect on the promoter and therefore should be excluded for maximum LCR promoter activity. The second part of the project involved site-directed mutagenesis studies performed on selected transcription factor binding sites with an attempt to further increase the level of LCR promoter activity and specificity towards HPV-infected cervical cancer cells. The results suggest that a GRE/YY1 mutation may significantly enhance promoter activity. In terms of promoter regulation, the E2BSs appeared to be responsible for promoter activation in the absence of viral E2 proteins. The findings of this study suggest a possible gene therapy approach towards the treatment of cervical cancer. By making use of the activity and specificity of the HPV-18 P105 promoter to induce cervical carcinoma-specific expression of appropriate therapeutic genes, suicidal phenotypes can be introduced selectively within HPV-positive cervical cancer cells while normal HPV-negative cells are unaffected.
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The use of Human Papillomavirus promoters to target Cervical Cancer cellsLung, Mandy Siu Yu, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2008 (has links)
Human Papillomavirus (HPV) is one of the most common causes of sexually transmitted disease worldwide. Infections by high-risk HPVs, such as HPV-18, have been associated etiologically with cervical cancer. The successful development of HPV vaccines may be beneficial to the HPV-na??ve population, but women that have already been exposed to the virus are still at risk of developing HPV-associated malignancies. A need for a systemic cure for HPV-infection therefore still exists. Gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers; however, few studies have explored this possibility for cervical cancer. The aim of this project is to construct a gene expression vector that can specifically target HPV-infected cervical cancer cells, by making use of the activity and selectivity of the P105 promoter which is determined by transcription control elements within the HPV-18 long control region (LCR). The first part of this study involved the construction of LCR deletion plasmids, and examining the subsequent level of gene expression induced within different mammalian cell lines. The results suggest the LCR to be capable in achieving cervical cancer-specific gene expression. The 3′-end of the viral L1 gene upstream of the LCR appeared to have a repressive effect on the promoter and therefore should be excluded for maximum LCR promoter activity. The second part of the project involved site-directed mutagenesis studies performed on selected transcription factor binding sites with an attempt to further increase the level of LCR promoter activity and specificity towards HPV-infected cervical cancer cells. The results suggest that a GRE/YY1 mutation may significantly enhance promoter activity. In terms of promoter regulation, the E2BSs appeared to be responsible for promoter activation in the absence of viral E2 proteins. The findings of this study suggest a possible gene therapy approach towards the treatment of cervical cancer. By making use of the activity and specificity of the HPV-18 P105 promoter to induce cervical carcinoma-specific expression of appropriate therapeutic genes, suicidal phenotypes can be introduced selectively within HPV-positive cervical cancer cells while normal HPV-negative cells are unaffected.
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Comparative Study of HPV 16 and HPV 18 Antibody Detection in Serum, Cervical Mucus, and Oral Mucosal TransudateBlalock, Emily Lauren 22 November 2008 (has links)
Measuring HPV exposure relies on detection of HPV type-specific antibodies, but methods are not standardized. Additionally, there is little information on the best sample type for HPV antibody detection. This study validated pseudovirion neutralization (PVN) assay for HPV antibody detection and compared it to IgG ELISA. Both assays were applied to paired serum and cervical mucus samples. Additionally, PVN assay was utilized to evaluate the feasibility of oral mucosal transudate (OMT) samples to monitor the HPV immune response. Serum was more likely to be positive on PVN assay than on IgG ELISA (p= 0.025). Both assays correlated with HPV-16 DNA status. HPV-18 PVN assay results correlated with HPV-18 DNA status. Few cervical mucus samples had detectable antibodies; no correlation with HPV DNA status was seen. OMT results were unsatisfactory. PVN assay was more sensitive than IgG ELISA; serum was a more reliable indicator of HPV-16/18 antibody status than cervical mucus.
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