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ramos_pmm_me_botib.pdf: 3067176 bytes, checksum: bb2655e0acf6fb1d0c707110baed9681 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os padrões anormais de metilação do DNA, especialmente a hipermetilação de genes com provável função supressora de tumor, representam um dos mais promissores marcadores moleculares do câncer por levarem à inativação funcional de genes críticos. Estudos prévios documentaram altos níveis de expressão do gene H19 em carcinoma de bexiga recorrentes. O gene H19 é regulado por imprinting, está localizado em 11p15.5 adjacente ao gene IGF2 (insulin-like growth factor 2 – somatomedin A) e codifica um transcrito não codificador de proteínas (micro RNA miR-675). Uma região que atua de forma coordenada no controle da expressão desses genes, chamada DMR (Differentially Methylated Region), atua na determinação do imprinting recíproco e na expressão mutuamente exclusiva dos genes IGF2 e H19. Ela encontra-se não metilada no homólogo materno e metilada no homólogo paterno e contém sete regiões de ligação da proteína CTCF (proteína bloqueadora do acentuador), que é sensível à metilação do DNA. Um relato prévio da literatura sugeriu que somente o sexto sítio de ligação do fator CTCF apresenta metilação parental específica. Este achado foi correlacionado com o padrão de expressão regulado por imprinting dos genes IGF2 e H19 em câncer de bexiga. No presente estudo, o padrão de metilação alelo-específico do gene H19 foi determinado em duas regiões distintas: no sexto sítio de ligação do fator CTCF contido na DMR e no primeiro éxon do gene H19 utilizando-se três abordagens diferentes: MSRE-PCR-RFLP (Methylation Sensitive Restriction Enzyme - Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), qMSP (quantitative real time Methylation Specific Polymerase Chain Reaction) para o sexto sítio e MSP-CTPP (Methylation Specific Polymerase Chain Reaction with Confontring Two Pair-Primers) para a região do primeiro éxon em 52 amostras de tecidos... / Abnormal patterns of DNA methylation, especially hypermethylation of genes demonstrating tumoral suppressor functions represent one of the most promising molecular markers of cancer because they can lead to functional inactivation of critical genes. Previous studies have documented high levels of H19 gene expression in recurrent bladder carcinomas. The H19 gene is regulated by imprinting, is located at 11p15.5 adjacent to the IGF2 gene (insulin-like growth factor 2 - somatomedin A) and encodes a non-coding transcript (micro RNA miR-675). A Differentially Methylated Region (DMR) region acts in a coordinated manner to control the expression of the IGF2 and H19 genes by determining their reciprocal imprinting and mutually exclusive expression patterns. The H19-DMR contains seven potential CTCF-binding sites. These sites are located upstream to the transcriptional initiation site, and the gamete-specific methylation acts as an insulator by precluding CTCF binding in the paternal allele. A previous literature report have suggested that only the sixth CTCF-binding site shows parental specific methylation. This finding was correlated with the imprinting expression pattern of IGF2 and H19 genes in bladder cancer. In the present study, the allele-specific methylation pattern of the H19 gene was evaluated in two distinct target regions: the sixth CTCF-binding site located in the DMR and the first exon of the H19 gene using three different approaches: MSRE-PCR-RFLP (Methylation Sensitive Restriction Enzyme - Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), qMSP (quantitative real time Methylation Specific Polymerase Chain Reaction) for the sixth CTCF-binding site, and the first exon of the H19 gene was analyzed by MSP-CTPP (Methylation Specific Polymerase Chain Reaction with Confronting Two-Pair Primers) in 52 samples of bladder tumors matched to normal adjacent tissues obtained... (Complete abstract click electronic access below)
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unesp.br:11449/92442 |
| Date | 20 May 2010 |
| Creators | Ramos, Priscila Maria Manzini [UNESP] |
| Contributors | Universidade Estadual Paulista (UNESP), Rainho, Cláudia Aparecida [UNESP] |
| Publisher | Universidade Estadual Paulista (UNESP) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | 114 f. |
| Source | Aleph, reponame:Repositório Institucional da UNESP, instname:Universidade Estadual Paulista, instacron:UNESP |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | -1, -1 |
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