Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-08-10T11:14:59Z No. of bitstreams: 1 JoaoFelipeBezerra_TESE.pdf: 746132 bytes, checksum: eaa5510288612768dad80b3efbef567c (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-08-10T13:50:11Z (GMT) No. of bitstreams: 1 JoaoFelipeBezerra_TESE.pdf: 746132 bytes, checksum: eaa5510288612768dad80b3efbef567c (MD5) / Made available in DSpace on 2017-08-10T13:50:11Z (GMT). No. of bitstreams: 1 JoaoFelipeBezerra_TESE.pdf: 746132 bytes, checksum: eaa5510288612768dad80b3efbef567c (MD5) Previous issue date: 2017-04-03 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / As fendas orais constituem um problema de sa?de publica atingindo cerca de 15% de todas as malforma??es, caracterizando-se pela forma??o incompleta das estruturas que separam a cavidade nasal e a cavidade oral com fatores gen?ticos e ambientais que contribuem para sua etiologia. Atualmente, estudos de microarray, utilizando pacientes fissurados identificaram diversas regi?es de susceptibilidade para o desenvolvimento das fendas orais n?o-sindr?micas, dentre essas alguns estudos demonstraram a regi?o do gene Interferon Regulatory Factor 6 (IRF6) associado ao aumento de risco para o desenvolvimento das fendas. O objetivo do presente trabalho ? pesquisar polimorfismos do gene IRF6 e as poss?veis associa??es com o desenvolvimento das fendas orais n?o-sindr?micas. Para isso, um total de 368 individuos (186 pacientes FL/P e 182 controles) foram selecionados no Servi?o de Atendimento ao Paciente Fissurado - HUOL/UFRN. Amostras de sangue foram coletadas para extra??o do DNA e an?lise dos polimorfismos do gene IRF6 (rs2235371, rs642961, rs2236907, rs861019, e rs1044516) por PCR em tempo real. Os pacientes foram classificados nos grupos Fenda L?bio-palatina (CLP), Fenda labial (CL) e Fenda Palatina (CP) e foi observada uma associa??o significativa de rs2235371 (OR: 11,24, 95% CI: 1.97-64.22, p = 0,016) no grupo com a fendas palatinas isoladas (CP). Al?m disso, a an?lise da combina??o al?lica mostrou um aumento do risco de fendas associado aos polimorfismos rs1044516 e rs2236907, uma vez que estavam presentes em todas as combina??es significativas. Assim, a associa??o do rs2235371 com pacientes do grupo CP mostra-se como um fator de risco para CP em nossa popula??o. A an?lise das combina??es al?licas mostram a influ?ncia de polimorfismos do IRF6 combinadas, principalmente (rs1044516 e rs2236907) sugerem que cada polimorfismo pode contribuir minimamente para aumentar o risco de desenvolver fendas orais n?o-sindr?micas em uma popula??o brasileira de Rio Grande do Norte. / Orofacial clefts are a public health problem accounting for approximately 15% of all malformations, characterized by the incomplete formation of structures that separate the nasal cavity and oral cavity with genetic and environmental factors that contribute to its etiology. Currently, microarray studies using fissured patients have identified several regions of susceptibility to the development of non-syndromic orofacial clefts among which some studies have demonstrated the region of the Interferon Regulatory Factor 6 (IRF6) gene associated with increased risk for non-syndromic orofacial clefts development. The aim of the present study is to investigate polymorphisms of the IRF6 gene and the possible correlations with the development of non-syndromic orofacial clefts. For this, a total of 368 individuals (186 FL / P patients and 182 controls) were selected in the Service of the Fissured Patient - HUOL / UFRN. Blood samples were collected for DNA extraction and analysis of the polymorphisms of the IRF6 gene (rs2235371, rs642961, rs2236907, rs861019, and rs1044516) by real-time PCR. Patients were classified into the CLP, CL and CP groups and a significant association of rs2235371 (OR: 11.24, 95% CI: 1.97-64.22, p = 0.016) was observed in the group with isolated palate clefts (CP). In addition, the analysis of the allelic combination showed an increased risk orofacial clefts associated with the polymorphisms rs1044516 and rs2236907, since they were present in all significant combinations. Thus, the association of rs2235371 with patients in the CP group is shown as a risk factor for CP in our population. The analysis of allelic combinations show the influence of combined IRF6 polymorphisms mainly (rs1044516 and rs2236907) suggest that each polymorphism may contribute minimally to increase the risk of developing non-syndromic orofacial clefts in a Brazilian population of Rio Grande do Norte.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-10-04T21:05:45Z No. of bitstreams: 1 ClelioDiogoSoares_DISSERT.pdf: 3089611 bytes, checksum: d4a3b5a01c7dc8a828197d92e650d07e (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-10-11T22:38:17Z (GMT) No. of bitstreams: 1 ClelioDiogoSoares_DISSERT.pdf: 3089611 bytes, checksum: d4a3b5a01c7dc8a828197d92e650d07e (MD5) / Made available in DSpace on 2017-10-11T22:38:17Z (GMT). No. of bitstreams: 1 ClelioDiogoSoares_DISSERT.pdf: 3089611 bytes, checksum: d4a3b5a01c7dc8a828197d92e650d07e (MD5) Previous issue date: 2012-08-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Introdu??o: H? evidencias consider?veis sugerindo que genes relacionados ao metabolismo do folato possuem um papel importante na etiologia das fendas orofaciais. A express?o de genes que possam estar ligados ?s fendas orais requer a presen?a de apropriadas causas ambientais em combina??o com fatores gen?ticos que culminam com a falha na fus?o dos processos faciais. Objetivo: Realizar estudo de associa??o da express?o dos genes da via do metabolismo do acido f?lico - MTHFR, MTR, RFC1, MTRR, com a ocorr?ncia das fendas orais. Metodologia: Foram estudados 50 filhos casos e suas respectivas m?es, e 50 indiv?duos controles e suas respectivas m?es. Inicialmente foram realizadas as an?lises bioqu?micas (Glicose, ALT, AST, Creatinina, Folato, Vitamina B12, Homociste?na), hematol?gicas (hemoglobina, hemat?crito, contagem de hem?cias, os ?ndices hematol?gicos, VCM, HCM e CHCM) e estudo de suas caracter?sticas cl?nicas. Para a realiza??o do estudo de express?o g?nica foi extra?do o RNA total a partir das c?lulas do sangue perif?rico, o qual foi quantificado e analisado quanto a sua pureza e integridade e encaminhado para a obten??o do cDNA a ser utilizado para o estudo de express?o utilizando ensaios pr?-desenhados. Finalmente foi avaliada a influ?ncia de determinados gen?tipos (MTRR A66G; MTHFR C677T; MTHFR A1298C; MTR A2756G; RFC1 A80G) sobre a express?o de RNAm dos respectivos genes estudados. A an?lise estat?stica dos dados foi realizada considerando o n?vel de signific?ncia de 95% (P<0,050) Resultados: Foi evidenciada a presen?a do consumo de ?lcool como fator de risco significativo presente para as m?es caso (P=0,001). Em rela??o as dosagens bioqu?micas n?o foram observadas diferen?as significativas entre os casos e respectivos controles os quais apresentaram valores de AST, ALT e creatinina dentro dos valores de refer?ncia. A dosagem de ?cido f?lico apresentou-se reduzida significativamente para o grupo dos filhos caso (P=0,010) e para suas m?es (P=0,001). Na an?lise hemat?logica n?o foram observadas altera??es em nenhum dos par?metros avaliados como hemoglobina, hemat?crito, contagem de hem?cias, os ?ndices hematol?gicos, VCM, HCM e CHCM dentre os grupos avaliados. A avalia??o da express?o g?nica para o grupo das m?es caso mostrou uma redu??o significativa na express?o do RNAm em todos os genes avaliados: para o gene da metionina sintase (MTR, p=0,008), da metionina sintase redutase (MTRR, p=0,015), do RFC1 (P=0,004) e da MTHFR (P=0,017) comparados com o grupo de m?es controle. No grupo de filhos fissurados, houve uma redu??o significativa na express?o do RNAm para os genes da metionina sintase (MTR, p=0,010) e da metionina sintase redutase (MTRR, p=0,034). Para a an?lise da influencia dos gen?tipos na express?o observou-se que o gen?tipo recessivo (CC) para o polimorfismo A1298C do gene MTHFR poderia estar associada a uma redu??o da express?o de seu RNAm. Conclus?o: Genes do metabolismo de folato relacionados s?o reduzidamente expressos em ambos os grupos caso deste estudo, uma vez que todos os quatro genes (RFC1, MTHFR, MTR, MTR), estavam reduzidos nas m?es e dois genes (MTR, MTRR) em seus filhos. A redu??o da express?o desses genes representa um aumento do risco associado com a presen?a de fendas orais nestes indiv?duos. / Introduction: There is considerable evidence suggesting that folate-related genes play a role in the etiology of oral facial clefts. Clefts are known to have a strong genetic component. The expression profile of genes involved on the pathway and folic acid metabolism which is linked to oral clefts requires appropriate environmental causes in combination with genetic factors that culminate in the failure of fusion of facial processes. Objective: The objective is to perform the association of differential gene expression of folate metabolism genes (MTHFR, MTR, RFC1, MTRR) with the occurrence of oral clefts. Methods: We studied 50 subjects with oral clefts and their mothers, and 50 individuals absent of oral clefts and their mothers, totaling 100 individuals referred cases and 100 controls respectively. For gene expression study, total RNA was extracted from peripheral blood cells, then it was quantified and analyzed for purity by absorbance relation and integrity in MOPS gel. The mRNA samples with good purity and integrity were transcribed to cDNA using reverse transcription kit. The cDNA was used in pre-designed gene expression assays. In a previous study performed by our group were evaluated by PCR-RFLP the MTRR A66G, MTHFR C677T, MTHFR A1298G, MTR A2756G, RFC1 A80G polymorphisms, in this study we evaluated the influence of these polymorphisms on gene expression. It was also performed biochemical, hematological analyzes and a clinical characteristics study of these individuals. We performed statistical analysis considering the significance level of 95% (P <0.050) Results: The consumption of Alcohol was reported as a significant risk factor for the present case mothers (p = 0.001). Regarding the biochemical there were no significant differences between children cases group and their controls which had values of AST, ALT and creatinine within the reference values. The folic acid dosage presented significantly reduced in case mothers group (P = 0.011). In haematological analysis was not observed significant changes in any of the evaluated parameters such as hemoglobin, hematocrit, erythrocyte count, and hematological indices, MCV, MCH and MCHC among the groups. The assessment of gene expression for case mother group showed a significant reduction in mRNA expression in all evaluated genes; for the methionine synthase gene (MTR, p = 0.008), the methionine synthase reductase (MTRR, p = 0.015), the reduced folate carrier 1 (RFC1, P= 0.004) and methylenetetrahydrofolate reductase (MTHFR, P= 0.017) compared with the control group mothers. In the case children group, similar results were obtained, with a significant reduction in mRNA expression of methionine synthase gene (MTR, p = 0.010) and methionine synthase reductase (MTRR, p = 0.034) analysis of genotypes in relation to expression was found that the recessive genotype (CC) for the MTHFR gene A1298C polymorphism is associated with reduced expression of its mRNA. Conclusion: Folate metabolism related genes are low expressed on both case groups of this study, since all four genes (RFC1,MTHFR, MTR, MTR,) were reduced on mothers and two genes (MTR, MTRR) in their children. These down regulated genes represent an increased risk associated with the presence of oral clefts in these individuals.