Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 microg (median 15 microg, mean 20.71 microg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.
Carbonic anhydrase isoenzyme VI (CA VI)plays an important role in the homeostasis of oral tissues participating in the processes of taste, protection of dental tissues against the loss of minerals, caries, and possibly in the formation of dental calculus in periodontal disease. This study aimed to verify the correlation between changes in the expression and activity of human salivary carbonic anhydrase VI and genetic polymorphisms in its gene (CA6). The study population consisted of 182 healthy volunteers (female and male, aged 18-22). Samples of total saliva were assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. CA VI catalytic activity was detected by a modified protocol of Kotwica et al. [J Physiol Pharmacol 2006;57(suppl 8):107-123], adapted to CA VI in saliva. Samples of genomic DNA were genotyped for polymorphisms rs2274327 (C/T), rs2274328 (A/C) and rs2274333 (A/G) by TaqMan® SNP Genotyping Assays. The concentration and catalytic activity of the salivary CA VI obtained for the different genotypes were analyzed using the Kruskal-Wallis nonparametric test and the Dunn test. The results showed that individuals with TT genotype (rs2274327) had significantly lower CA VI concentrations than the individuals with genotypes CT or CC (p < 0.05). There was also an association between polymorphism rs2274333 and salivary CA VI concentrations. There were no associations between the three polymorphisms analyzed and variations in CA VI activity. Our results suggest that polymorphisms in the CA6 gene are associated with the concentrations of secreted CA VI.
DEDICATÓRIAA Deus, este Ser Onisciente, que por Teu mistério nos torna Seres mais curiosos e vivazes.A minha mãe, Theresa (em memória) por ter sempre acreditado em mim, ao meu pai Chafique, por ter transmitido-me esta alegria de viver.Ao meu marido Elis (em memória) que ainda muito jovem me amparava em tudo e tenho certeza continua torcendo por mim.Ao meu filho, Filipe, que sempre me apoiou em meus desafios, me ensinando que ao me deparar com obstáculos devo olhar para eles e entender -são apenas conseqüências de novas trilhas! Te amo filho!!! A minha querida sogra Marion, mulher forte, um exemplo para todos nós.Aos meus irmãos Telma, Edázima, Edson e Cheila, pela amizade que nos une. vi AGRADECIMENTOS ESPECIAISAo meu orientador, Prof. Dr. Sérgio Roberto Peres Line, esta pessoa iluminada, que a natureza privilegiou com uma inteligência que impressiona a todos que o rodeiam, porém maior que este predicado que o torna um cientista nato e cheio de idéias é seu lado simples de ser e viver! Meu muitíssimo obrigada, querido professor! A minha auxiliadora, Dra. Maria Cristina Leme Godoy dos Santos. Imaginar o que um ser humano possa fazer ao outro, de uma forma incondicional, com um sorriso nos lábios, disposição, dedicação e não pensar na Crica, só vai acontecer para quem ainda não a conheceu. Querida amiga, bem aventurados serão teus alunos! Obrigada! vii
Biologia Buco-Dental Profa. Dra. Ana Paula de Souza Pardo, pela participação desta conceituada instituição no meu crescimento científico, profissional e pessoal.Ao Prof. Dr. Marcelo Rocha Marques, amigo e exemplo de dedicação, pelo apoio.
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