939 resultados para DNA EXTRACTION
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Avaliação clínica, microbiológica, imunológica e genética em pacientes com implantes osseointegrados
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
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This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Pós-graduação em Medicina Veterinária - FMVZ
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Pós-graduação em Ciências Biológicas (Genética) - IBB
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Pós-graduação em Zootecnia - FCAV
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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The human poliomavirus is the etiologic agente of Progressive multifocal leukoencephalopathy (PML), a disease characterized by focal lesions not expansives of the central nervous system that develops in imunocompromissed patients, specially people with aids. The main aim of the study was to evalute the prevalence of the JCV excretion in urine samples of patients with aids, without PML, to compare two JCV DNA detection techniques through of two diferents genomic regions and to evaluate the genotypic characterization of the positive samples. A total of 75 samples were colected in the Instituto de Infectologia Emílio Ribas, in Sao Paulo, Brazil, between may and november, 2009. To detect the JC virus it was made the DNA extraction and then the polimerase chain reaction (PCR). Firstly a fragment of 215 bp was amplified, which corresponds to the codifying gene of the strutural protein of de JC vírus capsid VP1. All the samples were later submitted to another PCR that uses a pair of primers complementaries to the early region of the JCV (T antigen) amplifying a fragment of 173 bp. Followed by the digestion of the amplified product with the restriction enzime BamH1, resulting in two smaller fragments (120 bp and 53 bp). The JC vírus was detected in 53 samples, for both techniques (70,7% for VP1 PCR, and the restriction enzime BamH1), 34/46 were men (73,9%) and 19/29 were women (65,5%). The JCV excretion was higher in individuals that were over 46 years old. Regarding the seven genotypes described in the literature, the ones that were more prevalent among the JC positive patients were 3B and 3A with 10 samples each (21,0%), the 2B with 9 samples (19,0%) and genotype 6, with six samples (13,0%). As in the brown patients as the white ones, the most prevalent genotype was 3B. In the present study it was observed a high prevalence of JCV DNA (70,7%) and the genotype 3 (43,0%)... (Complete abstract click electronic access below)
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It is believed that epigenetic mechanisms such as DNA methylation are important for the tumorigenesis and maintenance of the altered state of tumor cells. DNA methylation occurs by the addition of a methyl group to carbon 5 of cytosine, catalyzed by the enzyme DNA methyl-transferase, which can change the expression of a gene, including the tumor suppressor genes. In human squamous cell carcinoma, several features have shown the etiological role of genes in tumor development. Among them, FOXE1 gene (forkhead box E1 - thyroid transcription factor) is presented with an important role in susceptibility to disease. Similarly the FOXE1 methylation pattern could alter the expression of this gene in dogs and predisposed to tumor on. Therefore, this study aims to investigate in dogs, the validity of the strategy employed in humans to analyze the FOXE1 methylation status. DNA extraction from fresh frozen tumoral samples was performed by Wizard Genomic® DNA Purification Kit. The methylation status was determined by MSP-PCR (methylation-specific polymerase chain reaction), using 2.0 ng of DNA treated with sodium bisulphate. One hundred micrograms of bisulphite-modified DNA was amplified using primers specific for either methylated or unmethylated DNA (primers sequences are available at http://pathology2.jhu.edu/pancreas/primer.pdf). The analysis of fragments was loaded on to 7% polyacrylamide gels and silver nitrate staining. In this stage of technical approach, 60% were FOXE1 hypermethylated. In conclusion, it was observed that the standard technique for assessing the methylation pattern of gene FOXE1 in humans can be used for the same evaluation in dogs. The correlation of these molecular data with clinical and histopathological parameters may have diagnostic and prognostic value and still be used as a tumor marker for therapeutic decision and surgical approach