Cultivation of PCV2 in swine testicle cells using the shell vial technique and monitoring of viral replication by qPCR and RT-qPCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mitochondrial DNA control region diversity in a population from Espirito Santo state, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise de metilação do gene FOXE1 em carcinoma de células escamosas em cães: padronização metodológica

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

Estimativa da diversidade genética de Queixadas (Tayassu pecari) da região do Taboco (Corguinho, MS)

The Brazilian fauna has been constantly threatened by deforestation and forest fragmentation. As a result, many populations become isolated and small which negatively impacts their genetic diversity, putting them at a higher risk of extinction than large and stable populations. The aim of this work was to estimate the genetic diversity of white-lipped peccaries (Tayassu pecari) in the region of Taboco (Corguinho, MS), a fragmented area; and to compare these estimates with that obtained previously for two populations from Brazilian Pantanal, which is considered a relatively well-preserved biome and where the species is not threatened. A total of 18 blood and 72 hair samples of white-lipped peccaries had their DNA extracted and amplified for five polymorphic microsatellite loci. With the individuals identified, genetic diversity indicators (such as number of alleles, allelic richness, expected end and observed) and the inbreeding coefficient FIS were calculated. In addition, to verify if the population suffered a recent population bottleneck, we used the tests implemented in the program Bottleneck. The population of Taboco showed no evidence of recent population bottleneck (p > 0.05) or inbreeding (FIS = 0.008; p > 0.022). In addition, the levels of genetic diversity in this population (mean number of alleles = 2.60; mean allelic richness = 2.56 mean observed and expected heterozygosities = 0.45 and 0.47, respectively) were statistically similar to those found previously for the two populations from Pantanal (p > 0,05); although the region of Taboco is more impacted than the Pantanal. Even though we showed no evidence of loss of genetic diversity, it does not mean that the population is not suffering with fragmentation; but that there was not sufficient time to evidence the genetic changes. In addition, may be occurring gene flow with populations of nearby fragments, which is maintaining... (Complete abstract click electronic access below)

Identificação de DNA de Leishmania sp. no encéfalo de cães com Leishmaniose visceral

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Novel and cross-amplified microsatellite loci for the critically endangered São Paulo marsh antwren Formicivora paludicola (Aves: Thamnophilidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da diluição do DNA extraido de material parafinado para amplificação em PCR

Introduction: Several reasons may lead to the failure of polymerase chain reaction (PCR) using DNA purified from paraffin-embedded materials: presence of inhibitors and degradation of target DNA. DNA dilution will often reduce the concentration of potential inhibitors and still contain enough DNA to allow PCR amplification. Objective: To evaluate the dilution influence of DNA purified from paraffin-embedded materials on β-globin PCR amplification. Material and Method: Paraffin-embedded blocks from 30 patients with oropharynx squamous cell carcinomas, diagnosed and treated at the Oral Oncology Center were selected. DNA extraction was performed using QIAmp minikit (Quiagen). DNA was quantified and evaluated for purity by spectrophotometer analysis. Two groups were formed with different amounts of DNA: group I had the originally extracted DNA and group II had the same DNA, however diluted with ultrapure water addition. PCR was performed in both groups using oligonucleotides for human β-globin gene. Results: For Group I, amplification of the β-globin gene sequence was successful in 33.33% of the samples and for Group II, in 23.33%. Conclusion: Dilution of the DNA extracted of paraffin-embedded materials did not modify statistically the amount of positive samples β-globin gene amplified in PCR, although the results suggest that this is a way to increase the method for efficacy amplification of PCR.

Estudo citogenético em aranhas (Mygalomorphae e Araneomorphae) com ênfase na evolução dos genes de DNA ribossômico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aplicação de DNA Barcode na identificação de espécies dos gêneros Senna, Lantana e Casearia

Pós-graduação em Biotecnologia - IQ

Comparação de métodos para extração de dna na reação em cadeia da polimerase para detecção de Salmonella enterica sorovar enteritidis em produtos avícolas

The identification of Salmonella spp. in food samples by microbiological diagnosis is time consuming, with approximately five different stages, requiring about 120 hours until the final result. The utilization of the polymerase chain reaction technique (PCR) can reduce this time, but substances present in samples may affect the reaction. The present work aimed to compare DNA extraction by thermic treatment and by the use of cetyltrimethil ammonium bromide (CTAB), in products originated from poultry houses corresponding to raw material (meat meal) and experimentally contaminated drag swabs. Materials obtained from the extractions were submitted to PCR, utilizing a pair of initiator oligonucleotides for amplification of Sdf 1 gene fragments. Comparing the methods of extraction, it was observed that when CTAB was employed, SE was detected in 70% of meat meal and in 80% of drag swabs, while the thermic treatment method yielded positive results in 20% of meat meal and in 40% of drag swabs. SE was detected under both methods utilized for DNA extraction, but the use of CTAB detected a greater number of positive samples, compared with thermal treatment.