Orientador: Rejane Maria Tommasini Grotto / Resumo: O vírus Zika pertence a família Flaviviridae, gênero Flavivirus e tem seu genoma viral constituído de uma molécula de RNA de fita simples de polaridade positiva, com aproximadamente 10.700 pb. As interações de Flavivirus com compartimentos biológicos como as plaquetas foram relatadas pela literatura, sendo demostrado que o vírus é carreado para outros tecidos e sua viabilidade é mantida por um maior tempo, no entanto, este tipo de estudo ainda não foi reportado para o ZIKV. O objetivo deste trabalho foi comparar a carga viral do vírus Zika entre o plasma rico em plaquetas (PRP) e o plasma pobre em plaquetas (PPP). As amostras utilizadas foram coletadas de doadores saudáveis e posteriormente incubadas com o vírus ZIKA à 37°C na presença de CO2 durante 12 dias consecutivos, sendo retiradas alíquotas no momento do preparo e após 2, 4, 6, 8, 10 e 12 dias de incubação. Os resultados demostraram que a carga viral no PRP foi estatisticamente superior a do PPP depois de 12 dias de incubação com o vírus, sugerindo que as plaquetas podem carrear e preservar o vírus durante maior tempo comparado ao plasma isento de plaquetas. / Abstract: The Zika virus belongs to the Flaviviridae family, Flavivirus genus, and the viral genome consists of the molecule single-stranded, positive-sense RNA of approximately 10,700 bp. The interaction of Flavivirus with biological compartments such as platelets has been reported in the literature, and have shown that the virus is carried to other tissues and its viability is maintained for a longer time. However, this kind of evaluation was not reported for ZIKV yet. The aim of this study was to compare the viral load of Zika virus between platelet rich plasma (PRP) and platelet poor plasma (PPP). The samples were collected from healthy donors and subsequently incubated with ZIKA virus at 37 ° C with CO2 atmosphere for 12 consecutive days. Aliquots were evaluated at the zero time and after 2, 4, 6, 8, 10 and 12 days of incubation Results showed that the viral load in PRP is statistically higher than in PPP after 12 days of incubation with the virus, suggesting that platelets can carry and preserve the virus longer than in platelet-free plasma. / Mestre

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1a, IL-1b, IL-6, IL-8 and TNF-a were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1a, IL-1b, IL-6, IL-8 and TNF-a were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

Differently expressed genes e.g. in a disease may play a role in the etiology or progression of the disease. The traditional approach of finding differentially expressed genes is to compare the expression levels in the groups, and produce a list of differentially expressed candidate genes. With many pairwise comparisons, the risk of introducing type I and type II errors is high. One solution is to group together genes that are co-expressed into modules. Weighted gene coexpression network analysis (WGCNA) uses a topological overlap module approach and has been proved to find patterns that have been undetected by gene-to-gene comparison methods. qPCR has high sensitivity and specificity, and advances in technology has increased its throughput. The goal of the project was to construct WGCNA modules from qPCR data and evaluate the WGCNA method in five previously published qPCR data sets. There was little overlap between the differentially expressed genes found in the published articles and the candidates found by WGCNA. In three data sets WGCNA failed to produce any significant genes. In one of the data set significant genes were found where the original article failed. In one data set, 19 out of 60 genes that are top-ranked by the original authors were found in significant WGCNA modules. The biggest challenge with this type of comparison is to determine whether results that differ from the published studies are more or less biologically relevant. It is difficult to draw conclusions on whether the method is suitable for use for analysis of qPCR data based on this study.

The use of standards in quantitative PCR (qPCR) is essential, especially in forensic cases, to determine the concentration of DNA in an unknown sample. The standard curve for Quantifiler Trio is commonly made up of a ten-fold serial dilution of the known DNA standard in the qPCR kit. Due to the known concentration of the standard and the serial dilution, the threshold cycle values of the standards can be compared to the cycle threshold values of the unknown samples to determine their concentrations. Use of the Quantifiler Trio kit provides the concentration of total human DNA, including both small autosomal and large autosomal amounts which are used to calculate a degradation ratio, and also determines the concentration of male DNA in the sample. Each aspect has its own standard curve to determine the DNA concentration. However, serial dilutions cause variance between runs, even when using the same samples, due to small pipetting differences or errors which can make sample reruns and verifying data difficult. Creating a virtual curve by using data from multiple serial dilutions would minimize or eliminate the variance between runs. This is accomplished by taking the averages of slopes and y-intercepts of the standards to creature the virtual curve.

Molecular diagnostic tests have been developed and utilized to diagnose and to confirm diagnoses of many plant-parasitic nematodes. We evaluate the potential of a qPCR assay to detect and quantify Rotylenchulus reniformis in Mississippi directly from soil. A novel pipeline utilizing multiple databases containing nematode DNA and EST sequences was developed to aid in the selection of R. reniformis primers used in a PCR and qPCR assays. In vitro testing showed that the primers and probes developed from the novel pipeline for the qPCR assays could accurately detect the presence of R. reniformis. Subsequent testing resulted in a trend of increasing observed number of R. reniformis resulting in increasing estimates by qPCR

Introdução: Métodos quantitativos sensíveis e acurados para monitorar o quimerismo hematopoiético após Transplante de Células-Tronco Hematopoiéticas (TCTH) são necessários para verificar o sucesso do enxerto, uma vez que os resultados influenciam a abordagem terapêutica. Método: A técnica de detecção de DNA baseada em primers inserção-específicos de marcadores genéticos do tipo InDel (Inserção/Deleção) foi avaliada quanto a sua utilidade em quantificar quimerismo em amostras de DNA em baixa concentração, em PCR em Tempo Real (qPCR). Para isso, amostras de DNA de dez pacientes submetidos a Transplante de Medula Óssea (TMO) foram analisados para quantificar a concentração de DNA residual em diferentes períodos pós-transplante. Os resultados obtidos pela InDelqPCR foram comparados com a evolução clínica descrita nos prontuários. Resultados: As quantificações do DNA residual variaram de 0,021 ng/µL a 11,71 ng/µL, correspondendo a fração de 0,065% a 40,6% do DNA total presente na amostra. Os resultados obtidos (presença ou ausência do alelo inserção) foram condizentes com a evolução clínica dos pacientes, em alguns casos, evidenciando quimerismo prévio ao relatado nos prontuários. Conclusão: Nossos dados demonstram a utilidade do método InDel-qPCR, baseada em primers inserçãoespecíficos, no monitoramento de quimerismo hematopoiético. / Introduction: Sensitive and accurate quantitative methods to monitor hematopoietic chimerism after Hematopoietic Stem Cell Transplantation (HSCT) are necessary to evaluate engraftment since the results influence a therapeutic approach. Method: DNA detection technique based on insert-specific primers for Insertion/Deletion polymorphism (InDel) was evaluated for chimerism quantification of samples with low amounts of DNA, by quantitative real-time polymerase chain reaction (qPCR). Samples of patients undergoing Bone Marrow Transplantation (BMT) collected at different post-transplantation times were analyzed to quantify the residual DNA concentration. Then, the results found using InDel-qPCR were compared to the medical records. Results: DNA quantifications ranged from 0.021 ng/?L to 11.71 ng/?L, corresponding to a fraction of 0.065% to 40.6% of the total DNA. Our results, in some cases, shows chimerism presence previously to that reported in the medical records. Conclusion: Our data demonstrate the usefulness of the InDelqPCR method based on insertion-specific primers in the monitoring of hematopoietic chimerism.

A microbiota intestinal é um ecossistema complexo que desempenha um importante papel na gênese da obesidade. A ocorrência e participação dos Mollicutes na microbiota intestinal é praticamente desconhecida. Deste modo, o objetivo do presente estudo foi analisar a participação dos Mollicutes e dos Filos Firmicutes e Bacteroidetes na microbiota intestinal de mulheres obesas e eutróficas. A casuística foi de 20 mulheres obesas e 20 mulheres em eutrofia. Foram obtidas amostras de fezes, sangue e aplicado questionário semiestruturado sobre fatores relacionados com obesidade, microbiota intestinal e ambiente, além de Bioimpedância e questionário de frequência alimentar. Constatou-se uma associação positiva estatisticamente significante entre a presença de Mollicutes e mulheres obesas. Foi observada maior proporção de Firmicutes/Bacteroidetes na microbiota intestinal das mulheres obesas. Os resultados obtidos permitiram obter evidências importantes da participação dos micro-organismos da classe Mollicutes. As alterações da microbiota intestinal também contribuíram na definição de subconjuntos de indivíduos com diferentes perfis de risco metabólico e a da heterogeneidade associada a fenótipos humanos relacionados com a adiposidade. / The gut microbiota is a complex ecosystem that plays an important role in the pathogenesis of obesity. The occurrence and participation of Mollicutes in the gut microbiota is pratically unknown. The aim of this study was to analyze the participation of Mollicutes and Firmicutes and Bacteroidetes phylos in the gut microbiota of obese and normal weight women. For the study, it was collected samples of 20 women with obesity and 20 women of normal weight. It was collected stool samples, blood, semi-structured questionnaire on factors associated with obesity, gut microbiota and the environment, and anthropometric measurements using bioelectrical impedance and food frequency questionnaire. It was detected a statistically significant positive association between the presence of Mollicutes and obese women, and there was a higher proportion of Firmicutes/Bacteroidetes in the gut microbiota of obese women. The results provide important evidence about the participation of Mollicutes class in the gut microbiota of the population studied and interactions in intestinal microbiota can define subsets of individuals with different metabolic risk profiles and thus contribute to investigation of the heterogeneity associated phenotypes related to adiposity.