Navegando por Palavras-chave "Diversidade Genética"
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- ItemSomente MetadadadosAnálise da Diversidade Genética em Agentes da Esporotricose Usando Marcadores Aflp (Amplified Fragment Length Polymorphism)(Universidade Federal de São Paulo (UNIFESP), 2020-03-10) Carvalho, Jamile Ambrosio De [UNIFESP]; Camargo, Zoilo Pires De [UNIFESP]; Universidade Federal de São PauloSporothrix is a thermodimorphic fungus found in the environment which causes sporotrichosis, a subcutaneous mycosis that can be transmitted to humans directly from contaminated soil or plants through traumatic inoculum from fungal propagules or through bites and scratches from contaminated cats. The genus is divided into two clades, a clinical clade represented by S. brasiliensis, S. schenckii, S. globosa, and S. luriei, and an environmental clade represented by species rarely described as human pathogens. Considering the ongoing epidemic occurring in Brazil, mostly caused by S. brasiliensis, it is necessary to use techniques to explore diversity within the species. The existing techniques for this type of study have high costs, we aimed to standardize the AFLP technique for application in Sporothrix. AFLP is based on genomic DNA digestion using one or more restriction enzymes and selective amplification of the fragments generated using selective molecular primers with the addition of one or more nucleotides to the 3' region of these primers. The technique has been used for several microorganisms and has advantages as not requiring the knowledge of the sequence of isolates. To reduce costs and time, firstly, an in silico AFLP was performed to predict the fragments that would be generated and thus to choose the nucleotide combinations that presented more efficiency to separate the isolates by species. For this, we use two softwares: AFLPinSilico and ISIF. The two softwares were compatible with each other and both revealed six combinations that showed good results for studies in Sporothrix. These combinations were tested in vitro and all obtained good results, but only three were selected to continue the study with 188 isolates of Sporothrix from different geographic regions. In general, the study presented three new markers that demonstrated efficiency in separating Sporothrix by species and accessing the intraspecific diversity in the species of the clinical clade, clarifying questions of the expansion of S. brasiliensis.
- ItemRestritoLevantamento das espécies de Bostrychia dos manguezais de Bertioga, Santos - Cubatão e Complexo Estuarino-Lagunar de Iguape-Cananéia, com base em DNA barcoding(Universidade Federal de São Paulo, 2022-02-07) Santos, Juliana Moreira dos [UNIFESP]; Milstein, Daniela [UNIFESP]; http://lattes.cnpq.br/7442898844707725; http://lattes.cnpq.br/9069947770491880; Universidade Federal de São Paulo (UNIFESP)As macroalgas associadas aos mangues são organismos de suma importância ecológica nos ecossistemas estuarinos. Nos manguezais do Brasil, destacam-se as espécies do gênero Bostrychia. Estudos sobre a sua diversidade, quando baseados apenas em abordagens morfológicas, podem ser imprecisos pois os caracteres empregados para a identificação de espécies nem sempre são informativos. Além disso, algumas espécies são crípticas, o que muitas vezes leva a uma identificação duvidosa. No entanto, quando dados moleculares são associados a caracteres morfológicos, a delimitação em nível de espécie fica mais acurada. O presente projeto utilizou a técnica de DNA barcoding para identificar as espécies do gênero Bostrychia que ocorrem nos manguezais de Bertioga, Santos-Cubatão e Cananéia-Iguape. Os resultados obtidos com o sequenciamento dos marcadores moleculares da mitocôndria (COI-5P) e cloroplasto (rbcL), indicaram a ocorrência de quatro espécies distintas (B. montagnei, B. pilulifera, B. radicans e Bostrychia sp). Sendo, B. pilulifera uma nova ocorrência para os manguezais do Estado de São Paulo. A espécie identificada como Bostrychia sp. não obteve
- ItemAcesso aberto (Open Access)Otimização de técnica de PCR em tempo real para detecção das regiões pol e env dos subtipos B e F de HIV-1 e triagem de seus recombinantes(Universidade Federal de São Paulo (UNIFESP), 2009-01-28) Teixeira, Daniela [UNIFESP]; Diaz, Ricardo Sobhie [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Background: The discovery of 42 HIV-1 circulating recombinant forms (CRF) together with the innumerous unique HIV recombinants forms, makes clear the role of genetic recombination for the epidemic. In Brazil, clades B, F, and C co-circulate, with 5 recently described CRFs. Real Time PCR is a rapid and reliable tool capable of detecting different HIV-1 subtypes and recombinant profiles. Objective: The aim of this study was to develop real time PCR systems in order to detect the Brazilian CRF_28 and CRF_29, which are B/F recombinants, as well as detect B/F recombinants generated by in vitro competition assays. In future, these systems should be able to discriminate subtypes in clinical surveys. Methodology: MT-4 cells were separately infected with the viral strains BZ167 (subtype B) and BR020 (subtype F), and supernatant was collected in order to optimizing the real time PCR systems (TaqMan®) developed to detect the subtype profile of different genomic regions, including pol gene (protease, reverse transcriptase, integrase) and env gene (gp120 and gp41). The designed primers should be able to equally amplify the subtype B and F, which should be discriminated by subtype-specific probes. For future validation of these PCR systems, 157 clinical samples from the city of Santos were sequenced and phylogeneticaly analyzed in order to perform the clade assignment with Neighbor-Joining algorithm (Phylip software package v3.5). Results: The designed systems were able to differentiate the utilized viral strains. The estimated efficiencies for each system, for each probe, subtypes B and F separately, were respectively: 80,97 and 85,16% for protease region; 89,80 and 75,09% for reverse transcriptase region; 80,90% and 83,83% for integrase region; 93,49 and 98,93% for gp120 region; and 88,45 and 80,19% for gp41 region. For the co-infected cell culture, the detection of each subtype was performed in the first and fifth passages. Generally, the initial concentration of subtype B appeared to have decreased, some of them becoming undetectable, whereas subtype F seemed to increase with the passages, for protease, reverse transcriptase, gp120 and gp41 regions. The integrase region was an exception, since only the subtype B was detected, with increasing Cycle threshold (Ct) values over time. Sequencing results revealed that 65 out of 157 samples had the subtype profile defined for all regions. 43 out of 71 were defined as B, whereas 3 were F in all regions. 12 samples presented the CRF_28 profile, and 9 samples presented the CRF_29 profile. There were no subtype C samples in any genomic regions analyzed. Conclusion: The use of real time PCR technique for identification of fragments’ subtypes in cell culture and for evaluation of replicative dynamics of recombination in co-infected cultures warrants its potential use in future in vivo surveys. This methodology proved to be efficient, fast, less cumbersome and less expensive than DNA sequencing. The newly designed systems performed for supernatant of competition assays had suggested a divergent distribution of subtypes for the different regions, which reflects the possibility of genetic recombination. Results from clinical samples revealed a high prevalence of CRF_28/29 in this geographic region, thus reflecting the resulting consequence of different co-circulating strains and pointing to the need for a careful surveillance.
- ItemSomente MetadadadosRelação entre gravidade da Covid-19 e perfil genético do SARS-CoV-2(Universidade Federal de São Paulo (UNIFESP), 2021) Silva, Luiz Claudio Santana Da [UNIFESP]; Diaz, Ricardo Sobhie [UNIFESP]; Universidade Federal de São PauloIn December 2019, SARS-CoV-2 was detected in Wuhan, China, and in March 2020, the World Health Organization (WHO) declared it a pandemic named COVID-19. The study of the molecular epidemiology of SARS-CoV-2 with the evaluation of the relationship of viral mutations and coinfections with infection severity can help elucidate the disease's pathophysiology. In this study, SARS-CoV-2 positive nasal swab samples collected in early 2020 were subjected to NGS RNA metagenomic methodology to characterize the complete SARS-CoV-2 genome and viral coinfections. Twenty-four samples collected in early 2020 were analyzed: i) Mild infection (n=8), ii) Moderate infection (n=9) and iii) Severe infection (n=7). Eleven patients (45.8%) are female, and 13 are male (54.2%) with a mean age of 54 years. Of the 24 samples analyzed, it was possible to classify the variants (Pangolin) of 22 samples, which belonged to the variants B.1.1.28 (68.18%), B.1.1.33 (27.27%), and B.1.1 (5.4%). Mutations in 52 genome positions were identified, with a prevalence of non-synonymous substitutions of 57.69% mainly detected in the ORF1a gene (55.17%), without association with the disease severity. There was no relationship in the selective regimes as inferred by the synonymous/ non-synonymous substitution and the severity of the disease. It was not possible to identify viral coinfections of clinical relevance and association between mutations and infection severity. It was possible to identify the D614G variant, characteristic of the B.1 strain, which became the globally dominant form in June 2020 due to its greater transmission capacity. There was a statistically significant difference between the mean age of the mild and severe groups, with older age relating to the severe form of the disease. Ongoing molecular characterization of viral and host genetic profiles can lead to a greater understanding of the factors influencing the severity of COVID-19.