Desenvolvimento de metodologias eficientes de diagnóstico molecular dos fungos Phyllocista citricarpa e Phyllocista capitalensis

Orientador: Chirlei GlienkeMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : Diversas ferramentas para a detecção e identificação molecular de espécies vem sendo utilizadas no diagnóstico de fitopatógenos. Uma destas doenças, Mancha Preta dos Citros (MPC, agente causal fungo Phyllosticta citricarpa), é causa de perdas financeiras ao agronegócio citrícola – graças a depreciação visual e queda prematura dos frutos. Entretanto, outro fungo do gênero, P. capitalensis, não é patogênico a citros, mas filogeneticamente relacionado e morfologicamente semelhante, compartilha o mesmo hospedeiro e habita outras lesões similares a da MPC, gerando uma demanda pela distinção entre as duas espécies, dada a existência de barreiras fitossanitárias à primeira. Assim, o presente trabalho visou construir conhecimento sob duas regiões genômicas previamente utilizadas para a detecção de P. citricarpa (GCP) – até então não utilizada em qPCR – e P. capitalensis (GMF) – com problema de especificidade perante a novas espécies descritas dentro do gênero. Buscou-se a obtenção de sequências homólogas tanto a GCP em P. capitalensis quanto a GMF em P. citricarpa, P. brazilianiae, G. mangiferae e P. citribraziliensis. Também sua obtenção a partir de DNA extraído de tecidos foliares de pomares com sintomas de MPC e das próprias linhagens obtidas destes, pelo método de isolamento tradicional. Procedeu-se com a extração de ácidos nucléicos do respectivo material (kit MoBio UltraCleantm Microbial DNA Isolation), amplificação do fragmento GCP e GMF (em condições menos restringentes quando em outras espécies), clonagem no vetor pGEM®-T, avaliação dos transformantes e sequenciamento, ou o sequenciamento direto em alguns casos. Em seguida, foram desenhados e avaliados primers com base nas sequencias obtidas, visando detecção utilizando PCR quantitativa (qPCR) com o sistema TaqMan® para P. citricarpa e uma alteração na PCR convencional para P. capitalensis, com a substituição do primer reverso. Na região GCP foram observados polimorfismos escassos nas diversas linhagens de P. citricarpa, porém não foi encontrada uma região com alta homologia em P. capitalensis através do método utilizado. Esse resultado demonstra a especificidade destes primers para a espécie P. citricarpa o que diminui o risco de falsos positivos. Foi observada a amplificação de produtos diversos quando utilizado o material extraído diretamente de folhas cítricas, provavelmente associados a artefatos na reação de PCR, que justificam a tentativa de aperfeiçoamento da detecção, visando mais especificidade e sensibilidade. O ensaio de qPCR proposto mostrou-se mais específico entretanto menos sensível que o previamente disponível na literatura. Para a região GMF foram, igualmente, observados polimorfismos escassos nas linhagens de P. capitalensis e não foi encontrado um homólogo em P. citricarpa e P. citribraziliensis através do método utilizado. Tal ausência reforça a especificidade destes primers para P. capitalensis e diminui a possibilidade de falsos positivos para P. citricarpa. O diagnóstico via PCR convencional proposto foi capaz de distinguir o fungo P. capitalensis de todos os demais avaliados. Com o obtido pode-se concluir que os primers propostos para a detecção através de qPCR de P. citricarpa são uma alternativa aos previamente disponíveis e que a alteração no ensaio de detecção de P. capitalensis deixou o mesmo mais específico

Composition of endophytic fungal community associated with leaves of maize cultivated in south Brazilian field

The objective of this study was to conduct a survey about fungi associated with leaves from two different maize plant lineages and to analyze their microbiota diversity. Isolated fungi were identified by morphological analysis and molecular taxonomy was performed using ITS1-5.8S-ITS2 rDNA. About 27 fungi morphotypes were obtained, 15 of them were from the first maize lineage. About 86.7% of the individuals belonged to the Dothideomycetes class (Phoma sorghina, Epicocum nigrum, Cladosporium sp., Bipolaris zeicola, and Alternaria alternata complex) and 13.3% to the Sordariomycetes class (Diaporthe/Phomopsis sp. and Nigrospora sp.). This ratio was opposite in the other maize lineage with 25.0% of Dothideomycetes (E. nigrum and Pleosporales) and 75.0% of Sordariomycetes (Gibberella fujikuroi complex, Fusarium graminearum complex, Diaporthe/Phomopsis sp., and Nigrospora sp.). By concerning the analyses of morphological characteristics and molecular phylogeny, this study intended to identify the groups of saprophytic, phytopathogenic, and mycotoxin fungi, which differently co-inhabit leaf tissue of maize plants in both tested lineages

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genu

SARS-CoV-2 Delta and Omicron Variants Surge in Curitiba, Southern Brazil, and Its Impact on Overall COVID-19 Lethality

Screening efforts and genomic surveillance are essential tools to evaluate the course of the COVID-19 pandemic and assist the public healthcare system in dealing with an increasing number of infections. For the analysis of COVID-19 cases scenarios in Curitiba, Paraná, Brazil, we performed a diagnosis of positive cases, coupled with genotyping, for symptomatic and asymptomatic members of the Federal University of Paraná. We achieved over 1000 samples using RT-qPCR for diagnosis. The posterior genotyping allowed us to observe differences in the spread of strains in Curitiba, Brazil. The Delta variant was not associated with an infection wave, whereas the rapid Omicron variant spread became dominant in less than one month. We also evaluated the general vaccination coverage in the state, observing a striking reduction in lethality correlated to the vaccinated fraction of the population; although lower lethality rates were not much affected by the Omicron variant wave, the same effect was not translated in the number of infections. In summary, our results provide a general overview of the pandemic’s course in Paraná State and how there was reduction in lethality after a combination of multiple infection waves and a large-scale vaccination program

SARS-CoV-2 in saliva, viremia and seroprevalence for COVID-19 surveillance at a single hematopoietic stem cell transplantation center: a prospective cohort study

This prospective cohort study aims to analyze the surveillance of COVID-19 at a single hematopoietic stem cell transplantation (HSCT) center in Brazil, in 29 patients undergoing allogeneic HSCT and 57 healthcare workers (nurses and dentists), through viral shedding of SARS-CoV-2 in saliva and plasma and seroprevalence of anti-SARS-CoV-2 IgG. In addition, we report two cases with prolonged persistent detection of SARS-CoV-2 without seroconversion. The sample collection was performed seven times for patients and five times for healthcare workers. Only two patients tested positive for SARS-CoV-2 in their saliva and plasma samples (6.9%) without seroconversion. All healthcare workers were asymptomatic and none tested positive. Two patients (6.9%) and four nurses (8%) had positive serology. No dentists had positive viral detection or positive serology. Our results reflect a low prevalence of positive RT-PCR and seroprevalence of SARS-CoV-2 in patients and healthcare workers at a single HSCT center. Results have also corroborated how the rigorous protocols adopted in transplant centers were even more strengthened in this pandemic scenario

Transcribed Ultraconserved Regions Are Associated with Clinicopathological Features in Breast Cancer

Ultraconserved regions (UCRs) are 481 genome segments, with length longer than 200 bp, that are 100% conserved among humans, mice, and rats. The majority of UCRs are transcriptionally active (T-UCRs) as many of them produce non-coding RNAs. In a previous study, we evaluated the expression level of T-UCRs in breast cancer (BC) patients and found that 63% of transcripts correlated with some clinical and/or molecular parameter of BC. In this study, we delved into the expression levels of 12 T-UCRs and correlated them with clinicopathological parameters, immunohistochemical markers, and overall survival in two breast cancer cohorts: TCGA and Brazilian patients. We found that uc.268 is more expressed in TCGA patients under 40 years of age, associated with progesterone receptor (PR) and estrogen receptor (ER), and its high expression is found in luminal A. Lower uc.84 and uc.376 were respectively observed in metastatic and stage IV tumors associated with good prognostic in luminal B. Moreover, uc.84 was only related to the HER2+, while uc.376 was related to ER+ and PR+, and HER2+. A panel composed of uc.147, uc.271, and uc.427 distinguished luminal A from triple negative patients with an AUC of 0.9531 (sensitivity 92.19% and specificity 86.76%). These results highlight the potential role of T-UCRs in BC and provide insights into the potential application of T-UCRs as biomarkers

La Charente

24 juillet 18931893/07/24 (A22,N10116)-1893/07/24.Appartient à l’ensemble documentaire : PoitouCh

Guanylate-binding protein-1 is a potential new therapeutic target for triple-negative breast cancer

Background: Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen and progesterone receptor expression (ESR and PGR, respectively) and an absence of human epithelial growth factor receptor (ERBB2) amplification. Approximately 15-20% of breast malignancies are TNBC. Patients with TNBC often have an unfavorable prognosis. In addition, TNBC represents an important clinical challenge since it does not respond to hormone therapy. Methods: In this work, we integrated high-throughput mRNA sequencing (RNA-Seq) data from normal and tumor tissues (obtained from The Cancer Genome Atlas, TCGA) and cell lines obtained through in-house sequencing or available from the Gene Expression Omnibus (GEO) to generate a unified list of differentially expressed (DE) genes. Methylome and proteomic data were integrated to our analysis to give further support to our findings. Genes that were overexpressed in TNBC were then curated to retain new potentially druggable targets based on in silico analysis. Knocking-down was used to assess gene importance for TNBC cell proliferation. Results: Our pipeline analysis generated a list of 243 potential new targets for treating TNBC. We finally demonstrated that knock-down of Guanylate-Binding Protein 1 (GBP1), one of the candidate genes, selectively affected the growth of TNBC cell lines. Moreover, we showed that GBP1 expression was controlled by epidermal growth factor receptor (EGFR) in breast cancer cell lines. Conclusions: We propose that GBP1 is a new potential druggable therapeutic target for treating TNBC with enhanced EGFR expression17FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/11577-4; 2014/15968-3; 2015/25832-4; 2009/53853-5; 2012/09452-9; 2014/17820-3; 2014/18061-9; 2013/23510-4; 2014/06512-