Programa de Pós-graduação em Genética e Biologia Molecular
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Navegando Programa de Pós-graduação em Genética e Biologia Molecular por Por Orientador "Bailão, Alexandre Melo"
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Item Análise proteômica do fungo patogênico humano Fonsecaea pedrosoi, cultivado nas condições de temperatura de 22º C e 36º C(Universidade Federal de Goiás, 2017-09-11) Lima, Davi Vinícius de; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, lexandre Melo; Paccez, Juliano Domiraci; Novaes, EvandroThe Polymorphic fungus Fonsecaea pedrosoi is the main etiological agent of Chromoblastomycosis (CBM), characterized as a chronic cutaneous mycotic infection, occurring mainly in tropical regions. The limited availability of effective therapeutic protocols coupled with CBM clinical polymorphism lead to often prolonged and high relapse rate therapies. Results regarding the molecular biology of F. pedrosoi are scarce however, some virulence factors have already been described, evidencing the need to explore the mechanisms used by this pathogen during infection. The present work carried out in silico analyzes under specific culture conditions, with temperatures of 22º C and 36º C, aiming to better understand the proteome of this fungus, both intracellular and secreted. In order to identify these proteins, we used bioinformatic tools that are available online and allowed the prediction of protein sets that can be secreted by classical and non-classical routes. After the in silico prediction method, proteins were identified in the fungal extracts by the Ultraperformance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MSE) methodology. Those identified in the extracts that brought a more reliable interpretation corroborated the results in silico. The proteins identified stand out for being related to various biological processes, such as metabolism of carbon compounds, energy, proteins related to thermal and oxidative stress, defense, proteins related to the cell cycle, among other functions. The analyzes showed that the vast majority of identified proteins are secreted by alternative pathways, and the results point to the great importance of the biological roles that these secreted proteins can play during the onset of infection. Thus, we observed that the fungus presents variations in protein expression levels according to the condition addressed and may reflect on its behavior and responses to the successful establishment of the infection.Item Análise proteômica de paracoccidioides sp. isolado de um caso de fungemia(Universidade Federal de Goiás, 2014-03-20) Martins, Paulo Henrique Rosa; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, Alexandre Melo; Casaletti, Luciana; Barcelos, Rejane da Silva SenaParacoccidioides spp. are pathogens of Paracoccidioidomycosis a systemic mycosis that affects about 10 million people in endemic regions. The incidence of the disease is restricted to Latin America and most cases are found in Brazil. The disease is characterized by chronic granulomatous inflammation, and patients may present with a wide spectrum of clinical manifestations. After inhalation of conidia, the installation of fungus in the lungs, which subsequently through hematogenous route may cause an infection spreads. Hematogenous fungal infections represent a serious health problem, involving hospitalized patients with predisposing conditions that lead to a high mortality rate. Fungemia corresponds to isolation of fungi in the bloodstream and occurs mainly in immunocompromised patients. Yeasts have been increasingly present as etiological agents fungemia including Candida albicans and other species such as Candida non- albicans. In this study, Paracoccidioides spp. was isolated from a case of fungemia. So far this is the first molecular study of a case of fungemia caused by this fungus. In order to identify the molecular factors associated with this specific phenotype, a comparative proteomic analysis was performed. The samples were analyzed by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoUPLC - EPM), where the soluble proteins of fungemia were compared with strain Pb01 -like proteins. 206 proteins regulated positively and negatively regulated 183 were identified. Among the positively regulated protein, 22 % were related to cellular metabolism, 15 % related to protein synthesis and 12% energy. Of phenotypic characterization tests, which showed a better adaptation of isolated Pb9840 blood were also conducted. With this work we propose that the isolated Pb9840 developed mechanisms to better installing the bloodstream and causing the fungemia. The study of the proteomic profile of this isolate may elucidate the virulence mechanisms used by this fungus during fungemia and / or hematogenous spread.Item Análise proteômica do fungo patogênico humano Fonsecaea pedrosoi submetido à temperatura do hospedeiro(Universidade Federal de Goiás, 2021-09-29) Rosa, Gabriela Danelli; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, Alexandre Melo; Lima, Patricia de Sousa; Rocha, Thiago LopesChromoblastomycosis (CBM) is a chronic subcutaneous mycosis, very common in tropical and subtropical regions and affects many men related to rural activities. Lesions of this disease can appear in five clinical forms and treatments are difficult due to the recalcitrant nature of the disease. The main causative agent of CBM is the fungus Fonsecaea pedrosoi, which is a polymorphic, melanized fungus that presents a certain phenotypic plasticity in face of temperature variations. Thermotolerance is one of the virulence factors of virulence for this pathogen, since it survives the temperature increase, like that of the host. However, no work so far has searched to characterize the adapted cellular processes. Proteins are part of the strategies used in response to a variety of stressors, such as host temperature. In this work we mapped the intracellular proteomic profile of F.pedrosoi cultivated under different temperature conditions (28 and 37 °C) for 24 hours. It was possible to identify by means of Liquid Chromatography coupled to Mass Spectrometry (Nano UPLC-MSᴱ) a total of 486 differentially expressed proteins, 101 of which were up-regulated and 385 down-regulated. It was possible to observe that F. pedrosoi seems to respond to temperature stress by repressing central carbon metabolism pathways, such as glycolysis, tricarboxylic acid cycle (TCA) and glyoxylate cycle, cell wall biosynthesis and melanin and induced amino acid degradation enzymes and antioxidant proteins.Item Caracterização dos mecanismos de captação de ferro em Fonsecaea pedrosoi e Cladophialophora carrionii(Universidade Federal de Goiás, 2017-02-20) Silva, Kassyo Lobato Potenciano da; Bailão, Mirelle Garcia Silva; http://lattes.cnpq.br/8844829751376156; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Soares, Célia Maria de Almeida; Schrank, AugustoThe polymorphic fungi Fonsecaea pedrosoi and Cladophialophora carrionii are the main etiological agents of Chromoblastomycosis (CBM), a chronic cutaneous mycotic infection, which occurs mainly in humid tropical regions. The scarcity of effective therapeutic protocols added to the clinical polymorphism of CBM leads to prolonged therapies and a high relapse rate. There are few data on the molecular biology of F. pedrosoi and C. carrionii, although some virulence factors have already been described, revealing the need to explore the mechanisms used by these pathogens during infection. Iron is a vital micronutrient for all organisms, with the exception of some bacteria, participating in essential cellular processes, such as: DNA replication, oxidative stress, cellular respiration, oxygen transport, energy production, among others. During the infection, the host decreases the availability of iron to the pathogen to contain the advancement of the infection; while the pathogen activates mechanisms for obtain and use the metal to survive in the infected tissues, revealing a scenario of intense dispute. In order to characterize the presence of these systems in F. pedrosoi and C. carrionii, in sílico analyzes were performed, revealing the presence of orthologs for proteins involved in reductive iron assimilation, siderophore mediated uptake and regulators of capture and utilization strategies of the metal, these sequences containing conserved domains consistent with the predicted functions. The expression profile of transcripts demonstrated the induction of iron reducing scavengers, siderophore biosynthesis intermediates and the hapX positive regulator during iron deprivation, in addition to repressing the negative regulatory sreA. The production of siderophores was observed after qualitative approaches, and the siderophore ferricrocine was subsequently identified intra- and extracellularly in F. pedrosoi and C. carrionii. These siderophores were able to restore the growth of a mutant strain of ΔsidA from A. nidulans. Preferred iron sources were observed after growth assays and, in addition, some of these sources were used by F. pedrosoi and C. carrionii during infection in macrophages, demonstrating that these pathogens may have strategies for remodeling iron homeostasis for survival.Item Estudo da homeostase de zinco no fungo patogênico Histoplasma capsulatum var. capsulatum: Abordagens proteômica e funcional(Universidade Federal de Goiás, 2016-11-30) Siqueira, Janaina Gomes de; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Soares, Célia Maria de Almeida; Lima, Patrícia de SousaHistoplasma capsulatum var. capsulatum is a pathogenic fungus that causes the systemic disease known as histoplasmosis. This mycosis presents endemicity in the regions of the Mississippi and Ohio rivers in the United States, also found in several countries of South America. In Brazil, the number of people infected by this fungus has been growing in recent years, especially in immunocompromised people. H. capsulatum is a thermodymorphic microorganism, found in two morphotypes: mycelium at 25ºC and yeast at 37ºC. During the infection process the pathogenic microorganism needs to obtain nutrients so that it grows and develops within the tissue of the host. Among the essential nutrients, zinc is an essential metal ion, used in the maintenance of numerous metabolic pathways. Zinc is used as catalytic and structural cofactor of numerous metalloproteins. Zinc uptake is characterized as a virulence factor in H. capsulatum. In order to study the molecular mechanisms of zinc homeostasis in H. capsulatum, the in silico analysis of zinc homeostasis related proteins. The ZafA transcription factor, which acts on the regulation of genes in response to the zinc level in the medium and the transcription factor PacC that regulates genes in response to pH, were also found. ZrfA and ZrfC membrane transporters were also found ZrfF, ZrcA and ZrcC vacuolar transporters. The transcriptional profile analysis of these genes was measured by RT-PCR in infected macrophages and in vitro. During the infection the ZafA and ZrfA genes were induced, suggesting that these genes were expressed in order to supply the fungal cell with zinc, due to the metal deficiency caused by the infection. In vitro, ZafA, ZrfC, ZrfF, ZrcA and ZrcC were induced at zinc deprivation condition, compared with control condition. According with proteomic data of fungus under conditions of zinc deprivation, it was suggested the induction of proteins related to rescue, defense and virulence, related mainly to oxidative stress, besides other metabolic pathways such as gluconeogenesis. These data suggest that the fungus undergoes a metabolic remodeling in response to the oxidative stress caused by zinc deprivation.Item Identificação de metaloproteínas associadas a cobre, ferro e zinco codificadas pelos genomas de Paracoccidioides spp.(Universidade Federal de Goiás, 2014-03-11) Tristão, Gabriel Brum; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, Alexandre Melo; Soares, Célia Maria de Almeida; Staats, Charley CristianApproximately one third of all proteins have been estimated to contain at least one metal cofactor, being named metalloproteins. These represent one of the most diverse classes of proteins, containing metal ions provide catalytic, regulatory and structural functions. Bioinformatic tools have been developed to predict metalloproteins encoded by an organism based only on its genome sequence. Paracoccidioides complex includes termodimorphic pathogenic fungi that are found as saprobiotic mycelia in the environment and as yeast in host tissues. They are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis in Latin America. It is known that many metalloproteins are important for virulence of several pathogenic microorganisms. On this way, the present work aimed to predict the cooper, iron and zinc proteins encoded by the genomes of three phylogenetic species of Paracoccidioides spp. (Pb01, Pb03 and Pb18). Cu-, Fe- and Zn-proteins represents 7% of the total proteins encoded by Paracoccidioides spp genomes. Zinc-proteins were the most abundant metalloproteins representing 5.7% of the fungus proteome, while copper and iron proteins represent 0.3% and 1.2% respectively. Functional classification revealed that metalloproteins are related to many cellular processes. Furthermore it was observed that many of these metalloproteins play roles as virulence factors, in the biology of the fungus, and an analysis of the protein interactions revealed that many of them depend on each other to perform their functions. Thus, it is concluded that the Cu-, Feand Zn- metalloproteome of Paracoccidioides is of utmost importance for biology and virulence