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Caracterização molecular de genes preferencialmente expressos na fase leveduriforme patogênica de ´Paracoccidioides brasiliensis´ através das técnicas de ´Macroarray´ e de SSH (Suppression Substractive Hybridization) ; Molecular characterization of preferentially expressed genes in the yeast pathogenic phase of Paracoccidioides brasiliensis through the techniques of Macroarray and SSH (Suppression Subtraction Hybridization)

Marques, Everaldo dos Reis
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 22/12/2005 Português
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Paracoccidioides brasiliensis, um fungo termodimórfico, é o agente causador da paracoccidioidomicose (PCM), a micose sistêmica prevalente da América Latina. A patogenicidade aparenta estar intimamente relacionada com a transição dimórfica da forma de micélio para a de levedura, que é induzida pela mudança da temperatura do ambiente pela temperatura do hospedeiro mamífero. Há poucas informações disponíveis sobre genes de P. brasiliensis que são necessários durante a fase patogênica. Nós, então, realizamos as técnicas de SSH (“Suppression Subtraction Hybridization”) e de “Macroarray” com o objetivo de identificar genes que sejam preferencialmente expressos na fase leveduriforme do isolado Pb18. Genes identificados em ambos os procedimentos estão mais expressos na fase leveduriforme e estão envolvidos em metabolismo básico, transdução de sinal, crescimento e morfogênese e metabolismo do enxofre. Para testar se as mudanças observadas na expressão gênica refletem as diferenças entre as condições de crescimento usadas para obter as duas formas morfológicas preferivelmente às diferenças intrínsecas dos tipos celulares, nós realizamos experimentos com RT-PCR em tempo real utilizando preparações de RNA derivadas de ambas as fases...

Modulação da ativação de células dentríticas por Paracoccidioides brasiliensis; Modulation, activation of Dentritic cells by Paracoccidioides brasiliensis

Ferreira, Karen Spadari
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 15/12/2003 Português
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A paracoccidioidomicose (PCM) é uma micose sistêmica, endêmica na América Latina, causada pelo fungo dimórfico térmico Paracoccidioides brasiliensis (P. brasiliensis), cujo principal componente antigênico é a glicoproteína de 43 kDa (gp43). Diferentes formas clínicas podem ser desenvolvidas e estão diretamente associadas com vários graus de depressão da resposta imune celular. Considerando a importância das células dendríticas na interação dos sistemas imune inato e adaptativo, e na ativação de células T "naive", no presente trabalho estudamos se células dendríticas interagem com leveduras de P. brasiliensis, assim como seu principal componente antigênico (gp43). Foi demonstrado pela primeira vez que células dendríticas poderiam ser infectadas por leveduras de P. brasiliensis, e esse fungo permaneceu viável após fagocitose. Analisamos por citometria de fluxo a expressão das moléculas de superfície observando diminuição significativa da expressão das moléculas de MHC-II, CD80 e CD54 em células dendríticas quando estas foram incubadas com leveduras da cepa Pb18 ou com gp43. Esse resultado mostrou que a ação do P. brasiliensis em células dendríticas poderia ser mediada pela gp43. Ao analisarmos a síntese de IL-12...

Estudo da interação de linfócitos B-1 com antígenos de Paracoccidioides brasilienses; Study of the interaction of B-1 lymphocytes with antigens of Paracoccidioides brasiliensis

Noal, Vanessa Rosa
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 04/12/2009 Português
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Diversos dados na literatura têm demonstrado a participação de linfócitos B-1 em diferentes fenômenos imunológicos, tanto na resposta imune inata quanto na resposta imune adaptativa. Para melhor entendermos a ativação da resposta imune eficaz contra fungos patogênicos, pesquisamos a interação entre os linfócitos B-1 e o Paracoccidioides brasiliensis (P. brasiliensis), uma vez que este expressa moléculas antigênicas que podem ser reconhecidas pelo sistema imune. Utilizamos preparação antigênica do P. brasiliensis obtida de sua superfície leveduriforme denominada de CFA (antígeno livre da parede do fungo) e células leveduriforme do fungo. Observou-se que a maioria das células do sobrenadante da cultura celular de 4 dias de células totais aderentes peritoneais eram constituídos por linfócitos B-1; estas células expressam altos níveis de MHC-II (100%) e CD80 (90%). Contudo, não houve expressão significativa da molécula co-estimuladora CD86. Pela análise fenotípica, os linfócitos B-1 podem atuar como células apresentadoras de antígeno pois expressam CD80, CD86 e MHC-II; então realizamos o ensaio de proliferação celular utilizando linfócitos B-1 como células apresentadoras de antígenos e observamos proliferação celular de linfócitos TCD4+ significativa. Em relação às citocinas...

Caracterização e análise da expressão de genes (sod3 e ccp) envolvidos no estresse oxidativo em Paracoccidioides brasiliensis

Pessoa, Carine Ribeiro
Fonte: Universidade de Brasília Publicador: Universidade de Brasília
Tipo: Dissertação
Português
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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2006.; O fungo Paracoccidioides brasiliensis é dimórfico e patogênico ao homem. Ele é o agente etiológico da paracoccidioidomicose (PCM), a micose sistêmica de maior incidência da América Latina. O estabelecimento da infecção é dependente da transição de micélio para levedura e este processo in vitro é reversível e regulado pela mudança de temperatura de 22 °C para 36 °C. O projeto ¿Genoma funcional e diferencial do fungo Paracoccidioides brasiliensis¿ identificou 6.022 PbAEST, que correspondem a genes expressos no fungo, representando aproximadamente 80% do genoma do fungo. Na interação patógeno-hospedeiro, a resposta eficiente do patógeno contra o ataque oxidativo imposto pelas células fagocitárias do hospedeiro facilita a sua sobrevivência no hospedeiro. Os genes potencialmente envolvidos na resposta ao estresse oxidativo em P. brasiliensis, identificados no projeto transcriptoma, foram categorizados em 4 classes: enzimas antioxidantes (12 PbAESTs), biossíntese e metabolismo da glutationa e regeneração de NADPH (11 PbAESTs), homeostase de íons metálicos (3 PbAEST) e fatores transcricionais (7 PbAESTs). Entre as seqüências anotadas no transcriptoma...

Identificação proteômica, expressão heteróloga, citolocalização, estudos de regulação transcricional e traducional da Aconitase Mitocondrial de Paracoccidioides brasiliensis; Identification,characterization and regulation studies of the mitochondrial Aconitase of Paracoccidioides brasiliensis

BRITO, Wesley de Almeida
Fonte: Universidade Federal de Goiás; BR; UFG; Doutorado em Biologia; Ciencias Biologicas Publicador: Universidade Federal de Goiás; BR; UFG; Doutorado em Biologia; Ciencias Biologicas
Tipo: Tese de Doutorado Formato: application/pdf
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Paracoccidioides brasiliensis is a thermal-dimorphic fungus, the causative agent of Paracoccidioidomycosis (PCM), an important endemic mycosis in Latin America. A protein species preferentially expressed in yeast cells with a molecular mass of 80kDa and isoeletric point (pI) of 7.79 was isolated from the proteome of P. brasiliensis and characterized as an aconitase (E.C. 4.2.1.3). Aconitase is an enzyme that catalyzes the isomerization of citrate to isocitrate in both the Krebs cycle (KC) and the glyoxylate cycle (GC). We report the cloning and characterization of the cDNA encoding the aconitase of P. brasiliensis (PbACO). The cDNA showed a 2337 bp open reading frame (ORF) and encoded a predicted protein with 779 amino acids. A polyclonal antibody against the purified recombinant PbACO was obtained in order to analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in the extracellular fluid, cell wall, mitochondria, cytosol and peroxisomes of yeast cells as demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the Pbaco gene was performed through quantitative real time RT-PCR and results demonstrated increasing expression during differentiation from mycelium to yeast cells. Real time RT-PCR assays was also used to evaluate the Pbaco expression when the fungus grows on media with acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in highiron conditions.; Paracoccidioides brasiliensis é um fungo termodimórfico...

Inibição da isocitrato liase de Paracoccidioides brasiliensis pelo produto natural argentilactona; Inhibition of isocitrate lyase by Paracoccidioides brasiliensis natural product argentilactone

PRADO, Renata Silva do
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
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The fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomicosys (PCM), the most prevalent human systemic mycosis in Latin America. The toxicity of drugs and the appearance of resistant strains have imposition the search for new therapeutic approaches. Plants with reputed antimicrobial uses represent a rich source for the screening of potential antifungal compounds. In this work, we investigated the inhibitory action of argentilactone, extracted from the essential oil of Hyptis ovalifolia, and its analogues on P. brasiliensis yeast cells, during the differentiation from mycelium to yeast, on native and recombinant PbICL. Sensitivity tests on plates, which assessed the activity of argentilactone on yeast cells of P. brasiliensis showed both argentilactone as reduced argentilactone interfered in fungus growth, affecting the cells in a dose-dependent way. The experiments evaluating the minimum inhibitory concentration (MIC), which evaluated the influence of the compounds during the transition from mycelium to yeast in P. brasiliensis showed that argentilactone and reduced argentilactone interferes in this process, also in a dose-dependent way. A specific activity of isocitrate lyase of the fungus, both recombinant as native...

Clonagem e expressão do gene da tiorredoxina 1 de Paracoccidioides brasiliensis em Pichia pastoris; Cloning and expression of the thioredoxin 1 gene of Paracoccidioides brasiliensis in pichia pastoris

CINTRA, Lorena Cardoso
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
Português
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The termodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human systemic mycosis of high prevalence in Latin America. P. brasiliensis is exposed to oxidative stress (OS) caused by reactive oxygen species (ROS) produced by the defense cells of the human host. When the invasion by pathogens occurs, the host defense system generates ROS to fight the invader. Inside the human host, P. brasiliensis is phagocytosed by macrophages, facing an extremely hostile environment due to nitric oxide and hydrogen peroxide. The Trx1 is an intracellular redox protein, which participates in the maintenance of cell redox homeostasis, both in terms of OS as reducer. It is ubiquitous and is characterized by typical CXXC active site, responsible for oxidation, reduction, or isomerization of proteins disulfide bonds. In a previous work, it was isolated, characterized and cloned into expression vector pGEX-4T-3 cDNA coding for TRX1 of P. brasiliensis (accession number AY376435). The recombinant protein (recPbTRX1) was produced and partially purified and the yeast cells of P. brasiliensis showed increased expression of the gene coding for PbTRX1 in response to OS. This study aimed the heterologous expression of cDNA of a thioredoxin of the fungus P. brasiliensis in Pichia pastoris...

Análise da expressão de genes envolvidos na manutenção da homeostase de cobre no patógeno humano Paracoccidioides brasiliensis; Analysis of the expression of genes involved in the maintenance of copper homeostasis in the human pathogen Paracoccidioides brasiliensis

SANTOS, Rodrigo da Silva
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
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The fungus Paracoccidioides brasiliensis is a human pathogen with a wide distribution in Latin America. The fungus causes paracoccidioidomycosis when mycelia reachs the lungs. The success of the infection depends on the acquisition of essential micronutrients such as copper, which is required as cofactor for a variety of enzymes important in biological in several processes, such as respiration, growth and acquisition of iron. Previous studies, of the Laboratory of Molecular Biology showed that a high affinity copper transporter (PbCTR3) is a molecule highly expressed and probably necessary for the infection establishment of by P. brasiliensis. In the present study were isolated and characterized the genomic and cDNA sequences encoding for PbCTR3 of P. brasiliensis. The cDNA presented 582 base pairs and encodes for a protein with 193 amino acids, predicted molecular mass of 21.5 kDa and pI of 8.6. The genomic sequence has four exons interrupted by three introns. In silico analysis was performed on the database of the structural genome of P. brasiliensis (http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensisMultiHome.html) , where genes involved in maintaining the homeostasis of copper have been identified and used to design of a model of copper homeostasis in P. brasiliensis. The transcriptional behavior of Pbctr3 and genes involved in copper homeostasis were examined during exposure of yeast cells of P. brasiliensis to copper and iron depletion conditions...

Caracterização Molecular e Expressão Heteróloga de um cDNA Codificante para Tiorredoxina do fungo patogênico humano Paracoccidioides brasiliensis; Cloning expression and insulin reduction activity analysis of a thioredoxin homalogue of human pathologe Paracoccidioides brasiliensis

DOMINGOS, Fernanda de Castro
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
Português
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The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis (PCM), a human systemic mycosis highly prevalent in countries of Latin America. P. brasiliensis is subjected to different insults from human host, such as oxidative stress caused by reactive oxygen species produced by the host during the infection. Thioredoxin (TRX) is an intracellular redox protein that is required to maintain redox homeostasis in response to both reductive and oxidative stress conditions in several organisms. We report here the characterization of a 811 bp cDNA Pbtrx1, encoding a PbTRX1 of 116 amino acids, with a predicted molecular mass of 12 kDa and pI 5.2. This putative protein presented one highly conserved active site motif (WCGPC) between TRXs from several organisms. The phylogenetic analysis performed with PbTRX1 and TRXs from other organisms, putted P. brasiliensis in the fungi clade. We also performed the prediction of the secondary structure of PbTRX1 that shows a pattern characteristic of the open twisted alpha/beta, similar to TRX secondary structures described in other fungus. In order to obtain the recombinant PbTRX1, the expression construct pGEX-4T-3-trx1 was introduced into Escherichia coli cells and the expression and purification of the recombinant protein was obtained. The recPbTRX1 and PbTRX1 from yeast cells extract were found to catalyze the reduction of insulin. However the PbTRX1 from yeast cells extract treated with H2O2 showed highly insulin reduction activity than the yeast cells no treated. PbTRX1 was detected by Western blotting in the extracts from yeast cells growth and from mycelium to yeast transition. The yeast cells growth was significantly inhibited by H2O2; however the mycelium to yeast transition was little affected by this oxidant. Semi-quantitative RT-PCR was employed to analysis the expression of Pbtrx1 gene in response to H2O2. The level of Pbtrx1 transcripts was higher in yeast cells treated with H2O2 than in yeast cells no treated. To realize how P. brasiliensis deals with oxidative stress is essential to understand the mechanisms involved in its survival in the host. It may be possible that PbTRX1 enhances survival of P. brasiliensis in the host...

Caracterização e Análise Funcional da Beta -1,3-glicanosiltransferase 2 de Paracoccidioides brasiliensis; Characterization and Functional Analysis of Beta-glucanosyltransferases -1.3 2 of Paracoccidioides brasiliensis

LIMA, Patrícia de Sousa
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
Português
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Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a systemic disorder geographically restricted to Central and South America, and one of the most important endemic mycoses in these regions, especially among the male rural populations. The disease is most likely caused by the inhalation of asexual spores (conidia) produced by the mycelia form of the fungus, propagules that once in the lungs undergo differentiation towards the parasitic yeast form. The cell wall of P. brasiliensis is a dynamic structure, essentially composed of branched glucan (β-1,3 and β-1,6 glucans), chitin, lipids and mannoproteins. Many enzymes are responsible for cell wall remodeling. One of them is the Beta-1,3- glucanosyltransferase 2 (PbGel2p) presented here. The amino acid deduced sequence of PbGel2p presented similarity to others proteins involved in fungal cell wall biosynthesis and morphogenesis and it was characterized as a member of GH72 family, GH72_ subfamily. The recombinant rPbGel2p was overexpressed in Escherichia coli and the polyclonal antibody was obtained. The PbGel2p mRNA, as well as the protein, were detected at the highest level in the mycelium phase. The potencial role of PbGel2p in cell wall biosynthesis and morphogenesis was analyzed by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae GAS1Δ. The results indicated that PbGel2p is a cell wall-associated protein that probably works as a β-1...

Caracterização de um Antígeno Rico em Prolina(PRA/Ag2) do fungo patogênico humano Paracoccidioides brasiliensis; Characterization of a Proline-Rich Antigen (PRA/Ag2) of the human pathogenic fungus Paracoccidioides brasiliensis

CASTRO, Kelly Pacheco de
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
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The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a proline-rich protein (PRA/Ag2) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Coccidioides immitis. The protein, the cDNA and genomic sequences were analyzed. Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis. The cloned cDNA was expressed in Escherichia coli and the purified rPbPRA/Ag2 was used to obtain polyclonal antibody. The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbPRA/Ag2 in the fungal cell wall, linked through a GPIanchor. The expression of the Pbpra/ag2 gene was analyzed by real time PCR and results demonstrated developmental regulation in phases of P. brasiliensis, with a higher expression in the mycelium saprobic phase. The protein expression analyses corroborate the transcript levels.; O fungo dimórfico Paracoccidioides brasiliensis é o agente causador da micose sistêmica mais prevalente na América Latina. Em humanos...

Malato sintase de Paracoccidioides brasiliensis é uma proteína ligada à superfície que se comporta como uma anchorless adesina; The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

SILVA NETO, Benedito Rodrigues da
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
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The pathogenic fungus Paracoccidioides brasiliensis causative of Paracoccidioidomycosis (PCM), a pulmonary mycose acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside host, P. brasiliensis use the glyoxylate cycle for intracellular survival. Here, we provide evidence that malate synthase of P. brasiliensis (PbMLS) is localized on the cell wall, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By using Confocal Laser Scanning Microscopy and Western blot analysis, PbMLS was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis of mother and bud yeast cells. PbMLSr and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells A549. These observations indicated that cell wall-associated MLS of P. brasiliensis could be mediating the binding of fungal cells, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection.; O fungo de patogênico Paracoccidioides brasiliensis agente causador da Paracoccidioidomicose (PCM)...

Ação da fosfolipase B extracelular de Paracoccidioides brasiliensis na interação ex vivo com macrófagos alveolares; Action of extracellular phospholipase B of Paracoccidioides brasiliensis interaction with alveolar macrophage ex vivo

SOARES, Deyze Alencar
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Biologia; Ciências Biolóicas
Tipo: Dissertação Formato: application/pdf
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Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the most prevalent systemic mycosis in Latin America, paracoccidioidomycosis. The phospholipase B (PLB) enzyme is considered an important virulence factor in this dimorphic fungus, involved in the immune response of the host-pathogen interaction. Our objective was to determine whether a P. brasiliensis (Pb18) PLB is involved in adhesion / internalization of yeast and evasion of host immune responses. The effect of PLB was analysed using specific inhibition of PLB (alexidine dihydrochloride) and pulmonary surfactant in an ex vivo model (Pb18) of alveolar macrophage (MHS cells) infection. PLB enzyme assays and real time RT-PCR (qRTPCR) analysis of genes differentially expressed in the process of evasion: plb1 (phospholipase B1), icl1 (isocitrate lyase) and sod3 (Cu, Zn dismutase) and immune responses: clec2 (C-type lectin domain 2), cd14 (cluster of differentiation 14), tlr2 (toll-like receptor 2), nfkb (nuclear factor kappa B), nkrf (NF-kappaB repressing factor), il1β (inteleukin-1β) and tnfα (tumor necrosis factor alpha) were carried out using selective inhibition of PLB activity and pulmonary surfactant. The levels of cytokines inteleukin 10 (IL-10)...

An€álise da resposta de Paracoccidioides brasiliensis a diferentes tipos de agentes estressores e osmoreguladores e express o heteró‚loga e localizaçムo de β-1.3-glicana sintase; Analise response of Paracoccidioides brasiliensis to different types of stress agents and osmoreguladores and expresses Heteralogs and Location of the β-1.3-glucan synthase

TOMAZETT, Patrícia Kott
Fonte: Universidade Federal de Goiás; BR; UFG; Doutorado em Ciencias da Saude; Ciencias da Saude Publicador: Universidade Federal de Goiás; BR; UFG; Doutorado em Ciencias da Saude; Ciencias da Saude
Tipo: Tese de Doutorado Formato: application/pdf
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Paracoccidioides brasiliensis is a thermo-dimorphic human pathogenic fungus that lives at 23”C in the mycelium phase (infecting phase) and at 37”C in the yeast phase (parasite phase). In attempt to survive, the cell wall of fungi can change its composition and/or structure in response to environmental stress by compensatory mechanisms. The molecules involved in these mechanisms are possible target for the development of effective antifungal agents. In P. brasiliensis, the main components of the cell wall are glucans and chitin polymers. These polymers make a primary barrier that is responsible for the structural integrity and form of the cell wall. In this work the behavior of P. brasiliensis was evaluated against stress conditions with the aim of study, for the first time, the mechanisms used by this fungus in the maintenance of the cell wall integrity. Our results shown that P. brasiliensis yeast cells are sensitive to cell wall stressors calcofluor white (CFW), congo red (CR), SDS, KCl, NaCl and sorbitol. There was an increase in the PbFKS1 transcripts expression and in the content of cell wall β- 1,3-glicana after treatment with all stressor agents. After treatment with SDS and KCl the PbGFA1 transcripts expression and the cell wall GlcNAc residues also increased. The transcript expression of PbGEL3 was also evaluated being increased after treatment with CFW...

Análises transcricionais no processo de adesão por Paracoccidioides brasiliensis e caracterização funcional de adesinas; Transcriptional analysis of the accession process by Paracoccidioides brasiliensis and functional characterization of adhesins

NOGUEIRA, Sarah Veloso
Fonte: Universidade Federal de Goiás; BR; UFG; Doutorado em Medicina Tropical; Ciências da Saúde Publicador: Universidade Federal de Goiás; BR; UFG; Doutorado em Medicina Tropical; Ciências da Saúde
Tipo: Tese de Doutorado Formato: application/pdf
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Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis, prevalent in Latin America. Extracellular matrix (ECM) is a complex net where collagens, laminin and fibronectin can be found and, when exposed, is the first site for the fungus adhesion. Our aim was to study genes involved in the adhesion process using Representational Difference Analysis (RDA). RDA is a PCR-coupled subtractive method that allows the isolation of genes differentially expressed in two different cDNA populations. Hence, cDNAs were synthesized from RNAs extracted from P. brasiliensis yeast cells adhered to collagen and fibronectin to identify overexpressed genes. Genes involved in a wide range of cellular process were found and PbCtr3 (cooper transporter) and enolase (PbEno) were chosen to further studies. A synthetic peptide (PbCTR3) and the recombinant enolase (rPbEno) were utilized together with the anti-rPbEno polyclonal antibody in functional analysis with ECM components and plasminogen. The studies suggest that P. brasiliensis enolase, in the surface, is able to generate plasmin from plasminogen by plasminogen activator. Therefore, it was also demonstrated that this protein is secreted and able to promote fungus adhesion and invasion to cells. These findings clearly establish the role of enolase in the patogenicity of P. brasiliensis.; Paraccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM)...

Estratégias na identificação e caracterização de potenciais antifúngicos e seus alvos em Paracoccidioides brasiliensis; Strategies for identification and characterization of potential antifungal agents and their targets in Paracoccidioides brasiliensis

CARVALHO, Patrícia Fernanda Zambuzzi
Fonte: Universidade Federal de Goiás; BR; UFG; Doutorado em Medicina Tropical; Ciências da Saúde Publicador: Universidade Federal de Goiás; BR; UFG; Doutorado em Medicina Tropical; Ciências da Saúde
Tipo: Tese de Doutorado Formato: application/pdf
Português
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The termodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a systemic human mycosis geographically distributed in Latin America, being the eighth most common cause of death among chronic infections. PCM is acquired by inhalation of fungal propagules, which reach the lungs and is disseminate through the bloodstream and/or lymph to all parts of the body. The treatment of PCM is long, starting with a dosage of aggressive antifungal agents, extending for months or years. Resistance to antimicrobial drugs may limit the ability of effective treatment of patients, interfering with therapeutic efficacy. Thus, it is necessary to discover and develop new antifungal agents. Plants compounds have a great structural diversity, many of which are models for the synthesis of a vast number of drugs. The action of the oenothein B compound, purified from leaves of Eugenia uniflora, a plant from the Brazilian Savannah, was evaluated on growth, viability and expression of P. brasiliensis genes. The compound interfered with cell morphology and inhibited the transcripts of β-1- 3-glucan synthase. The synergistic effect between oenothein B and drugs used to treat PCM (amphotericin B, itraconazole...

Identificação e caracterização de moléculas envolvidas na interação de Paracoccidioides brasiliensis com o hospedeiro; Identification and characterization of involved molecules in the interaction of paracoccidioides brasiliensis with the host

DANTAS, Sabrina Fonseca Ingênito Moreira
Fonte: Universidade Federal de Goiás; BR; UFG; Doutorado em Medicina Tropical; Ciências da Saúde Publicador: Universidade Federal de Goiás; BR; UFG; Doutorado em Medicina Tropical; Ciências da Saúde
Tipo: Tese de Doutorado Formato: application/pdf
Português
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Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic L-amino acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the vivo-induced antigen technology (IVIAT) we identified immunogenic proteins expressed at high levels during infection. Quantitative real - time RT-PCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis.; Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM)...

CARACTERIZAÇÃO DA ISOCITRATO LIASE E METILISOCITRATO LIASE DO FUNGO PATOGÊNICO HUMANO Paracoccidioides brasiliensis; CHARACTERIZATION OF E METILISOCITRATO isocitrate Lias Lias The fungal human pathogen Paracoccidioides brasiliensis

TROIAN, Rogério Fraga
Fonte: Universidade Federal de Goiás; BR; UFG; Mestrado em Medicina Tropical; Medicina Publicador: Universidade Federal de Goiás; BR; UFG; Mestrado em Medicina Tropical; Medicina
Tipo: Dissertação Formato: application/pdf
Português
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Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. To cope with nutrient deprivation during the infection process, a number of pathogens employ the glyoxylate cycle (GC) to utilize fatty acids as carbon sources. The genes which constitute this pathway have been implicated in pathogenesis. An important aspect in the interaction between P.brasiliensis and your host is the ability to adhere to matrix extracelular components. In this work has shown that the isocitrate lyase of P. brasiliensis (PbICL) is located in the cell wall and also in the cytoplasm. PbICL recombinant and polyclonal antibody were able to inhibit the interaction of P. brasiliensis to epithelial cell cultures in vitro. Was also evaluated the ability of PbICL recombinant to connect the components of the extracellular matrix such as laminin, fibronectin, collagen type I and IV. These results suggest that PbICL is necessary to interaction between molecules of the extracellular matrix and P.brasiliensis...

Paracoccidioides brasiliensis: estudo micológico e imunoquímico de amostra isolada de tatu (Dasipus novencinctus); Paracoccidioides brasiliensis: a mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus)

Vidal, Monica Scarpelli Martinelli; Melo, Natalina Takahashi de; Garcia, Nilma Maciel; Del Negro, Gilda Maria Barbaro; Assis, Cezar Mendes de; Heins-Vaccari, Elizabeth Maria; Naiff, Roberto D.; Mendes, Rinaldo Poncio; Lacaz, Carlos da Silva
Fonte: Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo Publicador: Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; ; Formato: application/pdf
Publicado em 01/02/1995 Português
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Amostra de Paracoccidioides brasiliensis isolada de vísceras (baço e fígado) de um tatu (Dasipus novencinctus) foi estudada do ponto de vista micológico e imunoquímico. O tatu havia sido capturado em área da usina hidroelétrica de Tucuruí (Estado do Pará). Este já havia sido considerado como reservatório enzoótico do Paracoccidioides brasiliensis naquela região. Esta amostra, conservada na Micoteca do Instituto de Medicina Tropical de São Paulo sob o número 135, apresenta todas as características de Paracoccidioides brasiliensis, com elevado poder antigênico e baixa virulência para cobaios e ratos Wistar. A demonstração do exo-antígeno específico do P. brasiliensis, representado pela glicoproteína de peso molecular 43 kDa, foi evidente através das técnicas de Imunodifusão Dupla, Imunoeletroforese, SDS-PAGE e Imunoblotting.; A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135...

Paracoccidioides brasiliensis, nova amostra isolada de fezes de um pinguim (Pygoscelis adeliae); Paracoccidioides brasiliensis, a new strain isolated from a fecal matter of a penguin (Pygoscelis adeliae)

Garcia, Nilma Maciel; Del Negro, Gilda Maria Barbaro; Heins-Vaccari, Elisabeth Maria; Melo, Natalina Takahashi de; Assis, Cezar Mendes de; Lacaz, Carlos da Silva
Fonte: Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo Publicador: Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; ; Formato: application/pdf
Publicado em 01/06/1993 Português
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Os Autores apresentam os resultados obtidos com a amostra "pinguim" de Paracoccidioides, isolada por GEZUELE et al. (1989) na Antártica uruguaia. Das fezes de um desses animais, foi isolado um fungo considerado, recentemente, como nova espécie de Paracoccididoides - P. antarclicus. Os exames micológico e imunoquímico demonstraram tratar-se de Paracoccidioides brasiliensis, inclusive com a verificação da presença da glicoproteína 43 kDa pelos métodos de imunodifusão dupla, SDS-PAGE e imunoeletroforese. A possibilidade de se tratar de uma variedade do Paracoccididoides brasiliensis somente poderá ser confirmada através de outros estudos baseados na chamada taxonomía molecular, incluindo cariotipagem. Os Autores registram o significado epidemiológico deste achado, sugerindo uma revisão nos conhecimentos do nicho ecológico do P. brasiliensis.; The Authors show lhe results obtained through the study of a Paracoccidioides strain isolated from a penguin in the Uruguaian An-lartide by GEZUELE et al. (1989). From the fecal matter it was isolated a fungus which was recently considered as a new species of the genus Paracoccidioides - P. antarcticus. However, the mycological and immunochemical studies including the demonstration of the 43 kDa glycoprotein by immunodiffusion test...