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Item Avaliação do potencial in vitro e in vivo de tiossemicarbazida, tiossemicarbazida em nanopartícula de quitosana e tiossemicarbazida derivada do canfeno como antifúngico sobre Candida albicans(Universidade Federal de Goiás, 2016-05-06) Araújo, Deize Evangelista; Pereira, Maristela; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796262J0; Pereira, Maristela; Amaral, André Corrêa; Fernandes, Orionalda Fátima Lisboa; Ribeiro, Evandro leão; Borges, Clayton LuizCandidiasis is a serious health problem that affects a large number of people arround the world. The treatment of this disease is carried out with antifungals, which do not provide an effective cure, besides having resistance reports and high toxicity. In this context, it is necessary to study new antifungal compounds. In addition, the delivery form of the compounds in the living organism is also target of research, since the controlled release of compounds in biological systems can improve the mode of action of many drugs. Therefore, nanostructured systems, such as nanoparticles of chitosan, have been widely studied as controlled delivery of drugs. Then, this study aimed to evaluate the antifungal potential against Candida albicans of thiosemicarbazide (TSC), thiosemicarbazide-camphene (TSC-C) and chitosan nanoparticles incorporated with thiosemicarbazide (nanoTSC). In in vitro assays, the compounds were effective to inhibit nearly 100% of the fungus in concentrations of 1,37 mM to TSC, 0,02 mM to TSC-C and 0,27 mM to nanoTSC, and the most effective compound was TSC-C. NanoTSC and empty nanoparticles were also evaluated as to cell toxicity, and the concentration able to inhibit the fungus showed no cytotoxic activity. To confirm the values observed in in vitro assays, experiments were conducted using an animal model for vulvovaginal candidiasis. The results showed that TSC-C was more efficient in inhibiting C. albicans compared with TSC and nanoTSC, but was less effective than miconazole. However, histopathological analysis showed that the groups treated with the compounds under study, showed less intensity of damage to the vaginal epithelium and inflammatory infiltrates, compared with the positive control and the group treated with miconazole. Thus, the results suggest that TSC, nanoTSC and TSC-C are promising compounds in the treatment of vulvovaginal candidiasis.Item Efeitos de argentilactona sobre o perfil transcricional, parede celular e estresse oxidativo de Paracoccidioides spp(Universidade Federal de Goiás, 2014-06-02) Araújo, Felipe Souto; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Schrank, Augusto; Cravo, Pedro Vitor Lemos; Soares, Célia Maria de Almeida; Rocha, Juliana ParenteParacoccidioides spp, dimorphic pathogenic fungi, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problem. The drugs used against pathogenic fungi have various side effects and have limited efficacy, therefore there is an inevitable and urgent medical needing for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides spp exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 down-regulated. The cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, among them, heat shock protein 90 (hsp 90), cytochrome c peroxidase (ccp), hemoglobin ligant RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all those genes. Enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA, Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in Paracoccidioides spp responding to argentilacone. Paracoccidioides spp cell wall was also evaluated. The results showed that argentilactone causes a decrease in the levels of polymers of the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy.Item Identificação proteômica de alterações metabólicas em Paracoccidioides brasiliensis induzidas por derivado de chalcona(Universidade Federal de Goiás, 2021-09-30) Carvalho Júnior, Marcos Antonio Batista de; Silva, Kleber Santiago Freitas e; http://lattes.cnpq.br/3813868830071259; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Curcio, Juliana Santana de; Soares, Célia Maria de AlmeidaParacoccidioidomycosis is one of the most important systemic mycoses in Latin America. The infection occurs through the inhalation of fungal propagules belonging to Paracoccidioides genus that are present in soils, being largely associated with the contamination of rural workers, who are constantly exposed to this potentially contaminated material. Even more than a century after its discovery, the treatment of this infection with the currently available antifungal arsenal still represents a challenge, due to the long treatment time required, as well as the high toxicity of the drugs used. These questions highlight the need for research to develop and characterize new compounds with the potential to inhibit the growth of these microorganisms. In this sense, a class of molecules called chalcones has shown great versatility for demonstrating broad biological properties, including antifungal activity among them. Through a virtual screening methodology, a chalcone derivative named compound 3 was identified by our group as a promising inhibitor of Paracoccidioides spp. Thus, in order to understand the mode of action of compound 3, we applied a proteomic approach to identify the induced and repressed proteins of P. brasiliensis in the presence of compound 3. In addition, validation assays were performed on the results found. The analysis indicated that the compound can cause an imbalance in the fungal energy homeostasis by reducing the activity of the glycolytic pathway, beta-oxidation and citric acid cycle. In vitro validations also demonstrated that reactive oxygen species accumulate within the cells during exposure to compound 3, which can destabilize cellular components such as the plasma membrane. The molecular docking assay between compound 3 and the enzyme dihydropteroate synthase, which had its expression induced after the treatment, suggest that compound 3 may act as an inhibitor of this protein, impairing the folate biosynthesis that participates as a cofactor in synthesis of nucleotides and some amino acids. Therefore, these data support the efficiency of compound 3 in producing imbalances in key pathways for the P. brasiliensis' metabolic maintenance, contributing to its antifungal role.Item Clonagem e expressão heteróloga, modelagem e interações intermoleculares da enolpiruvilchiquimato 3-fosfato sintase de Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2017-08-07) Costa, Wanderson Lucas da; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Lacerda, Elisângela de Paula Silveira; Neves, Bruno JuniorParacoccidioides spp. are thermodymorphic fungi that when inhaled by humans, these conidia find a favorable environment, changing to the yeast phase and becoming pathogenic causing paracoccidioidomycosis (PCM), one of the most prevalent systemic mycoses in Brazil. Some antifungals are used in the treatment of PCM. Treatment depends on the patient's progression and tolerability of each drug, but their treatment may be for long periods and cause various side effects in the patient. The chiquimate pathway is coordinated by 7 enzymes that perform consecutive steps to convert erythrose-4-phosphate and phosphoenol pyruvate (PEP) into chorismate. In microorganisms, this pathway is involved in the production of the amino acids phenylalanine, tyrosine and tryptophan; These amino acids are essential to the maintenance of these organisms. In this work, pGEX4T3 vector cloning and heterologous expression of Pb18 EPSP synthase belonging to the chiquimate pathway were performed. This protein was expressed in E. coli (DE3) strain and purified. Antibodies were produced for expression analysis of the protein in Western blot. The modeling of EPSP synthase was performed aiming to identify the amino acids involved in the active site. The pull down-GST assay with soluble Pb18 proteins allowed the identification of 40 proteins that interact with EPSP synthase. These proteins belong to different functional categories, which are involved with the availability of phosphoenol pyruvate, the substrate necessary for the functioning of the chiquimate pathway.Item Estudo dos óleos naturais frente à Paracoccidioides spp.: atividade biológica de óleos de copaíba in natura e imobilizado em micelas(Universidade Federal de Goiás, 2017-08-10) Miguel, Meire Ane Costa; Pereira, Maristela; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4796262J0; Pereira, Maristela; Oliveira, Cecília Maria Alves de; Borges, Clayton LuizNatural oils add therapeutic values and are commonly used as herbal medicines. Copaiba oil, sucupira and andiroba, the janauba latex, have been extracted from native Brazilian trees and have been used against various pathogens. In this work we used natural oils, such caryophylleneas janauba compound against latex and Paracoccidioides spp.. This genus corresponds to filamentous fungi that cause systemic mycosis Paracoccidioidomycosis (PCM) with records in Latin America, with the highest number of cases they are in Brazil. From these compounds it was determined on the cytotoxicity in Balb/c 3T3 cells and for the oils that had inhibited the growth fungal interaction with the antifungal agents amphotericin B, sulfamethoxazole-trimethoprim (SXT) and itraconazole were evaluated. In addition, the copaiba oil encapsulated in Pluronic® F-127 micelle (oil-micelle) is a promising compound, not yet elucidated in Paracoccidioides spp. was also tested. The minimal inhibitory concentration (MIC) and the cytotoxicity of the compounds were evidenced by the microdilution technique. The minimum fungicidal concentration (MFC) was determined by the growth in plaque by the checkerboard technique was used to assay the interaction of the oils with the antifungal agents. Copaiba, oil-micelle and sucupira oil exhibited fungicidal activity with MIC values of 62.5 μg /mL, 125 μg/mL and 250 μg/mL, respectively. Andiroba oil, janauba-latex β-caryophyllene and did not inhibit the growth of Paracoccidioides spp. at the concentrations tested. Copaiba, sucupira and oil-micelle showed no significant hemolytic activity and cytotoxicity in the Balb/c 3T3 cells at MIC concentrations. The combination of amphotericin B was synergistic with copaiba oil and oil-micelle, having a fractional inhibitory concentration of 0.75 μg/mL. Copaiba oil and oil-micelle have been shown to be promising for the treatment of PCM, as they inhibit fungal growth and have no cytotoxic or hemolytic effects. In addition, copaiba oil and oil-micelle in combination with amphotericin B demonstrated additive activity, whereas sucupira oil had no synergistic activity.Item Clonagem, expressão heteróloga e interações intermoleculares da proteína aspartato semialdeído desidrogenase de Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2017-12-12) Nascimento, Thaylla Horbylon; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Rocha, Juliana Alves Parente; Bailão, Mirelle Garcia SilvaParacoccidioides comprises the genus of fungi causing paracoccidioidomycosis. The research of new treatments, especially those based on inhibition of metabolic pathways important for microorganisms has gained prominence and the aspartate pathway, necessary for the biosynthesis of threonine, isoleucine and methionine in microorganisms, is not found in humans. Catalyzing the second step of this pathway, we found aspartate semialdehyde dehydrogenase (ASADH), which has not yet been characterized in Paracoccidioides spp. and has no substituents in its catalytic function in the aspartate pathway. Thus, it is of interest the determination of its biological properties experimentally that their biological properties be determined experimentally. ASADH from Pb18 was cloned into vector pGEX-4T3, expressed in E. coli Stellar cells and purified on a glutathione-sepharose column. Then, antibodies were produced and used in Western blot assay to confirm protein expression. The pull-down-GST assay was performed and allowed the identification of complexes of interactions between ASADH and soluble proteins present in total protein extract of the yeast form of Pb18. Interactions with proteins of several functional categories, among them those related to the metabolism of threonine, isoleucine and methionine, were found, reinforcing the performance of ASADH in the amino acid biosynthesis of Pb18, as well as proteins related to substrate supply to the aspartate pathway and proteins that use metabolites of this pathway. Interactions with proteins involved in other metabolic pathways were also observed, as well as unprecedented interactions, suggesting the importance of ASADH in the functional processes of microorganisms.Item Clonagem, expressão heteróloga, modelagem e interações intermoleculares da proteína corismato sintase de Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2017-03-29) Santana, Andrea Leite Camargo; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Baeza, Lilian Cristiane; Parente, Juliana AlvesParacoccidioides fungi are the causative agents of paracoccidioidomycosis, an endemic disease in Latin America with great socioeconomic importance. Inhalation of spores, the infectious particles of the fungus, is a common way to get the infection. Its treatment is slow and generates long-term side effects making it necessary to study new metabolic pathways that can be potential targets for antifungal development. In this context, stands out the chiquimate pathway in Paracoccidioides spp., related to the production of chorismate and production of aromatic amino acids, and the chorismate synthase involved in the last stage of this pathway. The chorismate synthase from Pb18 was cloned into pGEX-4T3 vector, expressed in pLysS cells and purified on a glutathione-sepharose column. Antibodies were produced from the immunization of mice with the recombinant protein obtained and used in Western blot assay to confirm protein expression. For characterization of the enzyme, its modeling was carried out. Pull-down-GST assay was performed to identify interactions between the chorismate synthase and soluble proteins present in total protein extract of Pb18. The interactions found included proteins of different functional categories related to protein synthesis, related with metabolism of common intermediates such as folate and quinolinate or related with the availability of ATP, phosphoenolpyruvate and erythrose-4-phosphate required in the chiquimate pathway. Unexpected interactions with proteins of the cell cycle and also with regulating proteins of the cell division process were observed.Item Malato sintase de Paracoccidioides brasiliensis é uma proteína ligada à superfície que se comporta como uma anchorless adesina(Universidade Federal de Goiás, 2009-05-11) Silva Neto, Benedito Rodrigues da; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Soares, Célia Maria de Almeida; Lenzi, Henrique LeonelThe 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.