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Item Alterações genômicas e mutagênicas em duas espécies de anfíbios anuros(Universidade Federal de Goiás, 2015-02-20) Gonçalves, Macks Wendhell; Silva, Daniela de Melo e; http://lattes.cnpq.br/9895211901348365Amphibian populations have declined in many areas of the world. These declines seem to have worsened over the past 25 years and amphibians are now more threatened than other taxa, such as mammals or birds. Numerous factors contribute to this decrease; however, environmental pollution has recently been cited as a major factor. These vertebrates are important in natural control of several pests in natural and agricultural areas, where they are constantly exposed to large amounts of pesticides. Genotoxicity studies with amphibians are important to detect indicator species of environmental change. Amphibians are particularly sensitive to these variations due to their behavioral and physiological characteristics, such as, permeable skin (cutaneous respiration), low mobility and life cycle with simultaneous dependence of the aquatic and terrestrial environment. In this context, these animals could be used as bioindicators of the environmental quality in genotoxicity and mutagenicity studies. The aim of this study was to analyze the genomic damage in tadpoles of Dendropsophus minutus and Physalaemus cuvieri sampled in perturbed and natural areas, by the comet assay and micronucleus test. We found that the tadpoles of D. minutus and P. cuvieri collected in perturbed areas had the highest extensions of DNA damage compared to the tadpoles of natural areas. Tadpoles sampled in soybean crop areas had higher DNA damage, followed by those sampled in the cornfields. In contrast, the frequency of DNA damage in tadpoles of natural areas were significantly lower in relation to DNA damage suffered by tadpoles of agricultural areas. Thus, we can conclude that tadpoles of D. minutus and P. cuvieri sampled in this study can be considered excellent sentinel organisms, as they were sensitive to environmental changes. In addition, we can highlight the advantages of using the comet assay in environmental genotoxicity studies, such as its speed, high sensitivity in detecting DNA damage in the genome of amphibians, for a relatively low cost. However, it is necessary to invest in the standardization of methodologies for field studies, thus the results could be compared between different laboratories, and make environmental genotoxicity studies, involving aquatic organisms, more informative and applicable as a practical laboratory routine.Item Alterações metabólicas resultantes da fosforilação de Isocitrato Liase em Paracoccidioides lutzii(Universidade Federal de Goiás, 2015-04-08) Silva, Karla Christina Sousa; Tauhata, Sinji Borges Ferreira; http://lattes.cnpq.br/7082495215620618; Tauhata, Sinji Borges Ferreira; Bailão, Alexandre de Melo; Brito, Wesley de Almeida; Souza, Guilherme Rocha Lino de; Faria, Fabrícia Paula deParacoccidioidomycosis (PCM) is a systemic mycosis endemic from Latin America caused by the species encompassed in Paracoccidioides genus. This mycosis affects mainly economically active men, who live in rural areas. PCM may cause severe wounds to individuals and is highly incapacitating. This disease is considered a neglected disease by World Health Organization (WHO). When in a hostile environment, like deprivation of carbon sources (6C), fungi tend to use alternative strategies to assist the energetic metabolism. One strategy is using the glyoxylate cycle, which consists in 5 steps, 3 of them being common to the tricarboxylic acid cycle and the other 2 steps are catalyzed by exclusive enzymes from the glyoxylate cycle: isocitrate lyase (ICL) and malate synthase. There are reports in the literature suggesting that phosphorylation of ICL ofis a possible mechanism of enzyme regulation. Phosphorylation is a post-translational ubiquitous ubiquitous and known to modulate the activity / action of many molecules of diverse organisms. In this work, through the use of molecular interactions assays (pull down) using ICL of Paracoccidioides lutzii in its phosphorylated state and non-phosphorylated We found evidence, which suggest that the purine biosynthesis pathway is being used as an alternative way to produce energy to the organism and a high involvement of ICL with amino acids biosynthesis on its non-phosphorylated state. Apparently, phosphorylation works marking ICL to become degraded. This observation may open a new research field through the elaboration of new drugs for PCM treatment using phosphatase inhibitors.Item Análise da expressão e das interações da subunidade catalítica da PKA do fungo patogênico Paracoccidioides ssp.(Universidade Federal de Goiás, 2016-03-10) Teixeira, Mirian Vieira; Furlaneto, Silvia M. Salem Izacc; http://lattes.cnpq.br/5542866107144940; Furlaneto, Silvia M. Salem Izacc; Parente, Juliana Alves; Baeza, Lilian CristianeThe Paracoccidioides genus comprises a complex of pathogenic fungi that are the etiologic agents of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The infection begins after inhalation of fungal propagules, which reach the epithelium of the alveoli where the transition from the mycelial to the pathogenic yeast form. Host elevated temperature triggers the morphological switch, which is necessary for fungal pathogenicity. The cAMP/protein kinase A (PKA) signaling pathway has been shown to be important in controlling morphological changes and the pathogenicity of several pathogenic fungi. Evidence also highlights the importance of the cAMP/PKA pathway in the morphological transition of Paracoccidioides. PKA is the major effector of this signaling pathway. The protein is an inactive tetramer composed of regulatory subunit, encoded by the BCY1 gene; and catalytic subunit, encoded by the TPK2 gene. Upon binding of cAMP to the regulatory subunits, the catalytic subunits dissociate and become active. Activated PKA subsequently phosphorylates protein kinases, transcription factors, and other substrates to control several biological processes. In this study, we evaluated the expression and interactions of Tpk2 protein Paracoccididioides spp. The Tpk2 is present in mycelium decreased during the initial stages of transition phases, and increases again at the end of differentiation, with maximal levels in yeast. We analyzed the interactions of recombinant Tpk2p with Paracoccidioides proteins using pull-down assays followed by MS analysis. Two interacting proteins were identified: the heat shock protein 90 (Hsp90) and a conserved hypothetical protein with a MFS domain. Hsp90 is involved in the regulation of morphogenesis, development and virulence in several thermal dimorphic fungi. These data are important for understanding the mechanisms that trigger the transition phases in Paracoccidioides.Item Análise da genotoxicidade e da frequência de toxoplasmose em indivíduos diagnosticados com esquizofrenia(Universidade Federal de Goiás, 2018-03-09) Pedroso, Thays Millena Alves; Avelar, Juliana Boaventura; http://lattes.cnpq.br/2170858365257711; Silva, Daniela de Melo e; http://lattes.cnpq.br/9895211901348365; Silva, Daniela de Melo e; Parise, Michelle Rocha; Nunes, Hugo FreireSchizophrenia is a complex mental disorder in which the individual has difficulty distinguishing between what is real and what is imaginary. It is a multifactorial disease in which factors such as environment, genetic predisposition, toxoplasmosis and biochemical changes in the brain are closely related to the severity of the disease, both positive and negative symptoms. The present study aimed to evaluate the prevalence of toxoplasmosis and genotoxicity of Toxoplasma gondii in individuals diagnosed with schizophrenia. The ELISA immunoenzymatic assay was used to evaluate the seropositivity to T. gondii and the comet assay to detect genotoxic damage. In the group, 69 individuals and 45 individuals who did not have schizophrenia or any mental disorder were analyzed. A statistically significant difference (p <0.05) was observed when the mean age and tobacco use frequency were analyzed, the individuals diagnosed with schizophrenia showed higher seropositivity (p <0.01) when compared to the control group. Analyzing the mean ± SD (standard deviation), they showed greater genotoxic damage in the case group in relation to the control group. In addition, it was observed that the association between the genotoxic damage of the case group with sex, IgG, disease, smoking and alcoholic beverages presented values of p> 0.05. Thus, it can be concluded that there was no significant difference when comparing the genders to the prevalence of toxoplasmosis in the individuals diagnosed with schizophrenia. In the case group it was possible to verify greater genotoxic damage in the DNA than in the control group.Item Análise da herança do DNA do Trypanosoma cruzi em parentais e progênies de chagásicos de famílias de São Felipe – Bahia(Universidade Federal de Goiás, 2019-02-26) Santos, Eduardo José dos; Cardoso, Clever Gomes; http://lattes.cnpq.br/9545455455623006; Lacerda, Elisângela de Paula Silveira; http://lattes.cnpq.br/9390789693192751; Teixeira, Antônio Raimundo Lima Cruz; Zapata, Marco Túlio Antônio GarciaThe Trypanosoma cruzi, etiologic agent of Chagas disease is a flagellate protozoan transmitted by insect bugs (Reduvidae:Triatomine) to humans and wild mammals. The T. cruzi contains DNA in the nucleus (nDNA) and in the mitochondrion (kDNA). The kinetoplastid protozoan comprises maxicircles and and minicircles kDNA network sequences that integrate in the host’s cell genome. Insertional integrations of kDNA sequences bear high affinity to retrotransposable LINE-1 randomly distributed in the genome of somatic and germ line cells, and, thus generate an increasing diversity due to recombination and reshuffling. However, the profiles of kDNA integrations into the human genome in different Brazilian Ecosystems are unknown. In this regard, the ratio of transfer of T. cruzi minicircles sequences into the human genome was south. The study consisted of analyses of 36 members of two families from São Felipe-Bahia where Chagas disease is endemic. The study showed that 22% (8/36) families’ members had the diagnoses of the disease by immunofluorescence (IF) and enzyme-linked immunosorbant assay (ELISA). Yet, the PCR assays showed 61% (22/36) of families’ members had kDNA and nDNA assays positive for the T. cruzi infection. In order to detect the kDNA integration sites in the human genome, a tpTAIL-PCR (Targeted Primer Thermal Assymetric Interlaced-PCR), which consisted in combination of primers derived from T. cruzi minicircle conserved sequence with human LINE-1 sequence derived primers, was employed. The amplicon sequences obtained were subjected to homology analyses in Blast software. The hybrid kDNA-human DNA sequences revealed lateral and vertical transfer of T. cruzi minicircles into the genome of Chagas parents and progeny. Unravel of dynamics of mutations contributes to define variable profiles of kDNA minicircle sequence insertions into the human genome at different Ecosystems.Item Análise de celulases e xilanases por fungo isolado a partir do bioma cerrado(Universidade Federal de Goiás, 2013-05-06) Pereira, Douglas Endrigo Perez; Faria, Fabrícia Paula de; Bataus, Luiz Artur Mendes; http://lattes.cnpq.br/5637230378599476; Bataus, Luiz Artur Mendes; Faria, Fabrícia Paula de; Jesuíno, Rosália Santos Amorim; Fonseca, Marcio José PoçasLignocellulosic biomass is constituted by cellulose, hemicellulose and lignin in the plant cell walls. The conversion of lignocellulose to fermentable sugars mainly depends on the action of a complex of enzymes that hydrolyze cellulose and hemicellulose. This study was to analyze the production of cellulases and xylanases by fungi isolated from soil and decaying material in Cerrado. Initially, the fungi were analyzed for cellulase production capacity among the fungal cellulase-producing fungus capable of producing the highest levels of cellulase was selected for the experiments of xylanase and cellulase production by submerged fermentation and biochemical analysis of the enzyme secreted. Among the isolated fungi, fungus called IFBMD01 was that higher activity of cellulase in the supernatant when cultivated with wheat bran as carbon source. This was identified as Aspergillus cf niger. The enzymes studied with their respective results were: FPase with better production time of 7 days, showing activity of 0.10 U / mL, optimum pH 5.5, optimum temperature of 60 ° C; CMCase with better time production 6 days, showing activity of 8.19 U / mL, pH optimum of 5, optimum temperature of 70 º C; Avicelase with better production time of 9 days, showing activity of 0.01 U / mL, pH optimum of 5.5 and optimum temperature of 60 ° C; xylanase, with better production time 3 days, showing activity of 35.5 U / mL, and the optimum pH of 5 and the optimum temperature of 50 ° C, the β-Glycosidase, with higher production on the sixth day showing activity of 0.98 U / mL, with the optimum pH 5 and optimum temperature of 60 ° C. The activity of β-glucosidase showing in the culture supernatant was influenced by the ions Mn2 +, NH4, K +, Mg2 +, Ca2 +, Ba2 +, Al3 +, Na +, and Co2+, as well as EDTA, glucose, xylose and cellobiose. In the culture supernatant of the fungus A. niger IFBMD01 grown on FT for 3 and 6 days protein bands were visualized with activity against xylan and CMC molecular weight be 30 to be 66 kDa.Item Análise de moléculas envolvidas no metabolismo de nitrogênio no fungo patogênico humano Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2017-02-22) Silva, Lana OHara Souza; Paccez, Juliano Domiraci; http://lattes.cnpq.br/2350706025601982; Borges, Clayton Luiz; http://lattes.cnpq.br/8867708267053410; Borges, Clayton Luiz; Oliveira, Rosely Maria Zancopé; Reis, Angela Adamski da SilvaThe Paracoccidioides genus is composed of thermodimorphic fungus that causes paracoccidioidomycosis (PCM), an endemic human systemic mycosis in Latin America. These organisms grow as mycelium in temperatures below 28 °C and as yeast form in temperatures above 37 °C. Nitrogen is an important element in this microorganism’s nutrition that participates in the synthesis of proteins, nucleic acids and others biomolecules. In this regard, nitrogen uptake and metabolism are essential to growth and fungal establishment. When nitrogen levels and sources such as glutamine and ammonia concentration are limited, pathogenic fungus use a regulation system called Nitrogen Catabolic Repression that induces the expression of genes encoding permeases and enzymes required for the catabolism of secondary nitrogen sources, such as formamidase, gamma-glutamiltranspeptidase and urease. Gamma-glutamiltranspeptidase is an enzyme that catalyzes the first reaction of glutationa degradation and it has been the target of several studies about nitrogen starvation in various fungi. It has been observed that the expression of the gene encoding this enzyme was induced in limiting conditions of nitrogen and was repressed when the availability of nitrogen was high. Urease is an enzyme that catalyzes the degradation of urea in ammonia and carbonic acid. This enzyme is already known as a virulence factor in fungi such as Cryptococcus. neoformans, and also has been the target of studies about nitrogen starvation. In this study we expressed gamma-GT and urease proteins from Paracoccidioides brasiliensis, isolate Pb18, in Escherichia coli. The gene coding for Ggt and Ure were cloned in pET32a expression vector, and used for E. coli pLysS transformation. The recombinant proteins produced were shown to be catalytically active. Together, data obtained in this work could add knowledge about the role of gamma-GT and urease and can be used as a foundation for complementary experiments regarding nitrogen metabolism regulation, as well as in Paracoccidioides spp pathogenesis.Item Análise de polimorfismos em genes de detoxificação e reparo em indivíduos de municípios goianos(Universidade Federal de Goiás, 2017-03-10) Alves, Alessandro Arruda; Silva, Daniela de Melo e; http://lattes.cnpq.br/9895211901348365; Soares, Thannya Nascimento; Rodrigues, Flavia MeloIn Brazilian agriculture, the State of Goiás is one of the largest producers of corn, beans, cotton, sorghum, sugarcane and soybeans, with 75% of its exports composed of products related to agriculture and minerals. Thus, a large part of the economy of the State of Goiás is focused on rural activities, justifying the intense use of pesticides in the region. These pesticides will be metabolized in the liver in two main phases, the first one of bioactivation converting the original substances into more polar metabolites, through oxidation reduction reactions, hydrogenation and dehalogenation, most of the reactions are made by the cytochrome P450 (CYP) superfamily And the second phase in which conjugation reactions occur, in which metabolites are converted into non-toxic products. Agrochemicals are also known to cause various human health damage, such as cancer, cholinergic syndrome, Parkinson's disease and Alzheimer's as well as cytogenetic changes such as DNA damage and chromosome translocations. Thus, polymorphisms in enzymes acting in the path of detoxification and DNA repair may be important biomarkers of diseases in individuals exposed to pesticides. The present study sequenced 3 regions of the following genes: CYP2E1 (promoter region), CYP1A1 (exon 7) and OGG1 (intron region) of 260 individuals from Goiânia and Aparecida de Goiânia, GO. 20 ml of blood was collected, DNA was extracted using commercial kits and PCR was performed. Subsequently, the amplicons were sequenced by the automated Sanger method and compared to the 1000 Genome project. The population of this study was composed of 159 men and 101 women, mean age of 38 years. A total of 5 SNP's were found in the Goias population, being rs2031922 (change of C> T and frequency of the T allele of 0.05) of the CYP2E1 gene, rs1048943 (change of A> G and frequency of the G allele of 0.1 ) And rs1799814 (change of C> A and frequency of allele A of 0.07) of the CYP1A1 gene and rs1052133 (change of C> G and frequency of allele G of 0.2) and rs293795 (change of A> G and frequency Of the 0.18 T allele) of the OGG1 gene. All loci did not deviate from the Hardy-Weinberg equilibrium, nor was a statistically significant difference between age and genotype composition. The rs1048943 and rs1799814 loci of the CYP1A1 gene showed a moderate disequilibrium linkage and the rs1052133 and rs293795 loci of the OGG1 gene showed a strong disequilibrium linkage. The haplotype inference revealed a frequency of the haplotypes of loci rs1048943 and rs1799814 of A / C = 0.8, G / A = 0.12, A / A = 0.07 of the CYP1A1 gene. The haplotypes of the loci rs293795 and rs1052133 had A / G = 0.07, A / C = 0.8, G / C = 0.13. Due to the important participation in detoxification or DNA repair and relationship with pesticides, SNPs rs2031920 of the CYP2E1 gene, rs1048943 of CYP1A1 and rs1052133 of the OGG1 gene are potential biomarkers of susceptibility to disease development in individuals who have contact with these xenobioticsItem Análise de RNAs longos não codificantes do genoma de Arabidopsis thaliana (L.) Heynh(Universidade Federal de Goiás, 2017-03-07) Araújo, Vanessa Cristina da Silva; Novaes, Evandro; http://lattes.cnpq.br/0568272239145336; Novaes, Evandro; Vianello, Rosana Pereira; Coelho, Alexandre Siqueira GuedesLarge-scale sequencing of transcripts via RNA-Seq has been changing paradigms by demonstrating that transcription is prevalent throughout the eukaryotic genome. In these organisms, the vast majority of transcripts are non-coding (ncRNA). One type of RNA that has aroused great interest, given its prevalence, is long non-coding RNAs (lncRNAs), which are ncRNA with more than 200 nucleotides. However, little is known about the role and prevalence of these lncRNAs in plant genomes, even in model species such as Arabidopsis thaliana (L.) Heynh. The objective of this work was to identify lncRNAs in the Arabidopsis genome and to characterize their size, structure and nucleotide diversity. The sequences were obtained from previous work that sequenced total RNA from A. thaliana, grown under different light regimes, using Illumina Hiseq 2000 platform. These sequences were mapped into the reference genome with TopHat and assembled with Cufflinks. The assembled transcripts were compared with the genome annotation with Cuffcompare, to identify non-annotated transcripts. A total of 4,305 long putative RNAs were obtained, with 314 (7%) sense in relation to coding transcripts (mRNAs), 392 (9%) intergenic, 2,216 intronic (52%) and 1,383 (32%) antisense mRNAs. The lncRNAs obtained were filtered to eliminate those with coding potential, as well as those related to rRNA, tRNA and miRNA synthesis. A total of 3,710 high-confidence lncRNAs (HC-lncRNA) were obtained, of which 58.6% were not previously annotated. These HC-lncRNA emcompass a low proportion (~ 1%) lncRNAs in the genome of Arabidopsis thaliana. A functional enrichment analysis of Gene Ontology (GO) categories demonstrated that among genes containing lncRNAs there is a high proportion of categories linked to the localization and transport of proteins within the cell, as well as to nucleic acid binding. A gene expression analyses identified only 22 differentially expressed lncRNAs under the different light conditions in which samples were exposed. Using the SNP data from the 1001 genomes project, identified high nucleotide diversity within lncRNAs regions, indicating low conservation of the primary structure of these transcripts. The nucleotide diversity in regions of long noncoding RNAs is lower than in coding regions, but less than a diversity observed in neutral regions such as pseudogenes.Item Análise do secretoma de duas espécies filogenéticas de Paracoccidioides(Universidade Federal de Goiás, 2015-03-10) Oliveira, Amanda Rodrigues de; Borges, Clayton Luiz; http://lattes.cnpq.br/8867708267053410; Borges, Clayton Luiz; Weber, Simone Schneider; Lima, Patrícia de Sousa; Baeza, Lilian Cristiane; Casaletti, LucianaParacoccidioides is a termodimorphic fungus that causes paracoccidioidomycosis (PCM), the most frequent systemic mycosis that affects mainly the rural population in Latin America. The fungus grows as yeast when grown at 35-37° C and as mycelium at temperatures below 28° C. The establishment and severity of the disease depends on factors inherent to the fungus and the host. The diversity presented among isolates of the same genus has been explored between the microorganisms and attempt to clarify differences possibly related to virulence existing between isolates that cause the same disease. The secretion of proteins in a cell is a highly dynamic mechanism and is directly involved in the first pathogen contact with the host cells, enabling their survival, multiplication and dissemination. In order to try to elucidate this diversity the proteomic profile of secretome of two isolates of Paracoccidioides, PbEpm83 and Pb01, that in our experimental conditions of infection showed different behaviors was characterized. The use of Ultra Performance liquid chromatography coupled to mass spectrometry (UPLC-MSE) allowed the identification of 92 proteins / isoforms among the strains. Of the 92 proteins identified, 36 were preferentially secreted in PbEpm83 and 35 preferentially secreted in Pb01. Among the identified we can highlight proteins related to various biological processes: adhesion to the ECM, proteins related to thermal and oxidative stress, cell rescue, defense and virulence, proteins with immunogenic capacity, proteins related to defense against antifungal, heat shock proteins, among others. Our analysis showed that most of the proteins identified using non-conventional secretory pathways. Among the identified proteins there are already related proteins as virulence factors, such as, ATP synthase, mitochondrial Peroxiredoxin PRX 1, fructose bisphosphate aldolase, Didpeptidil peptidase, Thioredoxin, TCTP, Hsp70 and Hsp88. Our results highlight the importance of secreted proteins in the establishment of infection and that these species, within the same genus, maintain differences in the levels of protein expression that may reflect their behavior in the success of the infection.Item Análise dos polimorfismos RS1800871 e RS1800872 do gene da interleucina 10 em indivíduos diagnosticados com esquizofrenia(Universidade Federal de Goiás, 2019-02-28) Lopes, Alice Tâmara de Carvalho; Avelar, Juliana Boaventura; http://lattes.cnpq.br/2170858365257711; Silva, Daniela de Melo e; http://lattes.cnpq.br/9895211901348365; Silva, Daniela de Melo e; Franco, Fernanda Craveiro; Nunes, Hugo FreireSchizophrenia is an neurocognitive disorder that affects about 1% of the population. Evidence shows that increases in the risks of the onset of schizophrenia (EQZ) and other neuropsychiatric disorders are due to the imbalance of pro and antiinflammatory agents associated with environmental and genetic components. Cytokines are the major regulators of inflammatory immune reactions and brain development and emerge as a common pathway for elucidation of the disease. This study aims to evaluate whether there is an association between the rs1800871 and rs1800872 polymorphisms of IL-10 gene and the susceptibility to the development of schizophrenia by detection of polymorphisms (SNPs) by Sanger sequencing technique in individuals giagnosed with EQZ in Goiânia, Goiás. Samples were collected from 156 individuals, 95 individuals diagnosed with EQZ (case group) and 61 healthy individuals with no history of psychosis (control group). There isn’t association between rs1800871 (p=0.97) and rs1800872 (p=0.83) SNPs and the susceptibility to the development of EQZ. There was prevalence of smoking, alcohol use and chemical dependency (p<0.001). The haplotype T/A had a higher frequency in the case group (0,2).Item Análise proteômica comparativa do processo de diferenciação celular do fungo patogênico Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2014-07-31) Vaz, Alessandro Fernandes; Soares, Célia Maria de Almeida; Soares, Célia Maria de Almeida; Coelho, Alexandre Siqueira Guedes; Lima, Patrícia de SousaParacoccidioides spp. is the etiological agent of paracoccidioidomycosis, the most important endemic systemic mycosis in Latin America. Paracoccidioides spp. is a dimorphic fungus; mycelia is found in soil at temperatures below 25ºC, while in host tissues, and temperature around 36-37ºC the fungus takes the yeast form. Infection begins with the inhalation of conidia or mycelia propagules that upon reaching the host lungs differentiate into yeast, establishing disease. The morphological transition from mycelia-to-yeast is involved in the virulence of this pathogen and this aspect of morphogenesis deserves special attention due to its relevance to the fungal virulence. In the present study, we employed proteomic strategies using liquid chromatography coupled to mass spectrometry to evaluate the differential proteomic profile of cells ongoing transition from mycelia-to-yeast after 22 h of temperature shift from 22ºC to 36ºC (P. brasiliensis Pb-18 phylogenetic lineage S1). Nine hundred and ninety-one proteins were identified (350 in the mycelia, 288 in the transition and 353 in the yeast), and 251 were differentially regulated. The analysis of the functional categories to which those proteins belong provided us a comprehension on the metabolic reprogramming that occurs during the cell differentiation process, providing putative virulence factors.Item Análise proteômica comparativa entre Paracoccidioides brasiliensis e Paracoccidioides americana(Universidade Federal de Goiás, 2019-12-19) Portis, Igor Godinho; Ricart, Carlos André Ornelas; http://lattes.cnpq.br/1271167329803156; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Soares, Célia Maria de Almeida; Lima, Patrícia de Sousa; Curcio, Juliana Santana De; Pereira, Maristela; Rocha, Juliana Alves ParenteThe genus Paracoccidioides comprises termo dimorphic fungi that cause paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. These fungi can grow in yeast at 36 ° C and mycelium at temperatures below 28 ° C. Studies have shown that the fungus Paracoccidioides has molecular differences that allow the classification of species. Initially those fungi were classified in a single species, Paracoccidioides brasiliensis, but with the advancement of some molecular and genomic biology techniques, Paracoccidioides is currently classified into five species, P. lutzii, P. brasiliensis, P. americana, P. restrepiensis and P. venezuelensis. However, different species may have different proteomes and cellular responses. In this way in our study, we performed proteomic analyzes of two species of Paracoccidioides, P. americana and P. brasiliensis. iTRAQ labelling and mass spectrometery allowed the identification of 387 statistically significant proteins; two hundreds and two in P. brasiliensis and 111 proteins in P. americana. The identified proteins have different functions, such as amino acid metabolism, energy, cell defense, among others. The results showed differences in protein responses between P. americana and P. brasiliensis in proteins related to cell defense, mitochondria and energy metabolism, suggesting that these fungi may have different metabolism and pathways for growth, profliferation and virulence.Item Análise proteômica da fase leveduriforme do fungo patogênico Paracoccidioides sp durante a privação de nitrogênio(Universidade Federal de Goiás, 2014-03-13) Cruz-Leite, Vanessa Rafaela Milhomem; Borges, Clayton Luiz; http://lattes.cnpq.br/8867708267053410; Borges, Clayton Luiz; Bailão, Alexandre Melo; Brito, Wesley de Almeida; Casaletti, Luciana; Pereira, MaristelaThe fungi of the genus Paracoccidioides sp are etiologic agents of disease paracoccidioidomycosis (PCM), are termodimórficos and have the ability to move the saprobiótica form to yeast form at a temperature around 37 ° C. The uptake is essential for the growth and adaptation of the fungus in host tissue, where nitrogen-dependent pathways have close relationship with pathogenicity. Pathogenic organisms possess a regulatory system called Nitrogen Catabolic Represion which is induced when nitrogen availability is limiting in surround causing the expression of genes necessary for nutrient uptake when preferred sources such as glutamine and ammonia are scarce. This regulatory process comprises a complex system in the infectious process. This study aims to identify proteins regulated by nitrogen depletion, where the pathogenic fungus Pb01-like was analyzed as a proteomic response. Pb01-like was grown in minimal medium control MMcM (+N) and treated MMcM (-N) for 6 hours at 37 ° C, cytoplasmic proteins were extracted and subjected to tryptic digestion and quantification. The proteomic profile revealed an expression of 135 proteins, 40 proteins induced or identified the treaty, 58 repressed or identified control and 44 proteins were expressed constitutively. In silico analyzes showed that the gene is regulated by formamidase areA transcription factor that responds conditions of nitrogen depletion in Aspergillus nidulans and the surrounding medium the enzymatic activity for the gene of formamidase Pb01-like induced depletion of nitrogen during. The amino acid sequence of the regulatory gene areA important in the metabolism of nitrogen was aligned between Pb01-like, Pb18, Aspergillus nidulans and Neurospora crasssa where this alignment showed high identity and homology of conserved zinc finger domains and DUF 1752. Metabolic pathways such as fermentation, gluconeogenesis, protein synthesis, nitrogen metabolism were induced for the depletion of nitrogen, showing a possible modulation of the metabolism of the fungus in order to be able to adapt to environments with nitrogen limitation.Item Análise proteômica de Paracoccidioides brasiliensis após estresse oxidativo(Universidade Federal de Goiás, 2017-03-28) Fernandes, Rosiane Rodrigues; Soares, Célia Maria de Almeida; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793575Z5; Borges, Clayton Luiz; Pigosso, Laurine LacerdaParacoccidioides brasiliensis is a thermodymorphic fungus, the etiological agent of Paracoccidioidomycosis, a systemic mycosis of high incidence in Latin America. During the infectious process, this fungus is subjected to oxidative stress, produced by host defense cells. Therefore, we sought to analyze the proteomic responses of the fungus when exposed to oxidative stress. Menadione is a compound that decreases the viability of yeasts of Paracoccidioides brasiliensis. The main results were the induction of antioxidant enzymes, such as superoxide dismutase, cytochrome c peroxidase, thioredoxin and peroxisomal catalase, enzymes that help the fungus to fight reactive oxygen species. In addition, proteins involved in metabolic pathways such as glycolysis, tricarboxylic acid cycle and glyoxylate cycle had increased expression, demonstrating a possible remodeling of fungus metabolism. There was the induction of enzymes of the pentose phosphate pathway, which has a prominent role in cells under oxidative stress, since it is an important mechanism producing NADPH (nicotinamide adenine dinucleotide phosphate), an electron donor relevant to counteract the deleterious effects of reactive species of oxygen Heat shock proteins were also upregulated during the oxidative stress to which the fungus was exposed. In this work we investigated the response of a member of the species Paracoccidioides brasiliensis, Pb339 to oxidative stress, including the adaptive mechanisms of this fungus to this condition caused in vitro or by the defense system promoted by the host.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 Paracoccidioides brasiliensis sob privação de cobre(Universidade Federal de Goiás, 2020-04-27) Petito, Guilherme; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Soares, Célia Maria de Almeida; Castro, Agenor de; Silva, Lívia do Carmo; Curcio, Juliana Santana de; Silva, Kleber Santiago Freitas eCopper is an important micronutrient that acts as a cofactor in important enzymes that participate in processes of obtaining energy, reducing reactive oxygen species and metal uptake such as iron. Studies with different pathogenic fungi demonstrate the importance of an efficient copper homeostasis system, as well as the impact of this metal deprivation on the metabolism of these organisms. However, little is known about the impact of copper deprivation on pathogenic fungi of the genus Paracoccidioides. Thus, the objective of this study was to analyze the proteomic behavior of the fungus Paracoccidioides brasiliensis during copper deprivation, identifying metabolic changes in face of this condition. Proteomic analysis was performed using mass spectrometry and labelling the samples with iTRAQ (Isobariq tag for relative and absolute quantitation). In addition, methodologies such as qRT-PCR, fluorescence microscopy, enzymatic activities and Western bloting were applied in this study in order to support the proteomic data analyzed. We identified 183 differentially expressed proteins. Proteins related to gluconeogenesis, beta-oxidation and cell wall remodeling were up-regulated. We identified decreased expression of an important detoxification enzyme, Cu / Zn dependent Superoxide Dismutase (Sod1p). On the other hand, the enzymes Thioredoxin (Trxp), Glutathione S-transferase (GST) and Mn / Fedependent superoxide dismutase (Sod2p) were up-regulated. Also, NADPH production-related to the pentose-phosphate pathway enzymes. detoxifying enzyme reducing agent. An alternative oxidase (Aoxp) was up-regulated, while enzyme activity tests showed that the activity of the enzyme Cytochrome c oxidase (Coxp) was decreased. An iron uptake-related copper chaperone, Atx1 and described as important in response to oxidative stress, was up-regulated. We propose here an overview that presents the main metabolic changes in P. brasiliensis under copper deprivation and that include changes in the energy acquisition process, carbon metabolism flow, wall modeling, detoxification and respiration processes.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 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 em células leveduriformes do fungo Paracoccidioides sp. em excesso de cobre(Universidade Federal de Goiás, 2015-02-24) Portis, Igor Godinho; Lima, Patrícia de Sousa; http://lattes.cnpq.br/8330747475582900; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Soares, Célia Maria de Almeida; Borges, Clayton Luiz; Baeza, Lilian Cristiane; Tauhata, Sinji; Brito, Wesley de AlmeidaParacoccidioidomycosis is the most prevalent systemic mycosis in Latin America, caused by fungi of the genus Paracoccidioides. The fungus grows as mycelium in the saprophytic environment, with the infection caused mycelium propagules and/or conidia, that will differentiate into the yeast form. Copper is an essential micronutrient for eukaryotes and bacteria, is extremely important to various proteins, and cofactor of many enzymes and used in many biochemical processes. However, excessive, copper can cause damage to proteins, lipids and nucleic acids, inferring the importance of this metal homeostasis. The objective of this study was to evaluate the fungus Paracoccidioides sp behavior in the presence of copper excess, through proteomics analysis. In the presence of copper excess, the yeast colonies of Paracoccidioides sp. depicted brown color. Expression of transcripts encoding CTR3, a copper transporter and the copper chaperone ATX1 showed inhibition and induction, respectively in the presence of excess copper, as verified by qRT-PCR. Proteins related to stress responses, with melanin production and pathways for energy, are regulated, suggesting that Paracoccidioides sp. responds to copper excess with detoxification strategies, protection and induction of various pathways of obtaining energy.