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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.Item Diversidade genética de hilídeos do Brasil Central(Universidade Federal de Goiás, 2012-06-26) Oliveira, Hugo Henrique Pádua de; Silva, Daniela de Melo e; Cruz, Aparecido Divino da; http://lattes.cnpq.br/7868817504129985The anurans are a group of vertebrates with wide geographical distribution that obtained highly evolutionary success, being part of a lineage descendant of early tetrapods, the first vertebrates to conquer the terrestrial environment. Anurans are divided into two suborders: Archaeobatrachia and Neobatrachia. Within the suborder Neobatrachia there is Bufonoidea superfamily, which includes the three families of higher abundance in the neotropical region: Leptodactylidae, Bufonidae and Hylidae. The aim of this study was to analyze cytogenetically species of the family Hylidae of areas of the Cerrado of Goiás, by the technique of conventional cytogenetics and by silver nitrate staining. The metaphases were obtained directly from cell suspensions of spleen, liver and bone marrow. The slides were stained with Giemsa at 8%, to determine the diploid number and chromosome morphology. The species Hypsiboas multifasciatu and Hypsiboas raniceps presented the diploid number 2n=24 and fundamental number FN=48 with positive marks for NORs in chromosomes 8 and 10 respectively. The species Hypsiboas lundii presented diploid number 2n=24 and fundamental number FN=46 due to the presence of acrocentric chromosome in pair 6. The RONs for this species were identified in pericentromeric regions of short arms of chromosome 5 and in telomeric regions of short arms of chromosome 8. Within the genus Hypsiboas, only the species Hypsiboas albopunctatus presented diploid number 2n=22 and fundamental number FN = 44, and by silver staining technique we identified the presence of NORs on the short arms of the telomeric regions of chromosome pair 8. The species Pseudis bolbodactyla presented diploid number 2n=24 and FN=46 evidenced by the acrocentric pair 4. The species Scinax similis and Scinax constrictus showed different karyotypic notations. The species Scinax similis presented karyotype 2n=24 and FN=48 with methacentric and submethacentric chromosomes. We found for the species Scinax constrictus the karyotype notation 2n=22 and FN=44. Differences in the morphology of chromosomes with respect to literature data can be explained by the different criteria used for classification of the chromosomes. The morphology of some of the chromosomes found in this study differs from their representatives described in the literature, and such differences may be explained by adopting different criteria for the classification of chromosomes.Item Variabilidade genética de subpopulações de Mauritia flexuosa L.f (Arecaceae) em veredas do Estado de Goiás(Universidade Federal de Goiás, 2014-02-26) Corrêa, Kássia Marques; Chaves, Lázaro José; http://lattes.cnpq.br/9990967290797379; Soares, Thannya Nascimento; http://lattes.cnpq.br/5590256762396056Mauritia flexuosa is a large palm tree, commonly known as buriti that has wide distribution in the Cerrado, especially in association with fountains of waters and wetlands in formations known as palm swamps. The genetic variability of a species allows to elucidate the role of evolutionary processes in populations, contributing to the definition of efficient strategies for their use and conservation. Microsatellite markers are useful to quantify the level of variability among and within subpopulations. This study aimed to evaluate the genetic variability of six subpopulations of Mauritia flexuosa located in conserved and disturbed state of Goiás paths, based on microsatellite markers. 175 individuals were evaluated from six subpopulations. Genomic DNA was extracted from leaf tissue and subsequently quantified and diluted for performing polymerase chain reactions, using ten microsatellite loci. It was used an ABI3500 automatic sequencer, which acts through a capillary electrophoresis system, from which the genotypes were obtained. The array of genotypes obtained was used to estimate genetic diversity parameters among and within subpopulções. The analyzes were performed using GDA software. The average number of alleles per locus in subpopulations was 6,2, ranging from 5,6 to 7,0. The values of expected (He) and observed (Ho) heterozygosity were equal to 0,645 and 0,659, respectively, showing Ho value close to that expected by Hardy-Weinberg equilibrium. The analysis of population genetic structure revealed that subpopulations of M. flexuosa are moderately structured with θ = 0,099. The estimated value for f was not significant, as expected for dioecious species. The total fixation index considering all subpopulations was F = 0,08 , very close to the value of θ. This suggests that the genetic structure observed in these subpopulations is related to an effect of drifting associated with some restriction to gene flow between subpopulations. The limitation of gene flow between M. flexuosa subpopulations can be associated with the environment in which they occur, because the palm swamps have disjunct distributions and areas of dry land around them can act as barriers to gene flow. Another factor that could be related to this restriction to gene flow is the dispersion of seeds by zoochory, which becomes 13 difficult in fragmented environments and impracticable if it is kept in place unfavorable for seed germination. It was concluded that the sampled subpopulations showed a low level of inbreeding, and a relatively high genetic diversity. There is significant genetic structure, probably due to a slight isolation between subpopulations associated with the effects of genetic drift within subpopulations.Item Avaliação citogenética e molecular de indivíduos ocupacionalmente expostos aos agrotóxicos(Universidade Federal de Goiás, 2014-02-28) Batista, Mariana Pedrosa; Silva, Daniela de Melo e; http://lattes.cnpq.br/9895211901348365Rural workers are constantly exposed to pesticides they use on crops, and this exposure may be responsible for genetic damage causing a health risk. A problem with the use of pesticides is the genotoxicity, which can lead to the onset of disease. Little is known about the relationship between genotoxicity and the variation of genetic polymorphisms of xenobiotic metabolism that may modify individual susceptibility to the possible genotoxic effects of pesticides. Therefore, there is a need to study genes as glutathione-S-transferase mu (GSTM1) and theta glutathione S-transferase (GSTT1) encoding detoxification enzymes of genotoxic compounds. Another important assessment in individuals exposed to pesticides is the presence of chromosomal translocation t(14;18) (q31,q21), which can be investigated at the molecular level. Thus, this study evaluated the polymorphisms of GSTM1 and GSTT1 genes in 120 individuals occupationally exposed to pesticides and 115 controls (without exposure to pesticides), by real-time PCR. The frequencies of GSTM1 and GSTT1 genotypes were found in 49 % and 18 %, respectively in the exposed group and 37 % and 18 %, respectively, for the control group. It has been found that there is a greater possibility of poisoning in workers who have null genotypes. There was no statistically significant correlation between the increased risk of intoxication and alcohol consumption, smoking and use of PPE. In addition, 29 workers exposed to pesticides for more than 15 years and with null genotypes in GSTT1 and / or GSTM1, were evaluated for the presence of the t (14;18)(q31, q21), in 100 nuclei, by fluorescent in situ hybridization (FISH) . The translocation was observed in only one individual, which may have been caused by prolonged exposure to pesticides, increasing age or alcohol consumption. Thus, the study of genetic polymorphisms and translocations as biomarkers of susceptibility is of fundamental importance in understanding the processes involved in genotype distribution mutagenesis and carcinogenesis and could help minimize the risks for susceptible individuals who are exposed to pesticides.Item Estrutura genética molecular de uma coleção de Germoplasma in vivo e ex situ de Dipteryx alata Vog.(Universidade Federal de Goiás, 2014-02-28) Guimarães, Rejane Araújo; Chaves, Lázaro José; Soares, Thannya Nascimento; http://lattes.cnpq.br/5590256762396056The distribution patterns of genetic variability within and among subpopulations are important to know the performance of evolutionary processes, to adopt effective conservation strategies and to use genetic plant resources. The population genetic structure is focused on how the genetic variability is distributed within and between subpopulations. Microsatellite markers are especially useful for such analyzes because are highly informative. The Baru (Dipteryx alata Vogel), belongs to the Fabaceae family and is widely distributed in the Cerrado biome, also has a great potential for use in local cooking, as ornamental plant and in popular medicine, so being an important genetic resource plant. This study aimed to evaluate the genetic structure among subpopulations of D. alata which constitute a germplasm collection, maintained by the Escola de Agronomia, of the Universidade Federal de Goiás. The collection consists of 600 individuals from 150 progenies of 25 subpopulations from different regions of Cerrado biome. Nine pairs of primers were used, eight developed for the species D. alata and one developed and transferred from Phaseolus vulgaris for molecular analysis. The number of alleles ranged from 2 to 14 alleles, with an average of 5.5 allele per locus. The average of observed heterozygosity was equal to 0.340 and ranged from 0.102 (DaE06) to 0.711 (DaE41). The expected heterozygosity, under the condition of Hardy-Weinberg equilibrium was equal to 0.383, ranging from 0.162 (DaE06) and 0.809 (DaE41). The analysis of population genetic structure showed a level of medium relatedness between individuals of the same progeny (θs) of 0.169, which is equivalent to that kinship expected for a mixture of half-sibs and full sibs. From the value θs, it can be concluded that there is a high genetic differentiation between the progenies. When evaluated the subpopulations was found an estimated θp equal to 0.097, suggesting that there is a moderate genetic structure. The value of overall intrapopulation fixation index (f=0.122) indicated the presence of low inbreeding in subpopulations that composes the collection of germplasm. The estimated apparent outcrossing rate (ta) value was equal to 0.78. The overall F value was significant and equal to 0.27. The analysis of population genetic structure revealed the presence of genetic structure among subpopulations and among progenies within of the subpopulations. This reveals that the germplasm collection of D. alata presents genetic diversity within and among populations, indicating that the conservation strategies should consider both levels.Item Estudo filogeográfico de Micrurus lemniscatus (LINNAEUS, 1758) (SERPENTES: ELAPIDAE)(Universidade Federal de Goiás, 2014-03-10) Abreu, Tatianne Piza Ferrari; Silva Junior, Nelson Jorge da; http://lattes.cnpq.br/6544526824923185; Collevatti, Rosane Garcia; http://lattes.cnpq.br/9979596352166630; Collevatti, Rosane Garcia; Maciel, Natan Medeiros; Vaz-Silva, WilianMicrurus lemniscatus is a South American coral snake species, popularly known as “coral verdadeira”. It is widely distributed in Seasonally Dry Forests (SDF), Gallery Forests and Rainforests. The Tertiary events and the climatic oscillations of the Quaternary affected the distribution of these ecosystems altering, in turn, the distribution of animals associated to these habitats. We hypothesize that the forest expansion and contraction cycles caused by climate fluctuations may have influenced the current distribution and genetic structure of M. lemniscatus. This study aimed to study the evolutionary relationships and patterns of divergence among M. lemniscatus lineages and infer the historical biogeographic events that influenced the distribution and genetic variation. Twenty-nine individuals of M. lemniscatus were sampled from 16 localities in the states of Tocantins, Bahia, Goiás, Alagoas, Mato Grosso, Maranhão, Pará and Amazônia. Three mitochondrial regions (COI, 16S and ND4L) were sequenced, and together generated a fragment of 1595 bp and 23 different haplotypes. The analyses showed a very ancient lineage divergence ~ 4.5 Myr and high genetic differentiation among localities (FST=0.932; p<0.01), suggesting a limited gene flow among geographical regions. The demographic analyzes and neutrality tests indicated that there is no sign of expansion and that populations have constant size (Tajima’s D = 0.521; p = 0.763 and Mismatch distribution = 0.009; p= 0.779). In the analysis of the evolutionary relationship between haplotypes (by median-joining network method), no relationship between lineage and geographical space was found, suggesting incomplete lineage sorting. The results corroborate and give evidence that populations of M. lemniscatus were distributed in a more continuous region in the past, and the current distribution of this species may be the result of the reduction and separation of the geographical scope of their distribution, rather than having itself been an expansion event. This may be the result of cycles expansion of SDF and retraction of the rainforests during the cool and dry phases of the Quaternary.Item Identificação de metaloproteínas associadas a cobre, ferro e zinco codificadas pelos genomas de Paracoccidioides spp.(Universidade Federal de Goiás, 2014-03-11) Tristão, Gabriel Brum; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, Alexandre Melo; Soares, Célia Maria de Almeida; Staats, Charley CristianApproximately one third of all proteins have been estimated to contain at least one metal cofactor, being named metalloproteins. These represent one of the most diverse classes of proteins, containing metal ions provide catalytic, regulatory and structural functions. Bioinformatic tools have been developed to predict metalloproteins encoded by an organism based only on its genome sequence. Paracoccidioides complex includes termodimorphic pathogenic fungi that are found as saprobiotic mycelia in the environment and as yeast in host tissues. They are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis in Latin America. It is known that many metalloproteins are important for virulence of several pathogenic microorganisms. On this way, the present work aimed to predict the cooper, iron and zinc proteins encoded by the genomes of three phylogenetic species of Paracoccidioides spp. (Pb01, Pb03 and Pb18). Cu-, Fe- and Zn-proteins represents 7% of the total proteins encoded by Paracoccidioides spp genomes. Zinc-proteins were the most abundant metalloproteins representing 5.7% of the fungus proteome, while copper and iron proteins represent 0.3% and 1.2% respectively. Functional classification revealed that metalloproteins are related to many cellular processes. Furthermore it was observed that many of these metalloproteins play roles as virulence factors, in the biology of the fungus, and an analysis of the protein interactions revealed that many of them depend on each other to perform their functions. Thus, it is concluded that the Cu-, Feand Zn- metalloproteome of Paracoccidioides is of utmost importance for biology and virulenceItem Estrutura e variabilidade do promotor do gene do fator de necrose tumoral humano (TNF)(Universidade Federal de Goiás, 2014-03-12) Lopes, Mariana Paiva; Castelli, Erick da Cruz; http://lattes.cnpq.br/0992559318175235; Silva, Daniela de Melo e; http://lattes.cnpq.br/9895211901348365; Silva, Cláudio Carlos da; Soares, Thannya NascimentoThe gene for tumor necrosis factor (TNF) is located in the human MHC central region known as class III (Major Histocompatibility Complex). This gene encodes an important pro- inflammatory cytokine produced primarily by macrophages, and it has been associated with several diseases, such as autoimmune and degenerative ones. Studies indicate that TNF polymorphisms may influence their level of expression and activity and therefore could influence susceptibility to tumors. Whereas the bladder urothelium can constantly be the target of inflammatory processes and as the main forms of treatment of bladder tumors are immunotherapy, the genetic profile of an individual can influence susceptibility to this type of injury. In this study, we analyzed the structure and variability of the regulatory region of the TNF gene in Brazilian samples and the results were compared with data obtained by 1000Genomes project. A study of association between polymorphisms of the promoter region of TNF and bladder carcinoma patients in the state of São Paulo in Ribeirão Preto, in our analysis was conducted there was no relationship of the data found with bladder carcinoma. In all evaluated populations we found 15 variation points, found in 11 Brazilian and 15 variation points found in the 1000Genomes data, these variation points are arranged in 20 different haplotypes. These haplotypes with comparisons involving primate sequences indicated that one allele arose before the main speciation and human dispersion. Two strains were defined by haplotype relationships and are probably very old in human evolutionary history, since all populations evaluated showed haplotypes belonging to each of these strains. The frequency of haplotype H01 is the highest among all populations evaluated. However, the haplotype H12, although at reduced frequency compared to H01 is probably the oldest haplotype in part by the second line being shared with other primates.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 Isolamento, caracterização e transferibilidade de marcadores microssatélites de Byrsonima cydoniifolia A. Juss. (Malpighiaceae)(Universidade Federal de Goiás, 2014-03-13) Bernardes, Vanessa; Telles, Mariana Pires de Campos; http://lattes.cnpq.br/4648436798023532; Telles, Mariana Pires de Campos; Vianello, Rosana; Borba, TerezaMolecular markers are identifiable DNA sequences found at specific sites in the genome and transmitted by the standard laws of inheritance from one generation to the next. Markers known as microsatellite are indicated for genetic population analysis; due they are widely distributed in the genome and their high polymorphic information content per locus. To support different management practices, genebanks, conservation studies is important to perform the analysis of genetic variability in natural populations of the species of interest. In the biome Cerrado there are inumerous medicinal and fruit trees that are used in a extractive way, among them are those of the gender Byrsonima sp. popularly known as murici, belonging to the family Malpighiaceae. In Brazil presents about 70 species with wide distribution in the Cerrado. The murici is a multi- potential plant and although not be domesticated, its economic exploitation can be viable. In this context, the objective was to develop a set of microsatellite markers for species Byrsonima cydoniifolia A. JUSS. and test the potential transferability of this set of markers for species Byrsonima crassifolia L. Kunth. The microsatellite regions were isolated using genomic libraries enriched for repetitive regions and then they were used for primer design. Using the primers designed temociclagem conditions were tested and the products were characterized using the DNA of 90 individuals of B. cydoniifolia and 24 indivuduals of B. crassifolia. Of these, 14 loci were polymorphic and 3 monomorphic in three populations of B. cydoniifolia. The number of alleles ranged from 3 to 17, with an average of 10.45 alleles per locus. The observed heterozygosity was similar to the expected heterozygosity, the values ranged from 0.20 to 0.92 and 0.43 to 0.92; respectively. 39 private alleles were identified. This set of markers showed high combined probability of paternity exclusion (Q) equal to 0.999 and combined probability of identity (I) equal to I=7.84x10-1. The global values for the fixation index (FIS) were not significant (P<0.05), whereas the values of FST=0,082 and FIT=0,143 were significant. For Byrsonima crassifolia species, nine pairs of primers produced good patterns of cross amplification in the study population, with a mean of 7.56 alleles per locus. The observed heterozygosity ranged from 0.250 to 0.958, which was higher than the expected heterozygosity ranging from 0.223 to 0.905. The combined probability of paternity exclusion (Q) was equal to 0.99847 and high combined probability of identity equal to 6.42x10-9. The strategy of development of microsatellite markers from the genomic library enriched methodology was efficient to generate a panel with eleven polymorphic microsatellite markers for B. cydoniifolia and nine polymorphic microsatellite markers successfully transferred to B. crassifolia, providing a useful tool for population genetic studies not only for the species B. cydoniifolia and B. crassifolia, but possibly for other related species evolutionarily.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 Transferibilidade e desenvolvimento de sistema multiplex de genotipagem de marcadores microssatélites para Pterodon emarginatus Vogel (Fabaceae)(Universidade Federal de Goiás, 2014-04-10) Santana, Ariane Rolins de; Vianello, Rosana Pereira; Soares, Thannya Nascimento; http://lattes.cnpq.br/5590256762396056; Rodrigues, Flávia Melo; Silva, Daniela de Melo e; Soares, Thannya NascimentoMicrosatellite markers are widely used to assess the genetic variability in natural populations. These markers can be developed from derived Expressed sequence tag (ESTs) or genomic libraries. A feasible and economical alternative for obtaining of these markers for species that do not already have them is the transferability from primers designed for species evolutionarily close. The species Pterodon emarginatus Vogel (sucupira) belongs to the Fabaceae family and shows great potential for use timber and medicinal, which makes them a target of exploitation. Thus, studies that contribute to their use and conservation are needed, including the genetic population. Therefore, the aim of this study was to evaluate the potential for cross-amplification primers developed for the species Phaseolus vulgaris to P. emarginatus and to analyze the polymorphism of the loci transferred from multiplex genotyping in three populations of this species. For this, were evaluated 539 primers developed for Phaseolus vulgaris, with 345 derived from ESTs libraries and 194 genomics. Amplification tests were performed using three individuals, while the assessment initial of the polymorphism with eight. Finally, genetic diversity parameters were estimated using 88 plants from three natural populations of P. emarginatus. Primers that showed amplification products visualized on agarose gel were then evaluated in 6% acrylamide gel stained with silver nitrate. Polymorphic loci were synthesized with fluorescence for evaluation and genotyping by capillary electrophoresis in ABI3500 automatic DNA analyzer. The loci were transferred to P. emarginatus 23 (4 %) being 7 polymorphic. Polymorphic, 6 electropherograms profiles. These 6 loci were evaluated in populations along with three polymorphic genomic loci previously standardized sucupira, totaling nine loci suitable for genotyping. It was possible to develop two multiplex systems, a compound of 5primers and another for 4. The number of alleles per locus ranged from two to 13, with an average of five alleles per loci. The mean values of He and Ho for both populations were 0,563 and 0,463 respectively. The value of f was not significant for the total population and equal to 0,144. The values FIT and FST were significant and equal to 0,06 and 0,195 respectively. Microsatellite markers transferred and polymorphic for P. emarginatus showed polymorphism and can be used in studies genetic population more depth to the species.Item Caracterização funcional de proteínas hipotéticas do fungo patogênico humano Paracoccidioides sp.(Universidade Federal de Goiás, 2014-04-30) Silva, Paula Francinete Faustino; Salem-Izacc, Silvia Maria; http://lattes.cnpq.br/5542866107144940; Salem-Izacc, Silvia Maria; Novaes, Evandro; Brito, Wesley; Borges, Clayton Luiz; Georg, Raphaela de CastroNearly 60% of the Paracoccidioides genes encode proteins annotated as hypothetical proteins or predicted proteins (HPs). Transcriptomes studies revealed 2364 HPs expressed in mycelium and yeast form; during the transition from mycelium to yeast; and under conditions that mimic the infection pathway. In this study, we describe a global detection and functional inference for the HPs in Paracoccidioides. For these analysis we used computational methods based on sequence similarity, search for targeting signals, presence of known protein domains and also a functional classification based on Gene Ontology. Our analysis allowed the HPs to be classified into different functional categories such as metabolism, organelle organization, cell communication, protein localization, cell cycle, signaling and cell differentiation. We also performed a functional enrichment analysis of the transcripts expressed under different growth conditions. The best represented functional domains are those involved in the regulation of gene expression, suggesting that these HPs may be involved in regulation of the gene response and adaptation. The transcriptional profiling of six HPs confirms that they are highly expressed in the presence of human blood and plasma and also during phase transition, a crucial event for establishment of the infection process. Additionally, we have cloned and expressed the gene encoding the hypothetical protein PAAG_08614 from Pb01. The transcript and protein expression were evaluated by qPCR e western-blot, respectively. PAAG_08614 is preferentially expressed in mycelium and during mycelium to yeast transition. This work constitutes the first large-scale characterization of the proteins of unknown functional from Paracoccidioides spp. and helps the functional assignment of hypothetical proteins, as well as the understanding of the data generated from structural and functional genome.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 Transferibilidade e validação de marcadores microssatélites derivados de EST para duas espécies de Campomanesia (Myrtaceae) do Cerrado(Universidade Federal de Goiás, 2014-06-02) Miranda, Elen Amoreli Gonçalves Cintra; Telles, Mariana Pires de Campos; http://lattes.cnpq.br/4648436798023532The Savanna is highly diverse and has the richest flora and endemic in the world, comprising about 12,000 species of native plants. The family Myrtaceae is among the 10 families representative of this biome, comprising 14 genera. Among these, gender Campomanesia has several fruit species, known popularly as gabiroba. The species Campomanesia adamantium and Campomanesia pubescens have widespread occurrence in the Savanna. Its fruits can be consumed " in nature ", in the form of candy, ice cream , soft drinks and as flavoring in alcoholic distillates, besides having antidiarrheal properties, its bark and leaves are used in the form of teas. His exploration is by extractivism and can also be grown in small family orchards, being a source of extra income for some families. Thus, the study of genetic diversity and genetic structure of populations’ allies with other areas of knowledge can provide important information for planning and execution of native species conservation programs information. Currently, molecular markers have wide application for studies on the genetics of populations, among the types of markers used; microsatellites have been widely used in recent years by high power to detect genetic variability. Population - genetic studies of the genus Campomanesia sp. are scarce and so not found microsatellite markers available in the literature for any species of the Savanna o. In this context, the aim of this study was to test the potential for transferability of microsatellite markers derived gene regions (EST) developed for Eucalyptus sp., Campomanesia in two species (C. adamantium and C. pubescens), providing a panel of polymorphic markers for the species. To do so, we sampled two populations of each species and DNA was extracted from leaf tissue. The potential for transferability of 120 primers pairs were evaluated. The PCR products derived from the microsatellite markers were assessed in automatic transferred DNA analyzer. The array of genotypes was used to estimate the parameters of genetic variability for the panel of marker. Of the 120 primers tested, 87 were discarded for not having amplification products, many nonspecific fragments amplified 22 and 12 were considered successfully transferred the two species. The 12 markers transferred showed good discrimination power of the individual , with high probability of paternity exclusion (0,99939 to C. adamantium and 0,99982 for C. pubescens) and low probability of identity (5,718 x 10-10 for C. adamantium and 1,182 x 10-11 to C. pubescens). The average number of alleles at polymorphic loci was equal to 6,8 to C. adamantium, ranging from 2 (EMBRA1335 and EMBRA 1811) 16 (EMBRA 1364) and C. pubescens was 7,8 ranging from 2 (EMBRA1811 and EMBRA 1076) to 16 (EMBRA 2011) alleles per locus. Mean genetic diversity of Nei (1973) in populations was equal to 0,517 (C. adamantium) and 0,579 (C. pubescens). Mean observed heterozygosity (Ho) for the populations was equal to 0,505 (C. adamantium) and 0,503 (C. pubescens). For some markers for deviations expected for the Hardy-Weinberg equilibrium was observed due proportion’s estimates of intrapopulation inbreeding (f) were not significant for any population. Inbreeding full level (F) was significantly different from zero, suggesting some genetic variability structure populations. The structure of genetic variability in component among populations (θp) was equal to 0,105 (C. adamantium) and 0,249 (C. pubescens).Item Identificação de proteínas de superfície de Staphylococcus saprophyticus e análise de fatores de virulência(Universidade Federal de Goiás, 2014-06-09) Carvalho, Alex Jesus de; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Coelho, Alexandre Siqueira Guedes; Baeza, Lilian CristianeThe Gram-positive bacterium Staphylococcus saprophyticus, one of the coagulasenegative staphylococci, is the second most common causative agent of urinary tract infection, affecting mainly sexually active women. Staphylococcus saprophyticus can cause acute diseases as pyelonephritis, sepsis, nephrolithiasis, endocarditis, urethritis, epididymitis and prostatitis. This work aims to identify Staphylococcus saprophyticus surface proteins by using a proteolytic shaving approach, a methodology that was established to identify surface-exposed protein domains by tripsinization of intact cells. The peptides obtained were treated by trypsin, reduced, alkylated and identified by nano-chromatography using a nanoACQUITY UPLCTM system (Waters) coupled to a SYNAPT Q-TOF mass spectrometer (Waters). The homology analysis was performed using the software ProteinLynx 2.3 (Waters). Through the shaving, it was possible to identify 219 proteins, many of them, described as virulence factors. Of total, 01 is cell wall protein, 09 are extracelular proteins, 19 are membrane proteins and 190 are citoplasmatic proteins. Besides of the lysis process, the presence of cytoplasmic proteins on cell surface can be due to the activity of export pathways not yet identified and many of these proteins can be proteins with moonlighting function, in other words, proteins that plays more of one function, it can, in this case, plays functions on S. saprophyticus cell surface related to bacterial virulence. The main proteins with moonlighting function include metabolic enzymes of the glycolytic pathway; enzymes of other metabolic pathways, such as, glyoxalate cycle; chaperones and proteins related with the proteic folding. The prediction of cellular localization was performed through LocateP database. The results of this research help to elucidate the strategies and machineries used by proteins during the adhesion, infection and proliferation, leading us to understand the interaction between the pathogenic bacteria S. saprophyticus and the human host. The knowledge about the proteins present on the cell surface is of extreme importance, because many of these proteins represent targets to new drugs, therapeutic antibodies or vaccines, since the pathogen cell surface is the first to contact with the host cells during the infection process.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 Diversidade genética em população de melhoramento de mogno africano (Khaya ivorensis A. Chev.)(Universidade Federal de Goiás, 2014-08-04) Soares, Sabrina Delgado; Novaes, Evandro; http://lattes.cnpq.br/0568272239145336The strong demand for hardwood drives selective logging and degradation of forests ecossystems in Brazil. Due to its wood value and predatory logging, Brazilian mahogany (Swietenia macrophylla) is at risk of extinction. Currently, its trees are protected and banned from logging by the Brazilian Institute of Environment (IBAMA). Monoculture of the Brazilian mahogany is proven unsuccessful due to the attack of the Hypsipyla grandella larvae. An alternative to Brazilian mahogany plantation is the African mahogany (Khaya ivorensis), a species with similar high valued wood properties. The Universidade Federal de Goiás in partnership with Mudas Nobres Company started a breeding program with Khaya ivorensis. The objective of this study was to evaluate the genetic diversity and divergence among 53 superior trees selected and cloned as part of the breeding strategies. Clones were selected from three different populations, planted in two farms in Pará state and originating from Ivory Coast and Tanzania populations. Twelve seedlings of Khaya senegalensis were also used as a control group in the analyses of genetic divergence. The individuals were genotyped with eight microsatellite loci developed for K. senegalensis, by capillary electrophoresis on the ABI-3100 (Applied Biosystems) platform. The software GeneMapper (Applied Biosystems) was used to genotype the alleles. The average number of alleles per locus was 5.875. The average expected heterozygosity was lower (HE = 0.563) than the average observed heterozygosity (HO = 0.738). Therefore, the average intrapopulation fixation index was negative (f = - 0.314) indicating that high inbreeding depression is decreasing the frequency of homozygous when compared to what would be expected under Hardy-Weinberg Equilibrium. The average fixation index between populations was estimated at θ = 0.008, indicating that only 0.8% of the genetic variability is due to differences between populations. The dendrogram generated from the matrix of Rogers’ genetic distance showed two distinct groups, separating the control group with K. senegalensis from the K. ivorensis selected trees. The group composed of K. ivorensis had nodes with weak bootstrap consistency indicating weak genetic structure among selected trees. The lack of genetic structure was confirmed by a Bayesian approach on the Structure (version 2.3.4) program. The genetic diversity observed within the selected breeding population is comparable to that of natural populations of African and Brazilian mahoganies. The genetic distance estimated with this work will guide the selection of divergent progenitors to be crossed.Item Bioprospecção e caracterização de bactérias produtoras de ciclodextrina glicosiltranferase em solos de biomas brasileiros(Universidade Federal de Goiás, 2014-08-04) Ribeiro, Maycon Carvalho; Amaral, André Corrêa; http://lattes.cnpq.br/8801299423520104; Amaral, André Corrêa; Vieira, José Daniel Gonçalves; Fernandes, Éverton Kort KampCyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an important industrial enzyme for being the only one able to convert starch and related glucans in cyclic oligosaccharides called cyclodextrins (CDs). The arrangement of the glucose units in the formation of CD results in a molecule with the shape of a cone, with hydrophobic interior and hydrophilic surface. This arrangement of glucose molecules in CDs allows its use as a host molecule in the formation of inclusion complexes with organic and inorganic compounds. This mechanism is advantageous in protecting the guest molecule from light, heat and oxidizing conditions and also enable the "dissolution" of compounds of low solubility in aqueous media. Cyclodextrins are used from the food industry to the pharmaceutical, in controlled drug delivery systems and immobilization of toxic compounds for environmental protection. The CGTases are mainly produced by bacteria of the genus Bacillus, found degrading starch rich substrates. The aim of this study was to identify, isolate, select and characterize strains of CGTase-producing bacteria from soil samples from different regions of Brazil as well as calculate the enzymatic production of these bacteria on low-cost substrates. With this work, it was possible to identify 17 bacteria producing cyclodextrin glycosyltransferase enzyme, with nine of them had values above 1.5 for enzymatic production. Of these, all were characterized as gram positive Bacillus. Bioprospecting of bacteria in soils of different cultures led to the identification of bacteria that may be used in studies for the production of cyclodextrin glycosyltransferase and subsequent implementation by various industries.Item Transferibilidade e variabilidade genética de marcadores microssatélites gênicos em Egenia klotzschiana Berg (Myrtaceae)(Universidade Federal de Goiás, 2014-08-11) Siqueira, Mariana Natalice de; Telles, Mariana Pires de Campos; http://lattes.cnpq.br/4648436798023532Eugenia klotzschiana Berg is a species of Myrtaceae with restricted distribution in the Cerrado. Its fruits present nutritional potential and can be used in extractive way, as raw material for production of jams, jellies and juices. In this context, it is necessary to evaluate population-genetic aspects in order to provide information to assist in targeting strategies for management and conservation. To assess the genetic variability in a population context microsatellite markers have been used in recent years. The sequences flanking the microsatellite regions of the genome are highly conserved between related species, enabling the portability of these markers, reducing thereby the costs of developing the same for each species under study. In this context, the aim of this study was to test the potential for transferability to the genome of E. klotzschiana of genic microsatellite markers developed for Eucalyptus, as well as to characterize the genetic variability in their populations. For this purpose, DNA was extracted from leaf tissue of E. klotzschiana individuals and used to test the cross 120 pairs of amplification primers. Samples from seven locations were used to characterize the population genetic variability. The amplification products were analyzed on agarose and polyacrylamide capillary electrophoresis gel in different stages. Of total primers tested, 67 (55.83%) did not amplify, 42 (35%) had many nonspecific amplification products and 11 (9.2%) were successfully transferred. Of the 11 transferred eight showed polymorphism. For the eight polymorphic markers, the probability of identity was high (2.02 x10-3) and the power of paternity exclusion was moderated (0.74). In this population the average value of expected heterozygosity (He) was 0.28 and observed heterozygosity (Ho) was 0.44, as in subpopulations of He mean values ranged from 0.18 to 0.27 and the mean values Ho ranged from 0.36 to 0.53, respectively. No significant for the fixation index (f) were observed in the populations values indicating allele frequencies below the expected Hardy-Weinberg proportions.The extent of genetic divergence among subpopulations showed relatively high genetic differentiation, with a value of θ equal to 0.20, and peerto- peer values ranging between 0.00 and 0.34. The strategy of transferability of microsatellite markers was efficient to generate a panel of polymorphic microsatellite markers and learn a little of the genetic variability of the species. It was possible to verify that there is a low genetic variability within subpopulations for biomarkers. However, this work is a starting point for future studies to better understand the reproductive biology of the species Eugenia klotzschiana.