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Item Ocorrência de mutações em loci ligados ao cromossomo Y na prole nascida de indivíduos expostos à radiação ionizante(Universidade Federal de Goiás, 2009-05-13) ARRUDA, Jalsi Tacon; CRUZ, Aparecido Divino da; http://lattes.cnpq.br/7868817504129985In September 1987, in Goiânia-GO, Brazil, one of the most serious radiological accidents occurred at a radiotherapy unit involving a source of cesium-137. An area of 2,000 m2 was contaminated and 249 people were exposed, both externally and internally, to substantial doses of ionizing radiation, resulting in four fatalities due to acute radiation syndrome. The current study examined the occurrence of possible mutations in the Y chromosome of the exposed men and their male offspring divided into two groups: A) eight accidentally exposed men and eight boys; B) twelve occupationally exposed men and sixteen boys; and the control group with 8 men and 8 boys not exposed. DNA was isolated from peripheral blood lymphocytes and 30 loci (SRY, AMELY, ZFY, AZFa-Prox1, SY83, AZFa-Prox2, SY86, SY85, SY84, USP9Y, SY87, DBY, AZFa-Dist1, 12f2, AZFa-Dist2, UTYpe, SY106, SY124, SY127, SY134, SY135, SY143, SY1197, SY1291, SY1125, SY1054, YDAZ3, SY254, SY255, RH65618) were amplified by the polymerase chain reaction. All DNA tests had a probability of paternity of at least 99.99%. All analyzed individuals amplified STS; however, 4 fathers (8.4%) and 8 sons (21.2%) in group A, and 3 fathers (7.1%) and 3 sons (63.3%) in group B showed mutations. The total mutation rates were 0.11. The first generation of the accidentally exposed group showed 7 mutations in SY86, 12 mutations in SY84, and 1 mutation both in 12f2 and SY135. The first generation of the occupationally exposed group showed 2 mutations in SRY, AMELY, AZFa-Prox1, AZFa-Prox2, SY86, SY85, SY84, USP9Y, SY87, AZFa-Dist1, UTYpe, SY106, SY124, SY127, SY134, SY135, SY143, SY1125, SY1054, YDAZ3, SY254, SY255, and RH65618; and 4 mutations in 12f2. In the control group, only one son showed an SY84 deletion. Recombination events between repetitive regions are possibly the cause of the high incidence of de novo mutations in the Y chromosome. The mutations were possibly generated by intrinsic mechanisms that could have been increased by the ionizing radiation from cesium-137. The exposure to ionizing radiation from cesium-137 can be detected in offspring of exposed individuals, and the mutation rate can be attributed to radioactive exposure.Item Análises in vivo e in vitro de interações intermoleculares da Beta-1,3- glicanosiltransferase 1 de Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2010-01-28) BAILÃO, Elisa Flávia Luiz Cardoso; SOARES, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637The cell wall of pathogenic microbes acts as an initial barrier that is in contact with hostile environments. Besides functioning as a mechanical barrier, it harbours an immunogenic macromolecules arsenal. One of the ways that proteins can be associated to the cell wall, it is through GPI anchor. The hydrophobic C-terminal end of the β-1,3-glucanosyltransferase enzyme of the human pathogenic fungus Paracoccidioides brasiliensis is characteristic of GPI anchored proteins. The β-1,3-glucan assembling and rearrangement are essential since this molecule acts as a scaffold to support cell wall proteins and polysaccharides. In the thermodimorphic fungus P. brasiliensis, β-1,3-glucan is found predominantly in mycelium form and α-1,3-glucan is predominant in the yeast form. In this work, it was screened possible protein-protein interactions performed by β-1,3-glucanosyltransferase 1 of P. brasiliensis (PbGel1p). To obtain these results, a P. brasiliensis cDNA library was screened with PbGel1p using the Saccharomyces cerevisiae two hybrid system. In addition, pull-down assay was used as an in vitro complementary technique to isolate proteins that interact direct or indirectly with PbGel1p. It was screened 38 gene products using two hybrid system and it was identified 3 proteins using the pull-down assay associated with mass spectrometry. The PbGel1p role in the cell wall maintenance and remodeling was indicated through the analysis of screened interactions, like alpha-glucosides permease, acid phosphatase, GDSL lipase, septin, actin, tubulin, HSP90 and pyruvate kinase. Furthermore, nuclear localization of PbGel1p and its role in the locus-specific transcriptional silencing were suggested based on such interactions: Qde2 argonaute, transcription elongation factor spt6, others transcription factors and ATP-citrate synthase. Therefore, this study indicated, for the first time, that PbGel1p has multiple location and it participates either in roles classically described for glucanosyltransferases, as the cell wall remodeling, or in recently described functions for this family of proteins, as the locus-specific transcriptional silencing.Item Avaliação de critérios de compatibilidade entre pares de primers para otimização de sistemas multiplex de genotipagem(Universidade Federal de Goiás, 2010-01-12) BARBOSA, Ana Clara de Oliveira Ferraz; COELHO, Alexandre Siqueira Guedes; http://lattes.cnpq.br/0840926305216925The progress of Molecular Biology and Genetics provided the appearance of several molecular markers that detect the genetic polymorphism directly at DNA. Among these markers are the microsatellites (SSR), which are distinguished by their high degree of polymorphism. The use of these markers for individual genotyping has evolved into multiplex systems, which allow many SSR fragments to be detected and analyzed simultaneously. Currently there are several articles in literature discussing the criteria to be used in the primer design for use in PCR, as well as various softwares are available for this end. However, there are few studies and tools for the analysis of compatibility between pairs of primers for use in multiplex systems, where multiple fragments are simultaneously amplified using PCR. This paper evaluated different criteria for compatibility between pairs of primers. A set of 74 combinations of pairs of primers, involving the amplification of 94 SSR loci were evaluated in duplex systems. The same combinations were evaluated according to different criteria, including the degree of complementarity between primers, the magnitude of differences of denaturation temperatures (Tm) and the tendency to annealing between pairs of primers based on the Gibbs free energy resulting from the association between them. The comparison between the different criteria allowed the identification of a set of criteria with positive predictive value equal to 94%. These criteria were implemented for use in a software called Multiplexer, which from the analysis in sequence of pairs of primers, suggests compatible combinations for use in multiplex genotyping systems. Using this tool can significantly reduce the costs related to laboratory activities for genotyping using PCR.Item Análise dos níveis de poligalacturonases e glucanases expressas durante os processos de interação patogênica e saprofítica de Sclerotinia sclerotiorum(Universidade Federal de Goiás, 2008-05-30) BARBOSA, Silvio Romero Costa; SILVA, Silvana Petrofeza da; http://lattes.cnpq.br/6823998544968373The fungus Sclerotinia sclerotiorum can interact with a great range of vegetable species as well as to obey to the discharge especificity and patogenic specialization . It is capable to digest the cellular wall of host plants using for such a series of biochemical mechanisms and morfogenetics that optimize the invasion. Several enzymes are produced during the interaction plant-host and among them they stand out the family of the poligalacturonases (PGs) and them beta glucanases. PGs catalyze the hydrolysis of the connection glycosídic bond - 1,4 and them beta glucanases liberate glucans and oligossacarídeos during the hydrolysis. Our work tried to characterize, besides the pH, the action of these enzymes, as well as the gene expression during the interaction with bean (Phaseolus vulgaris) and under different cultivation means, pectin 1%, wall extract 1% and glucose 1%. The activities were measured by the method DNS where the amount was measured of you sugar reducers in the middle and the gene expression through electrophoretic profiles analyzed after the technique of RT-PCR. The results showed variation of the activity of PGs in the interaction being the middle with pectin with larger expression of them. Them beta 1,3 and beta 1,4 glucanases were expressed in both culture means proposed, however there was larger production of beta 1,3 during the invasion. The variation of the expression of such enzymes in different culture means suggests complexity of specific biochemical roles for your production raising new approaches for the recognition of the roads that they promote the development of the diseaseItem Expressão heteróloga do gene Hxyn2 do fungo Humicola grisea var. thermoidea em Pichia pastoris: Produção e purificação da enzima HXYN2r e aplicação em testes de panificação(Universidade Federal de Goiás, 2008-05-30) BASTOS, Fernando Medeiros; FARIA, Fabrícia Paula de; http://lattes.cnpq.br/3739169267521003Endoxylanases are the main group of enzymes involved in the hydrolysis of xylan. This enzymes have application in industrial proposes, like as drink, food, feed, clothes industries and for bleaching cellulose paper pulps. In bread making the xylanases have been used to improve processing and product quality of loaf, leading soft dough and loaf with larger volume as well as an improved crumb structure. The xylanolitic enzymes produced by filamentous fungi constitute an enzymatic pool with distinct activities which make their use in industrial process more difficult, the obtainment of recombinant microorganisms constitutes a strategy to achieve suitable enzymes to industrial process. The thermophilic fungus Humicola grisea var. thermoidea has proved to be a good source of xylanolitic enzymes. The gene Hxyn2 from H. grisea codes a xylanase with 23 kDa that belongs to G/11 family of glycosil-hydrolases. Its cDNA was cloned in the vector pHILD2 and expressed in yeast Pichia pastoris under the control of the promoter AOX1. The transformants were chosen trough genetic stability and capacity to produce and secrete the enzyme HXYN2r into culture medium. The purified HXYN2r showed a high xylanolitic activity with an optimum temperature of 60 ºC and an optimum pH value of 6.5. The aim of this project was the production, purification and application of HXYN2r enzyme in bread making tests. The production in the optimized medium obtained 100 mg of active xylanase per liter of medium BMMY-U. This quantify of enzyme presented 40% of the total proteins in culture supernatants. The proteins of culture supernatants was concentrated by liofylization and fractionated by into a chromatographic column of gel filtration Sephacryl S-100. The purified xylanase presented specific activity of 2250 U/mg and the purification profit was 6.4%. The 23 kDa protein was confirmed by the activity on Zymogram assay. The pure xylanase was added at the rate of 45 and 90 U/Kg of wheat flour in the bread making tests. In this rates it was not observed any effect of xylanase in specific volume either in crumb structure. The HXYN2r enzyme was partially purified by a heat treatment (45 min at 60 °C) and was concentrated by liofylization and a yield of 17.9% was obtained. The partially purified HXYN2r was added at the rates of 500, 1500, 3000, 4500 and 6000 U/kg of wheat flour. The added xylanase improved the specific volume and the crumb structure, but any effect was observed in the moisture content of the loaf. The best result was the rise of 16.0 % in loaf specific volume with the dose of 3000 U/kg of wheat flour. This effect was similar to the obtained with a commercial xylanase added to the dough in equal dose. The results presented suggest that the xylanase from H. grisea can be used in bread making to improve specific volume.Item Caracterização de um Antígeno Rico em Prolina(PRA/Ag2) do fungo patogênico humano Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2008-01-31) CASTRO, Kelly Pacheco de; SOARES, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a proline-rich protein (PRA/Ag2) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Coccidioides immitis. The protein, the cDNA and genomic sequences were analyzed. Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis. The cloned cDNA was expressed in Escherichia coli and the purified rPbPRA/Ag2 was used to obtain polyclonal antibody. The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbPRA/Ag2 in the fungal cell wall, linked through a GPIanchor. The expression of the Pbpra/ag2 gene was analyzed by real time PCR and results demonstrated developmental regulation in phases of P. brasiliensis, with a higher expression in the mycelium saprobic phase. The protein expression analyses corroborate the transcript levels.Item Estudo de associação do polimorfismo de base única no códon 72 do gene humano p53 e as características de pigmentação(Universidade Federal de Goiás, 2010-03-30) COSTA, Kárita Antunes; GUILLO, Lídia Andreu; http://lattes.cnpq.br/3401436781775091The p53 gene encodes a protein which has various functions such as monitoring of the cell cycle, role in repair mechanisms and apoptosis. New functions performed by this protein have been observed and studied as acting in the cascade of skin pigmentation by acting as transcription factor for genes important in this process as POMC (pro-opiomelanocortin) and MC1R (melanocortin receptor 1). Among the genetic polymorphisms, the codon 72 p53 gene is the most commonly studied and this variant has been associated with increased risk for various cancers, including skin. However, the association between this and other types of cancers has generated controversial results. The polymorphism occurs in a substitution G / C codon 72 p53 gene resulting in a change of amino acid sequence (CGC to CCC for arginine and proline). This polymorphism occurs in areas rich in proline generating morphophysiological changes as well as a difference in electrophoretic mobility of the protein. The aim of this study was to establish a possible association between the codon 72 polymorphism of p53 gene with the characteristics of pigmentation such as skin color, hair, eye and skin response after exposure to sunlight (reddening or bronze). The 96 healthy volunteers were recruited randomly and information on skin color, ability to tan and other characteristics of pigmentation were collected through a standardized questionnaire. Genomic DNA was extracted from venous blood and genotyping was performed by polymerase chain reaction (PCR). The polymorphism was investigated by photo documentation after agarose gel electrophoresis. Samples that generated doubts were confirmed by digestion with sitespecific action of the restriction enzyme BstUI. Allele frequencies of 96 volunteers to p53Arg/72 and p53Pro/72 were 67.70% and 32.30% respectively. Genotype frequencies for Arg / Arg, Arg / Pro and Pro / Pro were 50.00% 35.40% 14.60% respectively. We did not detect a significant association between reddening or tanning after sun exposure with the polymorphism (p = 0.678), on the other hand we observed an association between the genotype Pro / Pro and blue eyes / green (p = 0.01) among showing redness of the skin, demonstrating a disadvantage in relation to the sun when there is occurrence of this specific phenotype (eye color) combined with the genotype Pro / Pro.Item Caracterização Molecular e Expressão Heteróloga de um cDNA Codificante para Tiorredoxina do fungo patogênico humano Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2006-08-31) DOMINGOS, Fernanda de Castro; JESUÍNO, Rosália Santos Amorim; http://lattes.cnpq.br/5113656623817587The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis (PCM), a human systemic mycosis highly prevalent in countries of Latin America. P. brasiliensis is subjected to different insults from human host, such as oxidative stress caused by reactive oxygen species produced by the host during the infection. Thioredoxin (TRX) is an intracellular redox protein that is required to maintain redox homeostasis in response to both reductive and oxidative stress conditions in several organisms. We report here the characterization of a 811 bp cDNA Pbtrx1, encoding a PbTRX1 of 116 amino acids, with a predicted molecular mass of 12 kDa and pI 5.2. This putative protein presented one highly conserved active site motif (WCGPC) between TRXs from several organisms. The phylogenetic analysis performed with PbTRX1 and TRXs from other organisms, putted P. brasiliensis in the fungi clade. We also performed the prediction of the secondary structure of PbTRX1 that shows a pattern characteristic of the open twisted alpha/beta, similar to TRX secondary structures described in other fungus. In order to obtain the recombinant PbTRX1, the expression construct pGEX-4T-3-trx1 was introduced into Escherichia coli cells and the expression and purification of the recombinant protein was obtained. The recPbTRX1 and PbTRX1 from yeast cells extract were found to catalyze the reduction of insulin. However the PbTRX1 from yeast cells extract treated with H2O2 showed highly insulin reduction activity than the yeast cells no treated. PbTRX1 was detected by Western blotting in the extracts from yeast cells growth and from mycelium to yeast transition. The yeast cells growth was significantly inhibited by H2O2; however the mycelium to yeast transition was little affected by this oxidant. Semi-quantitative RT-PCR was employed to analysis the expression of Pbtrx1 gene in response to H2O2. The level of Pbtrx1 transcripts was higher in yeast cells treated with H2O2 than in yeast cells no treated. To realize how P. brasiliensis deals with oxidative stress is essential to understand the mechanisms involved in its survival in the host. It may be possible that PbTRX1 enhances survival of P. brasiliensis in the host, protecting the fungus against the reactive oxygen species and allowing, in this way, the progress of the infection.Item Avaliação histopatológica, histoquímica e morfométrica dos efeitos da toxicidade aguda do herbicida roundup® nas brânquias e no fígado do peixe Poecilia vivipara(Universidade Federal de Goiás, 2009-05-20) FARIA, Joana Cristina Neves de Menezes; SABÓIA-MORAIS, Simone Maria Teixeira de; http://lattes.cnpq.br/6723881044959716The indiscriminate use of agricultural pesticides, which contaminate the soil, water, and human beings, has become a problem that causes great concern nowadays. We studied the acute toxic effect of the herbicide Roundup® on animal behavior, tissue, and cells of the neotropical fish species Poecilia vivipara gills and liver in order to determine and compare their histoarchitecture, as well as to identify possible morphological alterations. The average lethal concentrations of Roundup® were calculated and, after that, the specimens were treated with acute exposure of this herbicide (24 h) at the concentrations of 0, 15, 25, and 35 μl per liter of water. The gills and livers were dissected, fixed in neuter formalin and Karnovsky s solution. The analyses, carried out using basic histological, classical histochemical, and morphometric tests, allowed the identification of alterations in the specimens treated compared to the control group. The alterations in animal behavior, tissue, and cells evidenced in this study confirmed the toxic effect of Roundup® on the model-test. Consequently, it is advisable to find a balance between the benefits of this type of product and the protection of the environment and human health.Item Identificação, caracterização molecular e avaliação da expressão do gene de uma proteína elicitora de defesa de trichoderma spp.(Universidade Federal de Goiás, 2011-06-30) FREITAS, Rachel Silveira; ULHOA, Cirano Jose; http://lattes.cnpq.br/8368469162867277Species of the genus Trichoderma have been used as biocontrol agents against different pathogens. The mechanisms employed by Trichoderma species against these pathogens ranging from competition for nutrients, production of non-volatile and volatile antibiotics in the production of hydrolytic enzymes, in a mechanism denominated mycoparasitism. In addition to its characteristics, many strains of Trichoderma are competent rhizosphere are able to colonize and grow in association with plant roots. The root colonization by Trichoderma spp., often is associated with the induction of local and systemic resistance. Whereas fungi of the genus Trichoderma have been described as inducers of defense responses and systemic resistance in association with maize (Zea mays), cucumber, tomato and cotton, it was our interest to analyze the interaction between Trichoderma spp. and beans. Therefore, this study aimed to identify and evaluate gene expression of protein elicitors of defense (SM1) in different isolates of Trichoderma spp. obtained from Cerrado soils. Eight isolates were selected and all showed a band of approximately 250pb, corresponding to the expected size of the gene Sm1 from T. Virens. The complete sequence of SM1 gene was obtained using as template cDNA and genomic DNA of T. harzianum. The amplification products containing an ORF of 417 bp and the protein predicted from this sequence has 138 amino acids. The ORF showed identity with sequences of other protein elicitors isolated from Trichoderma spp., and also proteins belonging to the family of cerato-platanin. Studies of the expression of SM1 with the isolate Trichoderma 37 showed that this protein is expressed in different carbon sources.Item Mapeamento de QTLs para teor de proteína em feijoeirocomum (Phaseolus vulgaris L.)(Universidade Federal de Goiás, 2006-12-15) LEÃO, Ariane Castro Mendes; CARNEIRO, Monalisa Sampaio; http://lattes.cnpq.br/2696490871291334The common bean besides being one of the basic meals of brazilian´s population, it is one of the main products that provide protein in the nutritional diet from the society share which is economically less favorable. The identification of molecular markers linked to controlling genes of the protein content in common beans (Phaseolus vulgaris L.) is a very important tool to help breeding programs, raising the efficiency and agility. This way, this work was made with two main goals: a) to map SSR and RAPD markers linked to loci (QTLs) that control protein content in two generations of a segregating population of common beans and b) to compare detection procedures of markers linked to QTLs using the ANOVA method and the process of interval mapping. For that reason, 94 families were taken from the F2 generation and 90 families from the F2:3 generation derived from the cross of genitors CNFC 7812 e CNFC 8056. Results indicated that there is the possibility of identifying molecular markers related to protein content in common beans, utilizing both detection procedures. The ANOVA method identified a greater number of QTLslinked markers than the process of interval mapping in both generations. There was coincidence between the identified loci obtained with the two methods for each generation. Loci that were associated with protein content were different for the F2 and F2:3 generations. However, there was a stable detection of a genomic region of the linkage group 4, indicating a possible role of this region of the common bean genome in the control of seed protein content. The proportion of the trait s phenotypic variation explained by QTLs varied from 5,5% to 9,5%, considering both generation.Item Estudo do potencial genotóxico, citotóxico e antitumoral do composto Cloreto de cis-tetraaminodiclororutênio(III) sobre diferentes células tumorais(Universidade Federal de Goiás, 2010-01-29) LIMA, Aliny Pereira de; LACERDA, Elisângela de Paula Silveira; http://lattes.cnpq.br/9390789693192751Current inorganic drugs such cisplatin and related compounds widely used in the treatment cancer, however its application is limited by its severe toxicity and drug resistence. These limitations have prompted a search for news metal-based antitumor agents. Ruthenium (III) complexes represent a new family of promising metal-based anticancer drugs. In the present study, was investigated in vitro the effects of the compound on cell viability, cintetics cell cycle phases, mechanisms of apoptosis and DNA damage on tumors cells. Results of the viability using MTT reduction test and the trypan blue exclusion assay on K-562 cells revealed that this compound significantly reduced the viability of the K-562 tumors cells (IC50 approximately 10.74 and 73.45 μM), respectively, moreover viability assays on A549 lung tumor cells showed that cis-(dichloro)tetrammineruthenium(III) induced effect moderate this cell lines (IC50 > 383 μM). Additionally was observed that this compound exhibits little cytotoxicity towards MRC-5 normal human fibroblast cells (IC50 > 383 μM) when compared to K562 tumor cell line (IC50 10.74 μM). Clonogenic Assay was performed on A549 cells, and observed that lower concentrations (0.38 and 3.8 μM) cis-(dichloro)tetrammineruthenium(III) diminished colony forming ability and highest concentrations (95 and 383 μM) no colony was observed. In cell cycle analysis on K-562 and S180 tumor cells cistetrammineruthenium( III) induced change in the distribution the cell cycle phase since that % of cells entering G1, S and G2 was decreased, correlating with increase in the proportion of cells in the sub-G1-peak (indicating apoptosis). In the analysis of damage to the DNA molecule of S180 cells using the comet assay, it was observed that the ruthenium compound induced damage to the DNA molecule to both treatments (24 and 48 h) as evidenced by an increase in damage index. In addition, cis-[RuCl2(NH3)4]Cl treatment induced apoptosis in cells K-562 as evidenced for increased DNA content in the sub-G1 peak (75.35%) and a significant increase in caspase-3, 8 and 9 activity. In tumor cells S180 the apoptosis was demonstrated for by a increase numbers of Annexin V-positive cells and fragmentation DNA. Taken together, these findings strongly demonstrate that cis-[RuCl2(NH3)4]Cl exerts antitumor activity and this activity correlates with DNA damage, process apoptotic and change cell cycle.Item Caracterização e Análise Funcional da Beta -1,3-glicanosiltransferase 2 de Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2010-01-29) LIMA, Patrícia de Sousa; SOARES, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a systemic disorder geographically restricted to Central and South America, and one of the most important endemic mycoses in these regions, especially among the male rural populations. The disease is most likely caused by the inhalation of asexual spores (conidia) produced by the mycelia form of the fungus, propagules that once in the lungs undergo differentiation towards the parasitic yeast form. The cell wall of P. brasiliensis is a dynamic structure, essentially composed of branched glucan (β-1,3 and β-1,6 glucans), chitin, lipids and mannoproteins. Many enzymes are responsible for cell wall remodeling. One of them is the Beta-1,3- glucanosyltransferase 2 (PbGel2p) presented here. The amino acid deduced sequence of PbGel2p presented similarity to others proteins involved in fungal cell wall biosynthesis and morphogenesis and it was characterized as a member of GH72 family, GH72_ subfamily. The recombinant rPbGel2p was overexpressed in Escherichia coli and the polyclonal antibody was obtained. The PbGel2p mRNA, as well as the protein, were detected at the highest level in the mycelium phase. The potencial role of PbGel2p in cell wall biosynthesis and morphogenesis was analyzed by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae GAS1Δ. The results indicated that PbGel2p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity. In addition, predicted protein-protein interactions between PbGel2p and others proteins of the fungus P. brasiliensis were assessed by using a S. cerevisiae two hybrid system and pull-down assay. The proteins that were found to interact with PbGel2p are: anthranilate synthase component 2 (involved in the tryptophan pathway), MYND domain protein SamB (related to fungal morphogenesis), mitotic spindle checkpoint protein Mad2B (required of the organization of the cytoskeleton and control of cell cycle), G protein complex beta subunit CpcB (organize the cytoskeleton of actin), WD repeat protein (involved in the control of cell cycle), phosphatidylinositol-4-phosphate-5-kinase its3 (required of the organization of the actin cytoskeleton), Hsp60 (related of stress conditions like temperature) and ATPase (localized in the plasma membrane / involved in the glucose metabolism). This suggested the relation of PbGel2p in other process to maintenance of cell wall integrity.Item Utilização de palhadas e extratos de Crotalária juncea L. e Brachiária decumbens Stapf.como alternativa no controle da germinação e emergência de sementes de algumas plantas daninhas(Universidade Federal de Goiás, 2009-05-01) LISBOA, Odicicléia Alves de Sousa; FERREIRA, Enderson Petronio de Brito; http://lattes.cnpq.br/6292879655540619; DIDONET, Agostinho Dirceu; http://lattes.cnpq.br/9432770938259181The allelopathy can be an alternative method to the control of weed plants. The use of cover crops straw as a mechanical barrier, or its chemical compounds resulted of staw decomposition, can inhibit the germination and growth of some weed plants. The objectives of this work were to evaluate the mechanical, allelopathyc and allelopathyc metabolite of straw decomposition effects on seeds germination of hairy beggarticks (Bidens pilosa L.), red morningglory (Ipomoea coccinea L.), goosegrass (Eleusine indica L.), milkweed (Euphorbia heterophylla L.) and bur grass (Cenchrus echinatus L.) weed plants. It were evaluated also the aquous straw effects on the germination of seeds of these weed plants and lettuce seeds, and to quantify the most important chemical metabolites in these aquous straw extracts, and the presence of phenols and flavonoids in the extracts stored during four weeks (0, 7, 14, 21 e 28 days). To obtair dormency supression and the best results in the germinations of the seeds of beggarticks (Bidens pilosa L.), red morningglory (Ipomea coccinea L.) and bur grass (Cenchrus echinatus L.), the seeds of the weed plants where immersed hot water (70 ºC) and in KNO3 solution (0,02%). Bur grass seeds were also exposed to 10 ºC for different periods. In conclusion, the crotalaria and braquiaria straws had both mechanical and allelopathyc effects on the seed germination and emergengy for the weed plants tested. When it had been observed the inhibition of the crotalaria and braquiaria aquous straws extracts on the radicular and aereal seedlings growth of red morningglory, milkweed and lettuce its effects were more pronounced at the higher used concentration. Phenols and flavonoids were found in the crotalaria and brachiaria aquous straw extracts, however its contents showed a reduction along the storing period. About the supression dormency methods utilized, It Was not observed supression dormency in all tested methods, wether some get affected the germination of the seed.Item Caracterização molecular, filogênica e enzimática de isolados de Trichoderma spp(Universidade Federal de Goiás, 2012-02-24) LOPES, Fabyano Alvares Cardoso; ULHOA, Cirano Jose; http://lattes.cnpq.br/8368469162867277Item Emissão de fluorescência e produção de massa seca em feijão e soja em função da aplicação de nitrogênio em cobertura(Universidade Federal de Goiás, 2009-06-29) MAFEI, Maloní Montanini; PORTES, Tómas de Aquino; Tómas de Aquino PortesNitrogen is considered an essential element for plants, since it is involved in the composition of various biomolecules such as ATP, NADH, NADPH, proteins, enzymes and chlorophyll. The chlorophyll is a pigment from the leaves of plants, which is directly associated with the potential of photosynthetic activity, as well as the nutritional status of the plant. The use of the variable fluorescence of chlorophyll has been widespread, especially in the study of photosynthetic capacity of plants, as a nondestructive method that allows qualitative and quantitative analysis of the absorption and utilization of light energy by photosynthetic apparatus. The objective of this research was to quantify the emission of the chlorophyll a fluorescence (F0, initial fluorescence; Variable-Fv, Fm-Maximum, Terminal-Ft and Efficiency Fotoquímica- Fv/Fm) in photosystem II and the dry matter in bean (Phaseolus vulgaris L .) and soybean (Glycine max (L.) Merr.) submitted the application of nitrogen in the form of ammonium sulfate in coverage. The experimental design was completely randomized in factorial formed by two legumes, two readings and two levels of nitrogen N0 = 0.000 g.kg-1 (control) and N1 = 0,060 g.kg-1. Nitrogen fertilization with ammonium sulfate in coverage influenced the minimum fluorescence (F0) and not influenced by other variables of fluorescence in bean and soybean. Increased the dry mass of branches for the bean, to have influenced the increase in soybean dry masses of shells of pods and number of podsItem Produção de celulas e xilanases pelo fungo termofílio Humicola grisea var. thermoidea em diferentes substratos lignocelulósicos(Universidade Federal de Goiás, 2010-08-30) MELO, Guilhermar Ramos de; FARIA, Fabrícia Paula de; http://lattes.cnpq.br/3739169267521003The vegetal biomass consists mainly of cellulose, hemicellulose and lignin. Cellulose is the most abundant polymer and xylan is the main component of hemicellulose. The conversion of cellulose and xylan to glucose and xylose can be realized by an enzymatic complex found in secretions of microorganisms such as fungi and bacteria. Enzymatic hydrolysis is an important step to the bioconversion of cellulosic and hemicellulosic fraction from lignocellulosic wastes. The thermophilic fungus Humicola grisea var thermoidea produces an efficient complex of cellulolytic enzymes (endoglucanases, cellobiohydrolases and β-glucosidase) and xylanolytic (endoxylanase and β-xylosidase) with high thermostability when grown on different lignocellulosic substrates. The aim of this study was to analyze the kinetics of production of cellulases and xylanase by the fungus H. grisea cultivated on medium containing rice straw (RS), corncob (CC), crushed cane sugar bagasse (CSB) and wheat bran (WB) as carbon source and subsequently analyze the profile of proteins with cellulolytic and xylanolytic activity secreted by the fungus when grown in minimal medium, by liquid fermentation, containing the substrates at concentrations of 1, 2 and 3% and maintained at 42 ° C, 120 rpm for different times. The best results were obtained when the fungus was grown in 3% BCA and FT, the peaks of FPase (0.17 U / mL) and CMCase (3.54 U / mL) were observed after 192 h of growth with 3% BCA , peak avicelase (0,195 U / mL) after 48 h with 3% FT and peak xylanase (23.75 U / mL) after 216 h with 3% FT. The results showed that the best inducer of enzyme production with FPase and CMCase activity was the CSB and the best inducer of enzymes production with xylanase and avicelase activity was the WB. In profile analysis of proteins secreted by H. grisea by SDS-PAGE (216 h) and zymogram (144 h), no band was seen when the fungus was grown in the presence of glucose, suggesting catabolite repression. However, two very strong protein bands corresponding to HXYN2 (23 kDa) and CBH1.2 (47 kDa) were visualized in the gels containing CSB (2 to 3%) and WB (2 and 3%). These enzymes are the main xylanolytic and cellulolytic systems of the fungus, respectively. Were monitored by recombinant enzymes from H. grisea (in gels), an endoxylanase HXYN2r (23 kDa), an cellobiohydrolase CBH1.2r (47 kDa). The masses full profile of H. grisea can be seen in Figures 13, 14, 15, 16, 18 and 19.Item Efeito de antioxidantes aos espermatozoides de equinos submetidos ao estresse térmico(Universidade Federal de Goiás, 2010-10-05) OLIVEIRA, Aline França Dias; GUILLO, Lídia Andreu; http://lattes.cnpq.br/3401436781775091The evaluation of the reproductive capacity of stallions and semen cooled and / or frozen is of fundamental importance in practical breeding of horses. Whereas predicting the fertility of a stallion is still a subjective decision, the present study was conducted to evaluate different staining techniques, as well as tests that assess the viability of semen in horses, studying the effects of the addition of cysteine and glutathione in plasma membrane integrity of sperm DNA, and to evaluate the effects of the addition of these antioxidants in preserving the viability of sperm undergoing incubation and refrigerated for short periods. We used semen from six stallions, wich were split into seven samples, one kept as control (Control Group) and other antioxidants cysteine was added at a ratio of 1.0 mM (Group C1, 0), 1.5 mM (Group C1, 5), 2.5 mM (Group C 2.5) and glutathione also at 1.0 mM (Group G1, 0), 1.5 mM (Group G1, 5) and 2.5 mM (Group G 2.5 ). All tests were performed every six hours. The three treatments were: Treatment I cooled to 12 ⁰C for 12 hours (zero hour; 6h resf.; 12h resf.) Chilled in boxes; Treatment II incubated in a water bath at 37 ⁰C for 12 hours (6h 37 ° C, 12h 37 ° C) and Treatment III - cooled and subsequently incubated, according to the treatments I and II. The evaluation of plasma membrane integrity was performed by staining eosine-nigrosin; acrosomal membrane by FITC-PSA and DNA by acridine orange. For each analysis were numbered 200 to 500 cells. The evaluation of the viability of sperm was performed by MTT assay according to Mosmann (1983). The results showed that antioxidants were effective (p <0.05) in keeping the DNA intact chromatin, especially glutathione. In the acrosome of antioxidants were protective, at times 18 and 24 hours, and in other treatments, no significant difference (p> 0.05) between control and treated groups. The MTT test showed that groups treated with antioxidants had absorbance values similar to those of control, showing positive effect (p <0.05) only when cooled by six o'clock in the cysteine group 2.5. In relation to the plasma membrane of spermatozoa stained with eosin-nigrosin, no protective effect of antioxidants in the samples. The values of their averages were close to the control group (p> 0.05). One factor was estimated that cooling per se, independent of the addition of antioxidants used, has been effective in protecting the sperm. And the incubation at 37 ⁰ C causes these cells, and the addition of cysteine and glutathione were efficient, if not protect, but to maintain the integrity of the factors evaluated, not causing more damage to sperm.Item Caracterização da expressão de inibidores de poligalacturonases (PGIPs) em resposta aos estresses biótico e abiótico em plantas de feijão comum (Phaseolus vulgaris L.)(Universidade Federal de Goiás, 2011-11-26) OLIVEIRA, Marília Barros; SILVA, Silvana Petrofeza da; http://lattes.cnpq.br/6823998544968373Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen that causes the disease known as white mold in many plants. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin. To counteract the action of PGs, plants express polygalacturonases-inhibiting proteins (PGIPs) that reduce the hydrolytic activity of endo-PGs and favor the accumulation of oligogalacturonides (OGs) with are elicitors of a variety of defense responses. PGIPs belong to the superfamily of leucine rich repeat (LRR) proteins and play important roles in resistance to infection of pathogens. In this study, real time RT-PCR was used to evaluate the expression of Pvpgip genes in dry bean plants (Phaseolus vulgaris L) submitted to different stress conditions. Transcriptional analysis showed that these genes are differentially expressed and activated by biotic (S. sclerotiorum infection) and abiotic (wound or methyl jasmonate treatment) stresses. Pvpgip1 was induced at early stages of the infection especially for plants treated with methyl jasmonate (MeJA) in which the transcript accumulation was higher. High levels of Pvpgip2 and Pvpgip3 expression were observed in infect plants treated or not with MeJA. All treatments showed induction of gene Pvpgip4. These results show that the/ four genes Pvpgip respond differently to treatment with the resistance inducer, fungal infection and wound.Item Avaliação do Potencial Citotóxico, Genotóxico e Antitumoral do Ditionato de cis-Tetraamino(oxalato)rutênio(III) em Diferentes Linhagens Celulares(Universidade Federal de Goiás, 2010-01-22) PEREIRA, Flávia de Castro; LACERDA, Elisângela de Paula Silveira; http://lattes.cnpq.br/9390789693192751Despite the resounding success of cisplatin and closely related platinum antitumor agents, the movement of other transition-metal antitumor agents toward the clinic has been exceptionally slow. Non-Platinum chemotherapeutic metallopharmaceuticals hold much promise for the future, and needs to be actively explored in a large variety of tumor types in combination therapies. The preparations of metallocomplexes with potential antitumor activity has been one of the main targets of transition metal chemistry since Rosenberg s discovery of cisplatin cisdiamminedichloridoplatinum (II), cis-[Pt(NH3)2Cl2]} cytotoxic activity in the 1960s. In 1978, cisplatin was approved as the first platinumbased drug for the oncology treatment, although several negative side-effects (nephrotoxicity, neurotoxicity, nausea, etc.) had been induced on treated patients. Nevertheless, cisplatin was followed by carboplatin {cis-diammine-1,1´ - yclobutanedicarboxylateplatinum(II), [Pt(NH3)2(cbdc)], approved in 1985} and oxaliplatin 1R,2Rdiamminocyclohexaneoxalatoplatinum(II), [Pt(dach)(ox)], approved in 1996}, which met requirements of improving antitumor activity and reducing disadvantages of cisplatin, carboplatin and oxaliplatin represent the second, and third platinum-based drug generations, respectively. Nowadays, not only platinum-bearing complexes are extensively studied with the aim to broaden a spectrum of transition metal-based complexes which could be used in the treatment of cancer. Ruthenium complexes have shown potential utility in chemotherapy and photodynamic therapy. Ruthenium complexes generally have lower toxicities compared to cisplatin attributed to their specific accumulation in cancer tissues. In vitro and in vivo studies show high anticancer activity of Ruthenium complexes and some of them are currently undergoing clinical trials. In the present work we studied the antitumor activity of the Ruthenium(III) compound cis-Tetraammine(oxalato)Ruthenium(III) Dithionate {cis- [Ru(C2O4)(NH3)4]2(S2O6)} against different tumor and normal cells lineages, analising cell viabilities, cell cycle distribution, apoptosis induction mecanistics and genome DNA damage. Correlation tests were performed to determine the effects of the time of exposure and concentration of Ruthenium complex on mitotic index (MI) and mitotic aberration index on Allium cepa root cells. A comparison of MI results of cis- [Ru(C2O4)(NH3)4]2(S2O6) to those of lead nitrate reveals that the Ruthenium complex demonstrates an average mitotic inhibition eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of Ruthenium complex concentrations did not display significant clastogenic effects. The cis- Tetraammine(oxalato)Ruthenium(III) Dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Results showed that Ruthenium(III) causes a significant reduction of proliferation of A549 cells with viabilities ranging from 55.5% to 24.6% when treated with 40 μM for 24 and 48h; and 32% to 18.2% when treated with 150 μM for 24 and 48h. The Ruthenium(III) compound induced a moderate (31.9% and 39.6% for concentrations 10 and 40 μM, respectively) to high degree (74% for concentration 32 μM) of cytotoxic activity against A549 cells (IC50= 33.72 μM). On the other hand, the normal lung fibroblast MRC-5 did not show significant reduction proliferation in the presence of Ruthenium(III) compound. Even when treated with higher concentrations of cis-Tetraammine(oxalato)Ruthenium(III) Dithionate for 48 hours, MRC-5 cells showed viabilities ranging from 85% to 78,4% for 40 μM and 150 μM, respectively. The antiproliferative and cytotoxic activity revealed that K562 cells cultured with concentrations 40 and 150 μg mL-1 of Ruthenium(III) compound showed significant reduction of proliferation after 72h of exposition, with viabilities ranging from 88.2% to 55.6% when treated with 40 μM for 24 and 72h; and 76.2% to 26.7% when treated with 150 μM for 24 and 72h. The Ruthenium(III) compound induced low [22.4% (24h) to 28.2% (48h) and 29.8% (24h) to 35.7% (48h) for concentrations 10 and 40 μM, respectively] to moderate [44% (24h) and 53% (48h) for concentration 150 μM] of cytotoxic activity against K562. After incubation for 48 h, the IC50 value was 18.28 μM. Compared to the cell cycle profiles of untreated cells, flow cytometric analysis indicated a sub-G1 arresting effect of Ruthenium compound on K562 cells, inducing a 1.7-fold, 2.2-fold and 2.4-fold increase in the number of sub-G1 cells for 24, 48 and 72 h, respectively, when compared to control. The compound also caused a significant increase in tailed cells in any of the concentrations tested compared with negative control that can be associated cytotoxicity with direct effect on K562 cells DNA.