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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 Avaliação da atividade da invertase de Saccharomyces cerevisiae imobilizada em polianilina sobre o caldo de cana(Universidade Federal de Goiás, 2010-02-19) BARBOSA, Eduardo Fernandes; FERNANDES, Kátia Flávia; http://lattes.cnpq.br/9737543228759171This work describes the immobilization of invertase on chemically synthesized polyaniline and activated with glutaraldehyde (PANIG) for production of invert syrup from sugarcane juice. Free invertase activity present in crude extract (E.B.) obtained from cells of Saccharomyces cerevisiae, was characterized for an evaluation of interferents present in the extract on enzyme activity (optimum conditions: temperature 50 ° C, pH of 4.5 in sodium acetate buffer 0.1 mol L-1 and reaction time of 10 minutes, with an activity of 11.31 ± 0.36 EU mL-1). We tested some parameters optimization of enzyme immobilization, such as amount of enzyme, immobilization time, pH and temperature of immobilization. The optimal immobilization was obtained in buffer sodium acetate 0.1 mol L-1 pH 4.5, immobilization time of 1 hour at 50°C and 169.55 EU mg-1 PANIG. The efficiency of immobilization was 0.86. The stability of the system PANIG-Invertase was tested against the storage time and thermostability, and after 75 days storage in buffer sodium acetate 0.1 mol L-1 pH 4.5 was obtained for 94% of initial activity with only 17% retained for the free enzyme. The immobilized invertase didn t change the optimal conditions compared to the free, but the immobilized was more stable in adverse conditions such as pH below and above optimum conditions showed an increase in thermostability. Some features of the hydrolysis product were evaluated (water activity, viscosity and color), compared to the sugarcane juice in nature, showing that the reactors allowed changes in sugarcane juice that expand the possibilities for using syrup obtained in the production of sweets, ice cream and syrups rich in fructose. The high stability of the system tested, along with its high retention of activity strongly suggests the use of the system in reactors.Item Morfo-anatomia e fitoquímica de Cymbopogon densiflorus (Steud.) Stapf e Cymbopogon nardus (L.) Rendle (Poaceae: Panicoideae)(Universidade Federal de Goiás, 2007-09-28) BARBOSA, Lília Cristina de Souza; PAULA, José Realino de; http://lattes.cnpq.br/3191837532986128; REZENDE, Maria Helena; http://lattes.cnpq.br/5093753722360659The genus Cymbopogon Sprengel belong to the Poaceae family and it has 40 species distributed in Tropical and Subtropical Africa, Asia and Australia, although some species went introduced in America. Many species of this genus are cultivated for the extraction of essential oil, from their leaves, with large medicinal, food and industrial importance. The species in focus, Cymbopogon densiflorus (Steud.) Stapf and C. nardus (L.) Rendle are originated from Africa and Asia, respectively. This research had as objective, to broaden the knowledge about the species C. densiflorus and C. nardus, by the morphological and anatomy studies from leaves and culms, phytochemical analysis and essential oil analysis from the leaves. Anatomical studies have been of relevant importance to the pharmacognosy researches, mainly for the identification of many vegetal raw materials. Several times, these raw materials are known by the same popular name or then, they are commercialized with contaminated agents or with other parts of the specie. Through of anatomical analysis, it was checked commons characters, such as leaf lamina and sheath amphistomata, stomatas with guard cells dumbbell and subsidiary cells dome-shape, rares in adaxial surface and abundant in abaxial surface, predominated in intercostal zones. The adaxial and abaxial surfaces had long cells and short cells: cork and dumb-bell and cross-shaped silica cells, these last it is placed in costal zones; macro-hairs and micro-hairs abundant in abaxial surface. In the leaf lamina, bulliforms cells are presents in adaxial surface, they were alternated with fibers in the costal zones and the mesophyll is homogeneous with chlorenchyma radiated to the bundle sheaths and arm cells with walls invaginated that they determined the intervenal distance by one or three cells, characterized Kranz anatomy. Bundle sheaths collateral, of 1st, 2nd and 3rd orders with single vascular bundle sheaths. The cap region is constituted by sclerenchyma and the epidermis has silica cells. However, both species had different anatomical features, as the form of midrib, in the leaf laminas; C. densiflorus showed colourless parenchyma cells in the mesophyll of leaf sheaths, that they do not exist in C. nardus. In the culms, numbers of metaxylem vessels in the each side of protoxylem vessels in vascular bundles: 1, in C. densiflorus, 2 and 3, in C. nardus; and the presence of sclerenchyma cylinder and fistula in C. nardus, absent characters in C. densiflorus. Moreover, in C. densiflorus, while C. nardus showed these characters. The preliminary phytochemistry analysis C. densiflorus and C. nardus leaves evidencied flavonoids, saponins, coumarins and traces of cardioactive glycosides. In the essential oil analysis, C. densiflorus leaves showed trans-p-mentha-1(7),8-dien-2-ol, trans-p-mentha-2-8-dien-1-ol, cis-carveol and cis-p-mentha-2-8-dien-1-ol as majority constituents; while C. nardus leaves had geraniol, citronellol and citronellal. The anatomical characters observed can be important to the taxonomic determinations of species studied, in the genus. Through the results found, it verifies the phytotherapics potential of both species. Future researches in isolation and purify of the secondary metabolites, pharmacologics and toxicologics analysis of extracts and of the essential oil, it will be important to assure the therapeutic efficiency of these.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 Implicações clinicopatológicas da proteína epCAM no carcinoma ductal infiltrante de mama(Universidade Federal de Goiás, 2008-07-31) CAIXETA, Gustavo Nogueira; SADDI, Vera Aparecida; http://lattes.cnpq.br/7496804650895441; AYRES, Flávio Monteiro; http://lattes.cnpq.br/1264753154131795Breast cancer is the second most common tumor in the world and the first among women. In the past two decades, several breast cancer studies focused on the expression and on the possible routes of cellular signaling for the epithelial cell adhesion molecule (EpCAM). This molecule was found to be overexpressed in various malignant tissues of epithelial origin, including breast cancer. The importance of EpCAM protein in tumor cells is still controversial. First, because it is believed that the cell adhesion role of EpCAM reduces the ability of metastasis. On the other hand, replacing the strong cell adhesion performed by cadherins instead of weak homophilic adhesion of EpCAM may be associated with the promotion of invasion and tumor metastasis. The aim of our study was to analyze the implications of EpCAM in breast ductal carcinoma infiltration. By using immunohistochemistry methods, we noticed the absence of EpCAM expression in 10 case (10,2%), low EpCAM levels expression in 45 cases (45,9%) and overexpression in 43 cases (43,9%). Unlike other published studies, the overexpression of the EpCAM protein did not correlate with the five-year overall survival of breast cancer patients, even those with axillary lymph node involvement. Similarly, there was no correlation between EpCAM s protein expression and classical prognostic factors, including the size of the tumor, axillary lymph node involvement, hormone receptors, c-erbB-2 overexpression and p53 immunodetection. The presence of distant metastasis , reported in 26 cases (26,5%), did not correlate significantly with the overexpression of EpCAMItem 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 Clonagem e expressão do gene da tiorredoxina 1 de Paracoccidioides brasiliensis em Pichia pastoris(Universidade Federal de Goiás, 2010-08-27) CINTRA, Lorena Cardoso; FARIA, Fabrícia Paula de; http://lattes.cnpq.br/3739169267521003; JESUÍNO, Rosália Santos Amorim; http://lattes.cnpq.br/5113656623817587The termodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human systemic mycosis of high prevalence in Latin America. P. brasiliensis is exposed to oxidative stress (OS) caused by reactive oxygen species (ROS) produced by the defense cells of the human host. When the invasion by pathogens occurs, the host defense system generates ROS to fight the invader. Inside the human host, P. brasiliensis is phagocytosed by macrophages, facing an extremely hostile environment due to nitric oxide and hydrogen peroxide. The Trx1 is an intracellular redox protein, which participates in the maintenance of cell redox homeostasis, both in terms of OS as reducer. It is ubiquitous and is characterized by typical CXXC active site, responsible for oxidation, reduction, or isomerization of proteins disulfide bonds. In a previous work, it was isolated, characterized and cloned into expression vector pGEX-4T-3 cDNA coding for TRX1 of P. brasiliensis (accession number AY376435). The recombinant protein (recPbTRX1) was produced and partially purified and the yeast cells of P. brasiliensis showed increased expression of the gene coding for PbTRX1 in response to OS. This study aimed the heterologous expression of cDNA of a thioredoxin of the fungus P. brasiliensis in Pichia pastoris, in order to obtain it in larger amounts for their subsequent biochemical characterization and application in biotechnological processes. The P. brasiliensis thioredoxin 1 (trx1) cDNA was obtained via PCR using the plasmid pGEX-Trx1 as template and cloned into expression vector pHIL-D2 and pPIC9 (for intracellular and extracellular expression). The insertion of the interested gene in the correct orientation was verified by sequencing and the homology was observed with Trx1 P. brasiliensis. These vectors were used to transform the P. pastoris yeast strain SMD1168 with his4- genotype. The presence of the cassette s expression was confirmed in the yeast s genome. No transformants able to secrete the protein from the building with the vector pPIC9 were detected and the intracellular production was carried from the pHIL-D2 vector.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 Resposta do feijão e da soja à inoculação com rizóbios e submetidos a diferentes níveis de sombreamento(Universidade Federal de Goiás, 2009-04-30) FERNANDES, Daiana Pereira; PORTES, Tómas de Aquino; Tómas de Aquino PortesThe soybeans and beans are of great economic and social importance to Brazil. From the nutritional point of view, they are very demanding in nitrogen and to supply its demand both species are able Biological Nitrogen Fixation (BNF). However, for the BNF, rhizobia require energy supply by the carbohydrates from photosynthesis. For this reason the availability of fotossintato is the biggest limiting factor in BNF. This work had as propose to evaluate the influence of inoculation with rhizobia in two phenological stages of plants under different levels of shading. In this context the inoculation was assessed by the following treatments: two species of legume (Phaseolus vulgaris, L. and Glycine max, L.) inoculated and not inoculated (Fi, Fni, Si and Sni) with specific strains (Rhizobium tropici and Bradyrhizobium japonicum and B. elkanii) in two phenological stages (before, Ep1, and after flowering, Ep2) and subjected to three shadow levels (no shade, 70% and 80% of shade). The experimental design was completely randomized in four replicates on the experiments 1, 2, 3 and 4 conducted in a greenhouse and three replicates for the experiments 5, 6, 7 and 8 in the external environment to a greenhouse. In experiments 1 and 3, both species have continued forming nodules after flowering, the plants of these treatments Fni Ep2, Sni Ep2 and Si Ep2 had the highest average for the number, volume, fresh weight and dry weight of nodules. As the activity of the nodules, the plants of treatments Fi Ep1 and Si Ep1 had the highest number of active nodules compared to treatments Fni Ep2 and Sni Ep2. Assessing the effect of shading in experiments 2 and 4, it was found that bean and soybean plants have behaved differently. The shading of 80% favored the formation of nodules in inoculated bean deferring between treatments of shading, in contrast to the soybean plants that there was no difference (p ≥ 0.05) among treatments. There was no statistical difference between treatments in the number of active nodes for the beans; the plants of the Sni treatment 80% had the lowest number of active nodules compared to the plants of treatment Si 0%. In experiments 5 and 7 plants of soybeans and reduced the number of nodules after flowering, and as in Experiment 1 and 3 there was a higher number of active nodes during the growing season compared to the reproductive period. The shading applied in bean plants (Experiment 6) was not enough to cause no difference between treatments on the number and activity of nodules; in the experiment with 8 soybean, the shading changed patterns of nodulation and activity of nodules (p ≥ 0.05). It is this work that the formation and maintenance of activity of the nodules were so distinct in vegetative and reproductive stages, but the shading of 70 and 80% applied was not sufficient to cause significant change in activity of the nodules, suggesting that the reduction the availability of fotossintatos is not the limiting factor in determining the efficiency of N, mainly in the beanItem Avaliação da atividade mutagênica e antimutagênica da Annona crassiflora Mart. (Araticum) pelo teste do micronúcleo em camundongos Mus musculus(Universidade Federal de Goiás, 2001-08-23) FERREIRA, Francinez Linhares; CHEN, Lee Chen; http://lattes.cnpq.br/4621907105842007RESUMO CAPÍTULO I The araticum (Annona crassiflora Mart.) is a typical brazilian plant found in cerrados of Brazil. This plant contains acetogenins that presents cytotoxic, antitumorigenic, antiparasitic and antimicrobial properties. Its leaves, barks, fruits and seeds are used by the population as therapeutic medicine to treat several diseases as diarrhoea, rheumatism and syphilis. In the present study we have evaluated the mutagenic activity of the crude ethanolic extract of leaves of araticum though quantification of micronuclei induced in mice bone marrow. Doses of 10 mg/kg (5% of LD50), 20 mg/kg (10% of LD50), 50 mg/kg (25% of LD50), 100 mg/kg (50% of LD50) and 160 mg/kg (80% of LD50) were applied i.p. in mice Mus musculus, in groups of 5 (five) animals for each dose. The cytological preparations were made according to Heddle`s methodology. For all the applied doses, frequency of micronucleated polychromatic erythrocytes (MNPCE) was evaluated after 24, 48 and 72 h of treatment. The cytotoxicity was evaluated by ratio the between numbers of polychromatic and normochromatic erythrocytes (PCE/NCE). Ours results indicated an absence of significantly increased of micronuclei for all the doses tested (p>0,01). Thus, we concluded that the phytoterapic araticum did not present mutagenic activity under our experimental conditions. However, the toxic activity was observed upon analysis of LD50 and PCE/NCE ratio. RESUMO CAPÍTULO II Annona crassiflora Mart. (araticum) is a typical brazilian plant found in cerrados of Brazil. This plant contains acetogenins that presents cytotoxic, antitumorigenic, antiparasitc and antimicrobial properties. Its leaves, barks, fruits and seeds are used by the population as therapeutic medicine to treat several diseases as diarrhoea, rheumatism and syphilis. In the present study we have evaluated the antimutagenic activity of the crude ethanolic extract of leaves of araticum through quantification of micronuclei induced in mice bone marrow. Doses of the extract (10mg/kg, 20mg/kg, 50mg/kg, 100mg/kg and 160 mg/kg) and MMC (4mg/kg) were co-applied i.p in mice Mus musculus in groups of 5 (five) animals for each dose. The cytological preparations were made in according to Heddle s methodology. For all the applied doses, frequency of micronucleated polychomatic erythrocytes (MNPCE) was evaluated after 36 h of treatment. The obtained results indicated that the crude ethanol extract from leaves ay doses of 20 mg/kg, 50 mg/kg and 100 mg/kg inhibited significantly reduced frequencies of micronuclei (P<0.01). Thus, we concluded that the phytoterapic araticum exerted an antimutagenic effect.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 Utilização do inibidor de papaína extraído de sementes de Adenanthera pavonina L. na purificação de proteases cisteínicas(Universidade Federal de Goiás, 2010-03-25) GAMBÔA, Adriane Guimarães; LOPES, Flavio Marques; http://lattes.cnpq.br/1423301895802989; FERNANDES, Kátia Flávia; http://lattes.cnpq.br/9737543228759171In this work papain inhibitor from A. pavonina was immobilized by covalent bond in polyaniline (PANI) modified with glutaraldehyde (PANIG) for be used as stationary phase for affinity chromatography and then applied in the purification of cysteine proteases bromelain and ficin. The extraction and purification of inhibitors protease from A. pavonina resulted in a yield of 3.9% in the last step of purification. Gel filtration chromatography performed in Sephadex G-75 resin as a purification step resulted in three protein peaks (F1, F2 and F3), but only F1 was used in the experiments of immobilization because of higher specific activity. Immobilization was performed using PANIG. To optimize the immobilization conditions the amount of PANIG in the reaction (5, 10 and 15mg), time (30, 60 and 90 min) and pH (5.0 to 8.0) were varied. The best conditions for immobilization of A. pavonina inhibitor, according to tests performed were 5mg PANIG, reaction time of 30min and pH 7.0. PANIG-I was used as bio-affinity stationary phase for separation of bromelain and ficin. Electrophoresis (SDS-PAGE) performed after the separation process revealed the presence of a single band for both bromelain and ficina, with 28 and 25 kDa, respectively. In this process, ficin was purified 2,60 fold and bromelain 0,89 fold, showing that the use of inhibitors of A. pavonina immobilized in PANIG were efficient in the purification of cysteine proteases.Item Análise de populações de Sclerotinia sclerotiorum em cultura de feijoeiro através de marcadores SSR.(Universidade Federal de Goiás, 2009-09-30) GOMES, Eriston Vieira; SILVA, Silvana Petrofeza da; http://lattes.cnpq.br/682399854496837379 Sclerotinia sclerotiorum isolates were collected under central pivot irrigations sistems, from Brazilian fields, divided in 4 populations (S2, S3, A and M) and analyzed to determine the genetic variability among and between populations using molecular markers based on microsatelittes. 10 primers were used and the amplification products were separate in poliacrilamida gels and the bands were silver stained. A total of 102 different haplotypes were identified, and the amount of haplotypes varied from 6 to 18 for locus. The genotypic diversity ranged from 65% to 91%. Analyses based on genetic diversity and fixation indices which indicate the variability between populations was 28.79% (FST = 28793) and the variability among populations was 71.21%. The Jaccard similarity index indicated that the populations S2 and A is genetically closer. The population S3 presented a similarity index of 0.44 compared with the populations S2 and A. The population M, originated from several collection sites, was considered genetically more distant showing a index of 0.46 compared with the others populations . The high variability between and among populations can indicate that, besides the possible introduction of new genotypes in the analyzed fields, could be having clonal and sexual reproduction in isolated of S. sclerotiorum from Brazilian cerrado.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.
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