Programa de Pós-graduação em Ciências Biológicas
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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 Ánálise de alterações no gene receptor de andrógeno em homens com infertilidade idiopática(Universidade Federal de Goiás, 2009-03-31) MESQUITA, Wyara Elanne de Jesus Castro; JESUÍNO, Rosália Santos Amorim; http://lattes.cnpq.br/5113656623817587Male idiopathic infertility is related to defects in normal spermatogenesis, due to genetic causes. The spermatogenesis is a dependent process on high levels of male sex hormones, the androgens. The androgen, in turn, perform its function when associated with the androgen receptor (AR), protein encoded by AR gene. Mutation in AR gene lead to a synthesis of non functional AR, which results in the failure of the process of spermatogenesis and, consequently, causes male infertility. This work has as its main objective the verification of the occurrence of mutation in the AR gene in patients with male idiopathic infertility who come from the HC-UFG Human Reproduction Center. Samples were analyzed from 206 patients. The result was that 95 patients were found to be normal while 111 with an altered result for the spermogram. The samples were amplified for exons 1, 4, 6, 7 and 8 of the AR gene and the results subjected to statistical analysis, Mann Whitney, logistic regression, and chi tests. The existence of the relationship between defects of sperm and AR gene mutation was verified. The analysis of the relationship between the spermogram and the AR gene mutation in five evaluated exons was significant only for exons 1 and 7. Patients with numerical unsettled spermogram had a higher frequency of mutations in exon 7, teratozoospermics in exon 1 and exon 7 in astenozoospermics patients. Exons 4, 6 and 8 showed no meaningful statistical relationship in reference to the alteration of the spermogram. Among results related to social custom, alcohol proved to be significant for mutation in the AR gene. This study has reaffirmed the relationship between the presence of mutation in AR genes as probable causes of defects in spermatogenesis. Consequently, male idiopathic infertility depends not only on the genetic factor, but also on the association between this factor and the environment where man inhabitsItem Identificação e descrição morfoanatômica e farmacognóstica das folhas de Solamum Scuticum M. Nee e bioatividade de extrato bruto em microorganismos e da fração alcaloídica em células cultivadas da linhagem vero(Universidade Federal de Goiás, 2008-04-16) MORAES, Leslivan Ubiratan de; PAULA, José Realino de; http://lattes.cnpq.br/3191837532986128; SABÓIA-MORAIS, Simone Maria Teixeira de; http://lattes.cnpq.br/6723881044959716The jurubeba was identified that such species was Solanum scuticum M. Nee, from which pharmacognostic and morphanatomic data were not available to Goiás. Because of that, we tried some complementary data. For the morphoanatomic evaluation of the leaves some cuts were carried out as described by Kraus and Arduin (1997). For the phytochemical trial, it was used some methodologies as described by Costa (2001). With the phytochemical screening, were identified alkaloids, flavonoids, coumarins, anthraquinones and saponins in prospecting phytochemical. Both in phytochemical trial, as in the phytochemical prospection, it was found alkaloids. Due to the biological activities of these secondary metabolites, we obtained the alkaloid fraction, using the dust of the leaves, and we got the ethanol extract. The fractions were divided by polarity and they were evaluated by thin-layer chromatography. Microbiological evaluation was carried out to verify possible contaminants. Such evaluation did not reveal the presence of microorganisms, and it was raised the possibility of antimicrobiotic activity of the raw extract in twenty three strains of bacteria and in two yeasts. According to the antimicrobial tests, the extract presented some features, as the difficult of solubility in aqueous medium, and the best dilution was achieved in dimethyl sulfoxide (DMSO) 50%. These tests also demonstrated the difficult of the solubilization of the fractions, which it will be used in the experiments of Vero cells cultures. It was verified the low antimicrobial activity of the raw hidroethanolic extract. Of all the fractions, the alkaloid fraction presented the best solubility in DMSO 0.3%. So, it was proceeded the dilution and the evaluation of the alkaloid fraction in Vero cells, and it was evaluated morphology, viability and cellular proliferation. We finally verified the citotoxicity of the alkaloid fraction in the statistical analyses, and in the vitality test carried out by Trypan blue, and also in the morphological alterations compatible to citotoxity alterations.Item Sequência completa e caracterização do plasmídeo crípico pVCM1 isolado de Salmonella enterica(Universidade Federal de Goiás, 2009-03-26) PENIDO, Ana Flávia Batista; BATAUS, Luiz Artur Mendes; http://lattes.cnpq.br/5637230378599476Samonella spp are Gram negatives bactérias belonging to Enterobacteriaceae family. S. enterica comprise about 2.500 sorovars. These sorovars can infect a broad range, including poultry, cattle, swins and humans, and are agent causative of salmonellosis an important public health issue worldwide. Small multicopy plasmids are frequently isolated from Gram negatives and Gram positives bacterias. In Salmonella, low molecular weight plasmids are found on 10% of Salmonella strains and their biological functions are unknown. However, many plasmids in Salmonella control important properties, such as, virulence factors, heavy metals and antibiotics resistance, and utilizations of alternative carbon sources. The pVCM1 plasmid was extracted from one strain of Samonella enteretidis isolated from broilers carcass. The strains were grown in liquid or solid Luria-Bertani broth at 37 °C. The plasmid was purified, separated on 1% agarose electrophoresis and visualized by ethidium bromide staining for analysis. Plasmid was digested with EcoRI enzyme and subcloned in the pUC18 vector.The plasmidal stability was evaluated, inoculating E. coli cells transformated with pVCM1 plasmid (cloned in pUC18) in liquid Luria-bertani broth supplemented with ampicillin. The pVCM1 was stable after 240 generations. The total DNA sequence of plasmid pVCM1 has 1981 pb. Genbank search resulted that pVCM1 showed 99% of identity with pB and 92% with pJ, which were isolated from Salmonella enteretidis. Only one ORF founded in pVCM1 showed significative similarity with others proteins of GenBank. The protein encoded by this ORF showed homology to Rep proteins of plasmids that replicates by rolling-circle mechanism. The pVCM1 posses three impotents elements: rep gene, single strand origin (SSO) and inverted repeat sequences. Such elements are importants for the rolling circle replication, suggesting that pVCM1 use this mechanism. The rep gene was amplified and cloned in the pGEMT-easy vector, but the heterologous expression of Rep protein wasn t gotten successfully.Item Ação antimicrobiana de enzimas hidrolíticas produzidas por Trichoderma asperellum e imobilizadas em blendas de polímeros biodegradáveis.(Universidade Federal de Goiás, 2011-01-28) SILVA, Barbara Dumas Santos; ULHOA, Cirano Jose; http://lattes.cnpq.br/8368469162867277; FERNANDES, Kátia Flávia; http://lattes.cnpq.br/9737543228759171The hydrolytic activity of enzymes produced by Trichoderma asperellum, immobilized biodegradable films, as growth inhibitor of microorganisms was tested. The inhibitory activity was demonstrated on Aspergillus niger, Penicillium sp. and Sclerotinia sclerotiorum, microorganisms usually related to the attack and/or food contamination at the field or packaged. We used two polymer blends with different compositions, cassava starch and poly-butylene adipate-co-terephthalate (Ecoflex®, BASF Chemical Company) and other composed for polyvinyl alcohol (PVA) and polysaccharide cashew gum (PEJU). T. asperellum was induced to produce enzymes involved in the attack mycoparasite (N-acetylglucosaminidases, β-1,3-glucanases, chitinases and proteases) by the addition of crude chitin in the growth medium. The enzymes produced in major quantity were N-acetylglucosaminidase and chitinase. The pool of enzymes produced in the experiments was then used for immobilization tests. The immobilization process was performed in films by two methods: covalent and ionic bonding. In both methods, the presence of immobilized hydrolytic enzymes resulted in reduced growth of microorganisms, but the covalent immobilization of the results were more expressive. S.sclerotiorum was the microorganism most sensitive, followed by A. niger and Penicillium sp. To confirm the action of hydrolytic enzymes produced by T. asperellum and evaluate the effects they produce cell wall of microorganisms and other structures, the films with enzyme immobilized by covalent bonding were subjected to scanning electron microscopy. The structures most affected were hyphae and spores. Overall, the synergistic action of all enzymes produced by T. asperellum, reduced the growth of microorganisms when immobilized on the surface of the films Starch-Ecoflex® and PVA-PEJU. Moreover, the polymer blends tested exhibited desirable characteristics for future use in food packaging and most importantly, also provide efficient systems for the immobilization of enzymes.