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Orientadores: Jose Andres Yunes, Paulo Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T03:29:24Z (GMT). No. of bitstreams: 1 Sousa_SylviaMoraisde_D.pdf: 2327103 bytes, checksum: 46b30ffc017a15b7cd82326bbf0adc53 (MD5) Previous issue date: 2006 / Resumo: A análise do banco de ESTs de endosperma de milho (MAIZEST) revelou que o gene da sorbitol desidrogenase (SDH) é o transcrito mais abundante no início do desenvolvimento da semente (aos 10 dias após a polinização - DAP). A SDH cataliza a redução NADH-dependente da frutose em sorbitol ou a oxidação do sorbitol em frutose. Em Rosaceae esta enzima tem um importante papel na translocação do sorbitol das folhas para os frutos e no armazenamento da frutose nos frutos. A semente de milho, todavia, não acumula frutose nem sorbitol, que portanto, deve ser um metabólito intermediário. A atividade bioquímica da SDH de milho já havia sido caracterizada, porém não havia informações sobre sua estrutura genômica. Visando estudar o gene da SDH, a seqüência genômica completa foi seqüenciada. A região codificante da SDH mostrou-se muito conservada, o que não ocorre com as regiões não codificantes. Inclusive, ocorrendo uma perda de introns entre as diferentes espécies de plantas. Análises de Southem blot e isoenzima indicaram que há apenas um loeus de SDH em milho. As análises de Northem blot e atividade enzimática confirmaram que a expressão está restrita ao endosperma, e que começa logo após a polinização, I atingindo o ápice aos 15 DAP e caindo a níveis baixos aos 25 DAP. A localização enzimática in situ mostrou que a atividade está restrita ao endosperma amiláceo, mais especificamente na região basal da semente, próxima ao embrião. Os mutantes sugary1 e shrunken2, que acumulam mais açúcares, têm uma maior atividade de SDH. A injeção de até 150 mM de sacarose na semente de milho causou um aumento de atividade de SDH, confirmando os resultados obtidos com os mutantes. Esse aumento parece ser ao nível transcricional e ser regulado pelo intron 1. O intron 1 parece controlar não apenas à reposta a sacarose, mas também a resposta à hipoxia. O papel do sorbitol e da SDH nas sementes de milho em desenvolvimento parece ser crucial, porém permanece indefinido. O sorbitol é encontrado nos embriões de milho, apesar de não ter sido encontrada atividade da SDH. Além disso, foi demonstrado que os embriões conseguem se desenvolver tendo como única fonte de carbono o sorbitol. Foram encontrados ESTs no MAIZEST de um transportador de sorbitol no endosperma e de aldose redutase (AR), capaz de converter sorbitol em glicose, no embrião. Foi mostrado também que o sorbitol pode ser transportado do endosperma para o embrião. Desta forma, podemos sugerir a partir dos resultados que o sorbitol pode ser translocado do endosperma para o embrião pelo transportador de sorbitol e ser convertido em glicose pela AR no interior do embrião. Nossos resultados indicam que o sorbitol pode atuar de três maneiras; primeiro como um açúcar que é transportado de modo não vascular do endosperma para o embrião; segundo, como um metabólito intermediário; e terceiro, servindo como um escape para o excesso de NAD(P)H formado no interior hipóxico da semente. Os resultados apresentados nesta tese mostram um mecanismo inédito de interação metabólica entre o embrião e o endosperma, mediado pelo sorbitol, cuja síntese na semente, até então, era tida como um caminho "sem saída" / Abstract: At 10 DAP (days after pollinization) sorbitol dehydrogenase (SDH) was found to be the most abundant transcript, as depicted by the number of reads in the MAIZEST database. SDH catalyzes the NADH-dependent reduction of fructose to sorbitol or the oxidation of sorbitol to fructose. In Rosaceae, this enzyme has an important role in the sorbitol translocation from leaves to fruits and fructose storage in fruits. Maize endosperm, however, does not store fructose or sorbitol. The biochemical activity of SDH was already characterized, but there is no information about SDH genomic structure. Trying to understand better this gene we sequenced a complete genomic SDH sequence. Amino acid sequence comparisons showed SDH to be highly eonserved, but the non-coding sequences were not, and an intron loss has occurred among plant species. Southern blot and isoenzyme analyses indicated that there is only one SDH loeus in maize. Northern blot and enzyme activity analyses confirmed that SDH expression is restricted to the endosperm, starting early after pollinization, reaehing a peak at 15 DAP and decreasing to low levels after 25 DAP. The in situ loealization of SDH activity revealed that the enzyme is expressed ali over the amilaceous endosperm, more especifically in the basal region. Sugary1 and Shrunken2 mutants, that store more sugar, had a higher SDH activity. Upon injection of up to 150 mM of sucrose in maize ear SDH activity incresead, confirming the mutant's results. Sueh increase seemed to be at the transcriptionallevel and regulated by the first intron. The first intron seems to control not only sucrose, but also hypoxia response. The role of sorbitol in developing maize kernels is potentially pivotal, but remains undefined. Suerose arriving at the kernel base is metabolized into fructose, which can be converted to sorbitol by SDH. This is a highly active enzyme in maize endosperm, but not in embryos. Still, there is considerable sorbitol in maize embryos. We verified that embryos eould grow having only sorbitol as a carbon source. We have found sorbitol transporter ESTs in endosperm and aldose-reductase, which can convert sorbitol into glueose, ESTs in embryo. We also demonstrated that sorbitol can be transported into the embryo. So, we hypothesize that sorbitol can be translocated into the embryo by sorbitol transporters, and inside the embryo, AR may convert sorbitol into glucose. In summary, SDH activity and sorbitol synthesis may contribute in three critica I ways during seed development; first as a non-vascular transport sugar moving from endosperm to embryo; second, as an intermediate metabolite largely isolated frem sugar-signaling paths; and third, as a much-needed shuttle for excess NAD(P)H forming in the hypoxic kernel interior. The presented results showed a new mechanism of embryo-endosperm interaction, mediated by sorbitol, which was considered a dead end, until now / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

published_or_final_version / Physiology / Master / Master of Philosophy

La protéine murine AKR1B7 appartient à la famille des aldoses réductases. Par son expression limitée à un petit nombre de tissus et son contrôle hormonal, elle constitue un modèle de choix pour l'étude des fonctions physiologiques de cette famille enzymatique. D'une part, ces travaux de thèse ont permis de démontrer ex vivo le rôle de l'activité prostaglandine F synthase des aldoses réductases murines et humaines dans la glande surrénale. Nous avons mis en évidence une nouvelle boucle de régulation négative des fonctions endocrines surrénaliennes qui utilise la prostaglandine F2alpha comme signal paracrine et/ou autocrine entre le cortex et la medulla. D'autre part, nous avons développé la première lignée de souris transgéniques exprimant spécifiquement la recombinase Cre dans la cortico-surrénale pour réaliser l'invalidation conditionnelle de gènes dans ce tissu. Cette lignée constitue un outil très puissant pour la création de modèles murins de pathologies tumorales surrénaliennes

Glucose isomerase (GI) catalyzes the conversion of D-glucose to D-fructose in vitro. It is one of the bulkiest commercial enzymes, essential for the mass production of high-fructose corn syrup (HFCS) and crystalline fructose. / In this study, the effects of nitrogen sources, carbon sources, expression vectors, host strains, bacterial (Vitreoscilla) hemoglobin, selective pressure, plasmid stability and fermentation process on the GI production were investigated. The results showed that E. coli could express cloned thermostable GI at high expression level. E. coli transformed with the recombinant plasmid P-lac-GI gave the best result in term of total GI production and expression level. Corn steep liquor could be used as a cheap alternative nitrogen source for what was in LB medium. The concentration of glucose affected the expression level of GI significantly. Replacement of the ampicillin resistance gene by kanamycin resistance gene improved the plasmid stability leading to high productivity of GI in fed-batch fermentation. A suicide system could further improve the plasmid stability resulting in a high productivity of GI. A feeding strategy for fed-batch fermentation with the optimized parameters was developed to result in the production of up to 3g/L recombinant GI, which constituted 50% of the total soluble proteins. The total yield was 5-fold higher than that from flask experiments and 7-fold higher than the highest ever recorded. The expression level was also 100% higher than it was in other reports. / Liu Zhaoming. / "August 2005." / Advisers: J. Wang; W. P. Fong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3780. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 129-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Glucose isomerase (GI) catalyzes the isomerization of glucose to fructose and consequently is one of the bulkiest industrial enzyme for the manufacture of high fructose corn syrup and crystalline fructose. The GI is used in industry mainly in the form of immobilized enzyme. / In this work, the immobilization of GI had been studied by several methods: ion exchange adsorption, covalent binding, alginate cells entrapment and cells cross-linking. Three kinds of carrier support (ion exchange resin, epoxy resin and amino resin) have been used in the immobilization of cells-free enzyme; the whole cells immobilization of GI by cross-linking agents polyethyleneimid and glutaraldehyde were critically examined. The results show that the cells cross-linking is the best method to prepare the immobilized GI products, as it is high in specific activity and thermostability, and low the cost. The method is likely to make significant contribution to the field of immobilization, its application has expanding rapidly in many walks of the society, including environment protection, food and pharmaceutical industries. / Jin, Caike. / "August 2005." / Adviser: Jun Wang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 125-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dependent cataract, chemically synthetized drugs were sometimes less than beneficial due to the severe side effects they cause. Recently a huge amount of study has been intensified on developing new drugs from natural compounds and even by utilizing plant extracts for their easily metabolizing polyphenolic compounds. In this study, BLAR, source of enzyme, was obtained as crude via differential centrifugation and ammonium sulfate precipitation. The enzyme assay conditions were optimized for the protein, substrate, coenzyme, and salt concentrations, also for the effects of pH and temperature. Ocimum basilicum, Lavandula stoechas, Melissa officinalis, Glycyrrhiza glabra L. and Tilia tomentosa were selected as commonly used alternative medicine plants. Plant extracts were prepared in ethanol and ethyl acetate and their inhibitory effects were tested on crude bovin lens aldose reductase enzyme. Fifty percent inhibitory concentrations (IC50) were found between values of 25.53 &micro / g/mL and 54.15 &micro / g/mL for ethanol extracts and between 41.55 &micro / g/mL and 82.96 &micro / g/mL for the ethyl acetate extracts of selected plants. In addition, the plant extracts were also characterized for their antioxidant activities by of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and test of total phenolic content (TPC) .

Aldose reductase, E.C.1.1.1.21, catalyzes the reduction of different types of aldehydes to their corresponding alcohols, and especially reduces various aldo-sugars using NADPH as the coenzyme. Under hyperglycemic conditions aldose reductase is involved in the development of diabetic complications. As a result, interest has been placed over the years on the development of potent aldose reductase inhibitors for possible use in the therapy of these severe diabetic complications. In this study, aldose reductase was isolated from bovine lens by differential centrifugation and ammonium sulfate precipitation. The conditions for the enzyme assay / such as substrate (DL-Glyceraldehyde) and coenzyme (NADPH) concentration, protein amount, effect of sulfate ions, temperature and pH on the enzyme activity were optimized. The inhibitory effects of Punica granatum, Spinacia olaeracea, Allium cepa Allium porrum, Malus flouribunda, Malus domestica extracts were tested on crude bovine lens aldose reductase. Four different types of organic fractions from each crude plant extract were obtained by solvent fractionation. The inhibitory activity of these organic fractions was calculated considering the aldose reductase activity without extracts as 100 %. All six plants were found to inhibit aldose reductase activity to different extent. Among these fractions obtained as / petroleum ether, diethyl ether, ethyl acetate, and n-butanol. Highest inhibitory activity was found for the ethyl acetate fraction. The IC50 values of ethyl acetate fractions of all these plants was calculated as, 25.46 &micro / g/ml, 20.5 &micro / g/ml, 18.5 &micro / g/ml, 12.32 &micro / g/ml, 6.45 &micro / g/ml, 5.4 &micro / g/ml, for Allium porrum, Malus domestica, Spinacia olaeracea, Malus floribunda Allium cepa, Punica granatum respectively.

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