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Thesis (Ph. D.)--Ohio state University, 1927. / Autobiography.

CARNEIRO, Rômulo Farias. Purificação e caracterização bioquímica de lectinas ligantes de galactose isoladas de invertebrados marinhos. 2017. 72 f. Tese (Doutorado em Biotecnologia de Recursos Naturais) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Coordenação PPGBiotec (ppgbiotec@ufc.br) on 2017-06-01T12:00:34Z No. of bitstreams: 1 2017_tese_rfcarneiro.pdf: 7523630 bytes, checksum: cb11806939e3065cba0c59a5ef0e109c (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-06-01T21:44:10Z (GMT) No. of bitstreams: 1 2017_tese_rfcarneiro.pdf: 7523630 bytes, checksum: cb11806939e3065cba0c59a5ef0e109c (MD5) / Made available in DSpace on 2017-06-01T21:44:10Z (GMT). No. of bitstreams: 1 2017_tese_rfcarneiro.pdf: 7523630 bytes, checksum: cb11806939e3065cba0c59a5ef0e109c (MD5) Previous issue date: 2017 / Lectins, which are found in all living organisms, are sugar-binding proteins that differ from immunoglobulins and glycoenzymes because they are not produced as an adaptive immune response, and they contain no site for the enzymatic modification of their ligands. Lectins have been studied for more than a century because of their ability to decipher glycocode. Excepting Crustacea (Arthropoda) and Bivalvia (Mollusca), marine invertebrates have been poorly studied compared to the distribution and presence of lectins. This work aimed to isolate lectins from groups of invertebrates with poorly studied, biochemically characterize such lectins and to evaluate its effect on the formation of bacterial biofilms. Lectins were isolated from the sponge Aplysina lactuca (ALL), the eggs gastropod Aplysia dactylomela (ADEL) and the sea urchin Echinometra lucunter (ELEL). ALL is a dimer formed by two identical 16 kDa subunits linked by disulfide bonds with affinity for lactulose, lactose and mucins. Its primary structure is similar to the Axinella polypoides lectin and its secondary structure is formed mainly by β-sheet. ADEL is a high mannose glycoprotein formed by two identical 27 kDa subunits linked by disulfide bonds. Its primary structure showed similarity with other lectins obtained from aplysid eggs. The secondary structure of ADEL is rich in β-sheet and sensitive to the presence of carbohydrates. ADEL showed affinity for several galactosides, especially for galacturonic acid (Ka = 1,5 x 107 M-1). The three-dimensional ADEL model reveals a new carbohydrate recognition domain. ELEL is a rhamnose binding lectin formed by two 11 kDa subunits linked by disulfide bonds. ELEL showed affinity for carbohydrates containing the same orientation in the hydroxyls of C-2 and C-4 as galactose. Its primary structure was similar to lectins found in eggs of sea urchins and fish. The three-dimensional ELEL structure model suggests the predominance of β-sheets in a β-sandwich domain. Ab initio calculations suggest the interaction of ELEL with a common receptor in tumor cells, Gb3. In addition, the three lectins showed ability to agglutinate bacteria and / or inhibitory effect of the growth of bacterial biofilms. In conclusion, this work reveals that invertebrates represent an excellent source of new galactose binding lectins and presenting inhibitory effect in the bacterial growth and/or antibiofilm, so these new lectins may be useful as biotechnological tools in the near future. / Lectinas animais podem ser definidas como proteínas ou glicoproteínas que reconhecem carboidratos de maneira específica, mas não participam do metabolismo dos mesmos e não pertencem a qualquer uma das principais classes de imunoglobulinas. As lectinas têm sido estudadas por mais de um século devido sua capacidade de decifrar o glicocódigo. Com exceção dos grupos Crustacea (Arthropoda) e Bivalvia (Mollusca), os invertebrados marinhos têm sido pouco estudados quanto à distribuição e presença de lectinas. Este trabalho objetivou isolar lectinas de grupos de invertebrados pouco estudados, caracterizar bioquimicamente as lectinas e avaliar seu efeito sobre a formação de biofilmes bacterianos. Lectinas foram isoladas da esponja Aplysina lactuca (ALL), de ovos do gastrópode Aplysia dactylomela (ADEL) e do ouriço do mar Echinometra lucunter (ELEL). ALL é um dímero formado por duas subunidades idênticas de 16 kDa unidas por pontes dissulfeto com afinidade por lactulose, lactose e mucinas. Sua estrutura primária é semelhante à da lectina da esponja Axinella polypoides e sua estrutura secundária é formada principalmente de folhas β. ADEL é uma glicoproteína do tipo high mannose composta por duas subunidades idênticas de 27 kDa unidas por pontes dissulfeto. A estrutura primária apresentou similaridade com outras lectinas de ovos de aplisídeos. A estrutura secundária de ADEL é rica em folhas β e sensível a presença de carboidratos. ADEL apresentou afinidade para diversos galactosídeos, em especial para ácido galacturônico (Ka = 1,5 x 107 M-1). O modelo tridimensional de ADEL revela um novo domínio de reconhecimento a carboidratos. ELEL é uma lectina ligante de ramnose formada por duas subunidades de 11kDa unidas por pontes dissulfeto, exibindo afinidade por carboidratos contendo a mesma orientação nas hidroxilas de C-2 e C-4 que a galactose. Sua estrutura primária foi semelhante a outras lectinas encontradas em ovos de ouriço e peixes. O modelo de estrutura tridimensional de ELEL sugere a predominância de folhas β em um domínio do tipo sanduíche β. Cálculos ab initio sugerem a interação de ELEL com um receptor comum em células tumorais, o Gb3. Ademais, as três lectinas apresentaram capacidade de aglutinar bactérias e/ou efeito inibitório do crescimento de biofilmes bacterianos. Em conclusão, este trabalho revela que invertebrados marinhos representam excelente fonte de novas lectinas ligantes de galactose e com efeito antibiofilme, sendo assim estas novas lectinas podem ser ferramentas biotecnológicas úteis em um futuro próximo.

Dissertação de Mestrado apresentada ao Programa de Pós Graduação em Ciências da Saúde, da Universidade do Extremo Sul Catarinense – UNESC, para obtenção do título de Mestre em Ciências da Saúde. / A galactosemia é uma condição causada pela deficiência de uma das quatro enzimas envolvidas no metabolismo da galactose: galactose mutarotase, galactocinase, galactose-1-fosfato uridiltransferase e UDP galactose - 4 - epimerase. Como consequência deste bloqueio, os indivíduos afetados pela galactosemia não podem efetivamente converter galactose em glicose, apresentando acúmulo de galactose e seus metabólitos em seus tecidos, especialmente nas células do tecido nervoso central, fígado e rins. Dentre os principais achados clínicos, destaca-se disfunção cerebral com atraso de desenvolvimento e deficiência de aprendizado e de memória, cuja fisiopatologia ainda não está esclarecida. Considerando-se o parco conhecimento a respeito dos mecanismos fisiopatológicos do dano cerebral apresentado por pacientes galactosêmicos, o presente trabalho avaliou o efeito da administração aguda de galactose sobre vias apoptóticas em cérebros de ratos jovens. Para tanto, vinte e quatro ratos machos da linhagem Wistar de 30 dias foram divididos em dois grupos: controle e galactosemia. Os animais do grupo galactosemia receberam uma única administração subcutânea de 5 μmol/g de peso corporal de galactose. Os animais do grupo controle receberam solução salina nos mesmos volumes por via subcutânea. Uma hora após a administração deste carboidrato, os animais sofreram eutanásia por decapitação com guilhotina e o córtex cerebral e o cerebelo foram isolados e limpos. Foram então determinados os imunoconteúdo de Bax, Bcl-2, AIF e p53, proteínas que fazem parte de vias de morte celular por apoptose, e a razão Bax/Bcl-2 nestes tecidos por western blotting. Não foram observadas diferenças significativas entre os grupos nos conteúdos das proteínas avaliadas, bem como na razão entre Bax/Bcl-2 em nenhuma das estruturas cerebrais estudadas. Os resultados do presente trabalho sugerem que a administração aguda de galactose não altera os processos de morte celular por apoptose via proteínas Bax, Bcl-2, AIF e p53 em cérebro de ratos jovens.

Orientador: Yong Kun Park / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos e Agricola / Made available in DSpace on 2018-07-14T13:47:16Z (GMT). No. of bitstreams: 1 Cruz_Rubens_D.pdf: 2455826 bytes, checksum: 549ccf877eb2741c83c96e97f2765e75 (MD5) Previous issue date: 1983 / Resumo: Foi estudada a produção de a-galactosidase por Aspergillus orizae , tanto em meio submerso como por fermentação semi-sólida. A enzima foi levemente induzida por rafinose e estaquiose, enquanto um extrato de oligossacárides da soja, fermentado por S, cereviciae (3.7% de açúcares totais), mostrou excepcional capacidade de indução. Simultâneamente, o microrganismo produziu invertase, esta de forma constitutiva. A a-galactosidase, parcialmente purificada, mostrou um Km de 2.04 mMol para PNPG e 140 mMol para melibiose. Para os mesmos substratos os valores de Vm foram de 0.36 µMol de PMP e 0.64 µMol de glucose/min/mg de proteína. O peso molecular foi estimado em 200 000 e a enzima foi inibida competitivamente por galactose e frutose e de forma não competitiva por glucose. Ag+, Hg+ e p-clororaercuribenzoato afetaram drasticamente a atividade enzimática, sugerindo a presença de grupo(s) -SH no sítio ativo. A enzima mostrou atividade máxima em pH 4,0 e mais de 70% da atividade máxima em pH 6,3. Nenhuma perda de atividade foi observada, quando tratada entre 35 a 50ºC, por 15 minutos. O preparado bruto contendo a-galactosidase e invertase diminuiu substancialmente os teores de rafinose e estaquiose do Extrato de Soja, o que sugere a possibilidade de seu emprego no tratamento de produtos da leguminosa, visando eliminar aqueles galastoligossacárides, apontados como causadores da flatulência / Abstract: The production, of a-galactcsidase by a strain of Aspergillus orizas was observed either by sub merged culture method or Koji method. The enzyme was slightly induced by raffinose and stacchyosef while in a previously fermented oligosaccharides extract from soybean with 3.7 per cent of total sugar the yield was greatly increased. The partially purified a-gaiactosidase showed a Km value of 2.04 and 140 mMole respectively for PNPG and melihiose. The Vm value was 0.36 µMole of PNP and 0.54 of glucose/rain/mg, of protein for the same substrates. The molecular weight was estimated in 200 000 by gel filtration in Sephadex G-200 and the enzyme was competitively inhibited by galactose and fructose and non competitively by glucose. Ag+, Hg+ and p-Chloromercuribenzoats remarkdly inhibited the enzyme activity suggesting the presence of SH groups in the active site. The optimum pH was 4,0 and more them 70% of the maximum activity was found in pH 6.3. No loss of activity was shown when submitted to heat treat: meant between 35 ¿ 50º C, for 15 minutes. Enzymatic filtrate substantially de creased the contents of raffinose and stacchyose from soybean milk suggesting the possibility of its application to eliminate soybean galactooligosccharydes responsible for flatulence troubles / Doutorado / Doutor em Ciência de Alimentos

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In the near future, biomass-derived energy is predicted to substantially complement that generated from petroleum. However, certain types of biomass employed as substrates in the microorganism-mediated production of renewable fuelethanol contain significant amounts of the recalcitrant hexose sugar galactose. The consumption of galactose in hexose sugar-fermenting yeasts is often delayed with respect to other sugars, such as glucose and mannose, because of an intrinsic preference for carbon sources requiring less energy in the preparatory reactions preceding glycolysis. This work comprised the search for, and characterization of anethanologenic yeast capable of efficiently assimilating galactose. Screening experiments conducted with wild-type Saccharomyces cerevisiae strains identified one isolate (Y-1528) exhibiting exceptionally fast galactose fermentation. The absence of conventional glucose repression, including a preference for galactose as carbon source and notable delays in the utilization of glucose and mannose, was demonstrated in mixed sugar fermentations. Endogenous extracellular glucose was observed during double sugar fermentations of galactose and mannose. This glucose was traced to supplied galactose by radioisotope labeling, suggesting involvement of UDP-galactose 4-epimerase in the responsible reaction mechanism(s).Sub-cellular fractionation was employed in an attempt to ascertain enzyme localization in Y-1528. Fermentations of lignocellulosic substrate mixtures by Y-1528 illustrated better performance than that accomplished by a reference yeast strain, and again showed a preference for galactose. Mixed cultures of Y-1528 and the same reference strain demonstrated accelerated hexose sugar consumption, and no detrimental effects from competition, during synthetic and lignocellulosic substrate fermentations. Glucose repression was absent in mixed culture fermentations. Fermentations of synthetic sugar mixtures augmented with lignocellulosic inhibitory compounds showed Y-1528 to have better performance than a reference yeast strain, despite a global detrimental effect relative to inhibitor-free fermentations. Cell recycle batch fermentations of spent sulfite liquor illustrated the toxic effect of the hardwood variant, as well as a net loss of performance from all strains tested. Y-1528 was taxonomically confirmed as S. cerevisiae. UDP-galactose 4-epimerase chromatographic purification was unsuccessful, but a partial sequence of the enzyme, showing complete identity with type sequence, was obtained by electrophoretic separation, liquid chromatography, and mass spectrometry. A significantly mutated UDP-galactose 4-epimerase gene was successfully sequenced.

In the near future, biomass-derived energy is predicted to substantially complement that generated from petroleum. However, certain types of biomass employed as substrates in the microorganism-mediated production of renewable fuelethanol contain significant amounts of the recalcitrant hexose sugar galactose. The consumption of galactose in hexose sugar-fermenting yeasts is often delayed with respect to other sugars, such as glucose and mannose, because of an intrinsic preference for carbon sources requiring less energy in the preparatory reactions preceding glycolysis. This work comprised the search for, and characterization of anethanologenic yeast capable of efficiently assimilating galactose. Screening experiments conducted with wild-type Saccharomyces cerevisiae strains identified one isolate (Y-1528) exhibiting exceptionally fast galactose fermentation. The absence of conventional glucose repression, including a preference for galactose as carbon source and notable delays in the utilization of glucose and mannose, was demonstrated in mixed sugar fermentations. Endogenous extracellular glucose was observed during double sugar fermentations of galactose and mannose. This glucose was traced to supplied galactose by radioisotope labeling, suggesting involvement of UDP-galactose 4-epimerase in the responsible reaction mechanism(s).Sub-cellular fractionation was employed in an attempt to ascertain enzyme localization in Y-1528. Fermentations of lignocellulosic substrate mixtures by Y-1528 illustrated better performance than that accomplished by a reference yeast strain, and again showed a preference for galactose. Mixed cultures of Y-1528 and the same reference strain demonstrated accelerated hexose sugar consumption, and no detrimental effects from competition, during synthetic and lignocellulosic substrate fermentations. Glucose repression was absent in mixed culture fermentations. Fermentations of synthetic sugar mixtures augmented with lignocellulosic inhibitory compounds showed Y-1528 to have better performance than a reference yeast strain, despite a global detrimental effect relative to inhibitor-free fermentations. Cell recycle batch fermentations of spent sulfite liquor illustrated the toxic effect of the hardwood variant, as well as a net loss of performance from all strains tested. Y-1528 was taxonomically confirmed as S. cerevisiae. UDP-galactose 4-epimerase chromatographic purification was unsuccessful, but a partial sequence of the enzyme, showing complete identity with type sequence, was obtained by electrophoretic separation, liquid chromatography, and mass spectrometry. A significantly mutated UDP-galactose 4-epimerase gene was successfully sequenced.