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Thesis (Ph. D.)--Ohio state University, 1922. / Autobiography. Includes bibliographical references.

Dissertação (Mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Curso de Pós-Graduação em Química / Made available in DSpace on 2012-10-19T03:49:51Z (GMT). No. of bitstreams: 0Bitstream added on 2014-09-26T00:07:45Z : No. of bitstreams: 1 182937.pdf: 10004171 bytes, checksum: cdcdece6908aa66d84bbc3ff96762194 (MD5) / O estudo de compostos modelos, dentro da Química Bioinorgânica, continua contribuindo significativamente no esclarecimento da participação dos metais de transição nos sítios ativos de enzimas. Os maiores avanços têm sido obtidos recentemente, proporcionando, por exemplo, o entendimento da relação estrutura/função dos sítios ativos em oxidases contendo cobre. Neste contexto, se encontra a galactose oxidase (GAO), uma metaloenzima mononuclear de cobre(II), que atua promovendo a oxidação de álcoois primários à aldeídos com a redução acoplada de O2 a H2O2. Neste trabalho, nós descrevemos as sínteses, caracterizações e o estudo das propriedades (análise elementar, IV, UV-vis, RPE, titulação potenciométrica, eletroquímica e estrutura por raios X) de novos complexos mononucleares de cobre(II) com novos ligantes hexadentados contendo átomos N,O-doadores, visando mimetizar o sítio inativo da GAO. Os complexos apresentaram geometrias octaédricas distorcidas e piramidais quadradas ao redor do átomo de cobre(II), com coordenações axiais e equatoriais dos grupos fenólicos (protonados e/ ou desprotonados), e equatoriais dos grupos amínicos e pirídinicos. Tais coordenações são similares as da GAO. As propriedades estudadas também podem ser correlacionadas com as da enzima, o que faz com que os complexos sintetizados possam ser considerados bons modelos miméticos para a mesma.

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. / Galactosemia is characterized by a deficiency in one of the three enzymes involved in the galactose metabolism galactosekinase (GALK), galactose-1-phosphate uridyltransferase (GALT) and uridine-diphosphate-galactose 4 epimerase (GALE). The main clinical findings observed in patients affected by galactosemia include neurodevelopmental delay, and learning and memory disabilities, whose pathophysiology is still unclear. Therefore, considering that a disruption in cholinergic neurotransmission has been related to the pathophysiology of brain damage in neurodegenerative diseases and in other inborn errors of the metabolism, in the present work we investigated the in vivo and in vitro effect of galactose on acethylcholinesterase (AchE) activity in different brain structures of developing rats. It was observed that acute galactose administration did not alter AchE activity in cerebral cortex, hippocampus and striatum of suckling rats (15-day-old). On the other hand, when administered to older rats, galactose provoked a significant increase in AchE activity in cerebral cortex. Finally, it was observed that high doses of galactose (5 mM and higher) in vitro also provoked an increase in this enzyme activity in cerebral cortex of 30-day-old rats. Our data demonstrate that acute galactose administration provokes an age-dependent increase in AchE activity in cerebral cortex of rats. It may be therefore concluded that a disruption in cholinergic homeostasis may be involved in the pathophysiology of brain damage observed in patients affected by galactosemia. / Galactosemia é caracterizada pela deficiência em uma das três enzimas envolvidas no metabolismo da galactose, galactosequinase (GALK), galactose-1-fosfato-uridiltransferase (GALT) e galactose- difosfato-uridine 4 epimerase (GALE). Os principais achados clínicos são disfunções do sistema nervoso central com atraso de desenvolvimento, deficiência de aprendizagem e de memória, cuja fisiopatologia ainda não está esclarecida. Dessa forma, considerando que alterações na neurotransmissão colinérgica foram relacionadas á fisiopatologia de outros erros inatos do metabolismo e doenças neurodegenerativas, no presente trabalho avaliamos os efeitos in vitro e in vivo da galactose sobre a atividade da enzima acetilcolinesterase (AchE) em diferentes estruturas de ratos ao longo do seu desenvolvimento. Observamos que a administração aguda de galactose não alterou a atividade da AchE em córtex cerebral, hipocampo ou estriado de ratos de 15 dias de vida. Por outro lado, quando administrada a animais com 30 e 60 dias de vida, a galactose provocou um aumento significativo da atividade da AchE em córtex cerebral. Por último, observamos que altas doses de galactose (5 mM e maiores) in vitro também provocam um aumento na atividade desta enzima em córtex cerebral de ratos de 30 dias de vida. Nossos resultados demonstram que a administração de galactose provoca um aumento na AchE de uma maneira idade-dependente em córtex cerebral de ratos. Dessa forma, podemos presumir que um desequilíbrio na homeostase colinérgica possa estar envolvido na fisiopatologia do dano cerebral observado em pacientes com galactosemia.

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. / Forestry, Faculty of / Graduate

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Pseudomonas aeruginosa (PA) représente un véritable problème de santé publique étant l'une des principales causes d'infections nosocomiales et de mortalité chez les patients atteints de fibrose cystique. Cette bactérie provoque des pathologies respiratoires chroniques qui persistent malgré une thérapie antibiotique agressive à cause de l'émergence de souches résistantes et de la formation du biofilm. Une stratégie prometteuse consiste à inhiber les facteurs de virulence de PA tels que PA-IL qui est une lectine soluble impliquée dans la reconnaissance des résidus galactose et qui semble jouer un rôle dans l'adhésion de la bactérie au glycocalyx autour de la cellule hôte ainsi que dans le développement du biofilm. Alors que les interactions lectine-carbohydrate sont caractérisées par une spécificité élevée, l'affinité entre les lectines et les saccharides simples est faible et une présentation multivalente des unités saccharidiques est généralement requise pour atteindre une interaction significative d'un point de vue physiologique. Ce manuscrit décrit la synthèse de glycooligonucléotides dont l'affinité envers PA-IL a été étudiées par DNA Direct Immobilisation microarray. Les blocs de construction saccharidiques ont été assemblés sur des échafaudages phosphorylés en utilisant une combinaison de synthèse en phase solide d'ADN et « click chemistry » (cycloaddition 1,3-dipolaire azide/alcyne). Grâce à la technologie glycoarray, les glycomimétiques ont été analysés à une échelle nanomolaire. Les résultats expérimentaux ont permis d'établir des relations structure-activités précises. En outre, des études des docking ont confirmé les résultats expérimentaux. La synthèse des candidats les plus affins envers PA-IL a été conduite sans l'étiquette d'ADN et à plus grande échelle pour vérifier par des analyses biologiques leurs propriétés anti-adhésives ou inhibitrices du biofilm. / Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. It often leads to chronic respiratory infection despite aggressive antibiotic therapy due to the emergence of resistant strains and to the formation of biofilm. A promising approach is to inhibit the virulence factors of PA such as PA-IL which is a soluble lectin implicated in the recognition of galactose residues that seems to be involved in the adhesion of the bacterium to the glycocalyx surrounding host's cells as well as in the biofilm development.If carbohydrate-lectin interactions proceed with high specificity, the affinity between lectins and simple saccharides is low and a multivalent display of saccharidic units is generally required to attain physiologically significant association. This manuscript reports the synthesis of high affinity glycooligonucleotides toward PA-IL for its inhibition and their bindings properties were studied on a DNA direct immobilisation microarray. Glycoside building blocks were assembled on phosphorylated scaffolds using a combination of DNA solid phase synthesis and microwave assisted « click chemistry » (copper (I) catalyzed 1,3-dipolar cycloaddition). Thanks to glycoarray technology the glycomimetics were studied at the nanomole scale. The experimental results have been used to assess structure binding relationships. In addition, docking studies have confirmed ours experimental results. The synthesis of the best hits as anti-biofilm or anti-adhesive molecules have been synthesized at upper scale without their DNA tag for biological studies as anti-adhesive compounds and biofilm inhibitors of PA.

Le lactosérum est un résidu liquide laitier qui a lieu pendant la fermentation du fromage. Il est composé par lactose (le solide principal de substance sèche), protéines, vitamines et minéraux. À cause de ces éléments, sa demande biologique et chimique d’oxygène (DBO) et (DCO) est grande (30 < DBO < 50 g/L; and 60 < DCO < 80 g/L). Il est nécessaire donc de traiter le lactosérum avant d’en jeter dans les lacs, les rivières, etc. La valorisation du lactosérum par des bactéries est d’un grand potentiel technique non seulement pour la réduction de DBO et DCO mais aussi pour obtenir des produits comme le 2,3-butanediol (2,3-BD). Des bactéries comme Enterobacter cloacae, Klebsiella pneumoniae et Paenibacillus polymyxa consomment et transforment des saccharides comme lactose en 2,3-BD. Il en est d’autres, comme Escherichia coli, qui doivent être génétiquement modifiées car elles n’ont pas le chemin enzymatique pour produire 2,3-BD. De cette manière l’objectif principal de cette recherche est celui de tester l’habilité d’une souche génétiquement modifié d’E. coli pour transformer le lactose (un disaccharide) en 2,3-BD afin de savoir le potentiel du lactosérum comme une source de lactose. La souche d’E. coli JFR12 (ECGM12) a été utilisée pour fermenter trois concentrations de glucose, galactose et lactose (12.5, 25 and 50 g/L) enrichirent M9. Le rendement de 2,3-BD le plus grand (36% environ, g 2,3-BD/g saccharide) fut obtenu en présence de 25 g/L de glucose et lactose; quoique l’usage de n’importe quelle concentration de galactose produisît des rendements plus pauvres de 2,3-BD. En plus, 2 mélanges de glucose-galactose ont été testés (1:1, w/w) à une concentration finale du 25 et 50 g/L de mélange. Les rendements de 2,3-BD obtenus ont été très similaires à les obtenus avec du galactose comme l’unique source de carbone. Par conséquent, une hypothèse a été formulée: l’usage de galactose entrave la formation de 2,3-BD, alors que les enzymes impliquent dans l’hydrolyse du lactose pourraient neutraliser l’effet du galactose et de cette manière, les rendements de 2,3-BD ont pu être hauts / Whey is a dairy effluent generated during the cheese manufacturing. Its composition presents lactose (the main solid of dry matter), proteins, vitamins and minerals. Due to these compounds, its biological (BOD) and chemical oxygen demand (COD) are high (30 < BOD < 50 g/L; and 60 < COD < 80 g/L). Therefore, it is necessary to treat the whey before releasing it into lakes, rivers, etc. The valorization of whey by bacteria is a potential technique not only for reducing the BOD and COD, but also to obtain products like 2,3-butanediol (2,3-BD). Bacteria like Enterobacter cloacae, Klebsiella pneumoniae and Paenibacillus polymyxa consume and transform saccharides like lactose into 2,3-BD. Other bacteria such as Escherichia coli have to be genetically modified since they do not possess the enzymatic pathway to produce 2,3-BD. In this way, the main objective of this research is to test the ability of a genetically modified strain of E. coli to transform lactose (a disaccharide) into 2,3-BD in order to know the potential of whey as a lactose source. In this way, the E. coli JFR12 (ECGM12) was used to ferment three concentrations of glucose, galactose and lactose (12.5, 25 and 50 g/L) supplemented M9. The highest 2,3-BD yield (around 0.36 g 2,3-BD/g saccharide) was obtained in the presence of 25 g/L of glucose and lactose; whereas the use of whatever galactose concentration provided poorer yields of 2,3-BD compared to those obtained using glucose and lactose. Moreover, 2 mixture of glucose-galactose was tested (1:1, w/w) at a final concentration of 25 and 50 g/L of mixture. The 2,3-BD yields obtained were very similar to those using galactose as a sole carbon source. Therefore, it was hypothesized that galactose impedes the formation of 2,3-BD, whereas enzyme involved in the lactose hydrolysis might counteract the effect of galactose, leading to high 2,3-BD yields

O hidrogênio (H2) tem sido considerado uma fonte de energia limpa bastante promissora, pois sua combustão origina apenas moléculas de água, sendo uma alternativa ao uso de combustíveis fósseis. Entretanto, os métodos atuais de produção de H2 demandam matérias-primas finitas e uma grande quantidade de energia, tornando a sua obtenção não sustentável. Mais recentemente, a via fermentativa tem sido considerada para a produção de H2, utilizando como matérias-primas efluentes industriais, materiais lignocelulósicos e biomassa de algas, denominado de bio-hidrogênio de primeira, segunda e terceira geração, respectivamente. Neste trabalho foi isolada uma bactéria anaeróbia a partir de uma cultura mista (lodo) de um sistema de tratamento de vinhaça, após pré-tratamento do lodo a pH 3 por 12 horas. Este microrganismo foi identificado com 99% de similaridade como Clostridium beijerinckii com base na sequência do gene RNAr 16S denominado de C. beijerinckii Br21. A temperatura e o pH mais adequados para o crescimento e produção de H2 por esta cultura foi 35 °C e pH inicial 7,0. A bactéria possui a capacidade de utilizar ampla variedade de fontes de carbono para a produção de H2 por fermentação, especialmente, monossacarídeos resultantes da hidrólise de biomassa de algas, tais como glicose, galactose e manose. Foram realizados ensaios em batelada para a produção de H2 com a bactéria isolada empregando diferentes concentrações de glicose e galactose, visando a sua futura utilização em hidrolisados de alga. Os parâmetros cinéticos dos ensaios de fermentação estimados pelo modelo de Gompertz modificado, como a velocidade máxima de produção (Rm), a quantidade máxima de hidrogênio produzido (Hmáx) e o tempo necessário para o início da produção de hidrogênio (fase lag) para a glicose (15 g/L) foram de: 58,27 mL de H2/h, 57,68 mmol de H2 e 8,29 h, respectivamente. Para a galactose (15 g/L), a Rm, Hmáx e foram de 67,64 mL de H2/h, 47,61 mmol de H2 e 17,22 horas, respectivamente. O principal metabólito detectado ao final dos ensaios de fermentação, foi o ácido butírico, seguido pelo ácido acético e o etanol, tanto para os ensaios com glicose, como com galactose. C. beijerinckii é um candidato bastante promissor para a produção de H2 por fermentação a partir de glicose e galactose e, consequentemente, a partir de biomassa de algas como substratos. / Hydrogen (H2), considered an alternative to fossil fuels, is a promising source of clean energy because its combustion originates water molecules only. However, the current H2 production methods require finite raw materials and a large amount of energy, which makes them unsustainable. The fermentative pathway has been considered for H2 production from renewable raw materials such as industrial wastewater, lignocellulosic materials, and algal biomass, the so-called first, second, and third bio-hydrogen generation, respectively. In this work, after pre-treatment at pH 3 for 12 h, a H2-producing bacterium was isolated from a mixed culture (sludge) collected from an anaerobic bioreactor used to treat sugarcane vinasse. The microorganism was identified as Clostridium beijerinckii based on the sequence of the 16S rRNA gene; it was named C. beijerinckii Br21. The most appropriate temperature and initial pH to achieve H2 production by this strain was 35 °C and 7, respectively. The bacterium was able to use a wide variety of carbon sources, especially the monosaccharides glucose, galactose, and mannose resulting from hydrolysis of algal biomass. Batch assays using different concentrations of glucose and galactose were performed to produce H2. The kinetic parameters of the tests were estimated by the Gompertz modified model. The maximum production rate (Rm), the maximum amount of produced H2 (Hmáx), and the phase lag () for glucose and galactose, both at 15 g/L, were 58.27 and 67.64 mL of H2/h, 57.68 and 47.61 mmol of H2, and 8.29 and 17.22 h, respectively. The main metabolite detected at the end of fermentation tests was butyric acid, followed by acetic acid and ethanol. The results indicated that the new C. beijerinckii isolate is a promising candidate for fermentative H2 production from algal biomass.