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Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Eugene G. Mueller, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (PhD)--University of South Australia, 2001.

Detoxification of compounds occurs in two phases. In phase I, a functional group on the toxicant is made available for subsequent Phase II reactions. In phase II, the functional groups are conjugated to a compound that will increase the solubility of the toxicant, enhancing its elimination. Uridine diphosphate glucuronyl transferase (UDPGT) is one microsomal enzyme involved in phase II reactions. It catalyzes the conjugation of toxic compounds with glucuronic acid in reactions in which uridine diphosphate glucuronic acid (UDPGA) is the donor substrate. A new purification procedure for UDPGT has been developed. This procedure includes a Polyethylene glycol fractionation, ion exchange chromatography with DEAE Bio-gel A and affinity chromatography with UDP-hexanolamine-Sepharose. The purification was monitored for three different substrates, bilirubin, 4-nitrophenol (PNP) and 7-hydroxy-4-methylcoumarin (HMC). For this last substrate, HMC, a new continuous fluorometric assay was developed. The purification fold and activity recovery, respectively, towards each substrate was as follows: bilirubin, 31 and 80%; PNP, 31 and 80%; and HMC, 24 and 60%. The significance of these results is discussed with reference to the activation of UDPGT in microsomes by detergents and the reactivation of purified UDPGT by phospholipids. / M.S.

In this study, high-performance liquid chromatography (HPLC) was employed to detect and quantify pyrimidine salvage enzymes by monitoring the disappearance of substrates or formation of products.

A esquistossomose humana, doença causada pelo S. mansoni e com 6 milhões de infectados somente no Brasil, possui uma única estratégia terapêutica eficiente atualmente disponível. Esta se baseia na utilização de praziquantel e relatos de cepas resistentes à essa droga tem despertado o interesse da comunidade científica sobre o desenvolvimento de novas estratégias terapêuticas. Uma melhor caracterização dos processos metabólicos do parasita podem auxiliar nestas buscas. Diante desse contexto, nosso grupo tem trabalhado na caracterização estrutural e funcional das enzimas que compõem a via de salvação de purinas e pirimidinas deste parasita, com dez enzimas já caracterizadas. Uma das enzimas remanescentes é a uridina fosforilase (UP) (EC 2.4.2.3), cujo a qual o genoma do parasita apresenta duas isoformas, a smUPa e smUPb (92% de identidade entre elas). Com o objetivo de caracterizar estruturalmente estas enzimas, ambas foram obtidas via expressão heteróloga, purificadas e submetidas a ensaios de cristalização e co-cristalização (para obtenção das estruturas interagindo com diferentes ligantes). Após coleta de dados de difração de raio-x, processamento e refinamento adequado foram obtidas seis estruturas da smUPa (smUPaapo, smUPa+Timidina, smUPa+timina, smUPa+uracil, smUPa-5fluorouracil) e duas da smUPb (smUPbapo e smUPb+citrato). A análise das estruturas revela que as duas isoformas apresentam essencialmente a mesma estrutura, no entanto, apesar das poucas divergências em nível de sequência de aminoácidos, existem diferenças significativas entre os sítios ativos. A smUPa apresenta o sítio com as mesmas características de UPs conhecidas, em contrapartida a smUPb apresenta duas mudanças significativas que elimina a capacidade de interagir com a base nitrogenada (Q201L) e a cavidade que acomoda a base nitrogenada (G126D), o que torna as smUPs um caso único de isoformas de UP em um mesmo organismo conhecidas. É plausível que a smUPb não seja capaz de catalisar a fosforólise reversível da uridina, sendo ou um pseudogene ou alguma outra enzima com atividade catalítica diferente da UP. Para a completa caracterização destas enzimas, testes de atividade enzimática serão realizados e deverão auxiliar a determinar a real função da smUPb. / Human schistosomiasis, a disease caused by Schistosoma sp., is estimated to affect six million individuals in Brazil alone and there is currently only one therapeutic strategy available. This is based on the use of praziquantel and reports of the appearance of strains resistant to the drug has motivated the scientific community towards the search for new possible therapies. Biochemical characterization of the parasites metabolism is essential for the rational development of new therapeutic alternatives. Based on this,reasoning our group has been working on the structural and functional characterization of the enzymes involved in the pyrimidine and purine salvage pathways of S. mansoni. One of the remaining enzymes to be characterized is uridine phosphorylase (UP) (EC 2.4.2.3), for which there are two isoforms present in the parasite genome, smUPa and smUPb, which share 92% sequence identity. In order to structurally characterize these enzymes, both smUPs were produced by heterologous expression, purified and submitted to crystallization e co-crystallization assays, in the latter case in order to obtain the structure of different enzyme-ligand complexes. After data collection, processing and refinement, five structures of smUPa (smUPaapo, smUPa+Timidina, smUPa+timina, smUPa+uracil and smUPa+5fluorouracil) and two structures of smUPb (smUPbapo and smUPb+citrato) were obtained. Analysis of the structures revealed that the isoforms have the same fold, but despite the high sequence identity, significant differences are observed at the active site probably profoundly affecting enzyme activity. Whilst SmUPa presents an active site similar to that of other known UPs, smUPb is predicted to lack the ability to interact with the nucleoside base due to the presence of a leucine in place of a glutamine at position 201 and an aspartatic acid in place of glycine at position 126. These differences turn the smUPs into a unique case of UP isoforms. It is plausible that smUPb is unable to catalyze the reversible phosphorolysis of uridine and could be either a pseudogene or a different enzyme altogether of unknown catalytic activity. A complete functional characterization in vitro will be necessary in order to determine its real function.

In duplex DNA, guanine radical cations react with water to form mainly 7,8-dihydro-8-oxoguanine (8-OxoG). Understanding for the mechanism for migration of a radical cation (hole) from the site of initial DNA oxidation to a remote guanine is an important step in the process that will lead to a thorough understanding of DNA damage and its repair. The vast majority of charge migration in DNA experiments utilize guanine oxidation as a monitor for charge transfer. The synthesis of a potential monitor for charge transfer through DNA that is independent of guanine oxidation is reported herein. The system is a carbazole moiety covalently attached to the 2O position of uridine which was successfully incorporated into a DNA strand. Carbazole has a low oxidation potential, and will create a deeper trap than guanine during DNA charge transfer. One electron oxidation of carbazole should lead to the formation of its radical cation. The high extinction coefficient of carbazole radical cation should make it clearly observable with UV analysis. Hence a monitor for charge migration in DNA independent of guanine oxidation is obtained.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2003. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Fahrni, Christoph, Committee Member ; Collard, David, Committee Member ; Schuster, Gary, Committee Chair.