LA GALACTOSE OXYDASE EST UNE METALLOENZYME DU CUIVRE QUI CATALYSE L'OXYDATION AEROBIQUE DES ALCOOLS PRIMAIRES EN ALDEHYDE AVEC REDUCTION CONCOMITANTE D'UNE MOLECULE DE DIOXYGENE EN PEROXYDE D'HYDROGENE. SON SITE ACTIF RENFERME L'ASSOCIATION ORIGINALE CUIVRE(II)-RADICAL TYROSINYLE QUI EMMAGASINE DEUX EQUIVALENTS OXYDANTS.<br />A PARTIR DE COMPLEXES DU CUIVRE MODELES, NOUS AVONS REPRODUIT CETTE ASSOCIATION ET L'AVONS ETENDUE A DES STRUCTURES PLUS COMPLEXES POUVANT EMMAGASINER TROIS ET QUATRE EQUIVALENTS OXYDANTS.<br />EN PREMIER LIEU, NOUS AVONS PREPARE DES COMPLEXES DU CUIVRE A PARTIR DE LIGANDS TRIPODAUX A SPHERE DE COORDINATION DE TYPE N3O. LES COMPLEXES RADICAL PHENOXYLE-CUIVRE ONT ETE ENGENDRES PAR OXYDATION ELECTROCHIMIQUE DES COMPLEXES PRECEDENTS. NOUS AVONS PU OBSERVER DES EFFETS DE LA STRUCTURE DU LIGANDS SUR L'ENVIRONNEMENT AUTOUR DU CUIVRE ET SUR LA STABILITE DES COMPLEXES RADICALAIRES CORRESPONDANTS.<br />DANS UN SECOND TEMPS, LA CHIMIE EN SOLUTION DES LIGANDS EN PRESENCE DE CUIVRE(I) OU (II), SOUS DES CONDITIONS AEROBIQUES OU ANAEROBIQUES, A ETE ETUDIEE AFIN DE MIMER ET COMPRENDRE LE PROCESSUS DE MATURATION DE L'ENZYME. UNE ETUDE PAR RMN DU FLUOR 19 A PERMIS D'APPORTER UNE CONTRIBUTION NOUVELLE A LA COMPREHENSION DE CETTE CHIMIE EN SOLUTION: DIFFERENTS TYPE DE MARQUAGE PAR LE FLUOR ONT ETE UTLILISES: MARQUAGE PAR F OU CF3 SUR UN PHENOL OU F SUR UNE QUINOLEINE.<br />DANS LA DERNIERE PARTIE, DES COMPLEXES DESTINES A STOCKER PLUS DE DEUX EQUIVALENTS OXYDANTS ONT ETE MIS AU POINT: POUR LE STOKAGE DE TROIS EQUIVALENT OXYDANTS, NOUS AVONS OBTENU LES DEUX PREMIERS COMPLEXES COMPORTANT UN RADICAL PHENOXYLE PONTANT DEUX IONS CUIVRIQUES

M.S. / Biochemistry / The EPR spectrum of the Tyr-Cys· free radical in oxidized apoGAOX has been investigated, using a combination of approaches. Power saturation analysis has been used to resolve two unique spectra through Evolving Factor Analysis (EFA) global fitting, indicating the presence of two distinct free radical species in the sample. The component that dominates at low microwave power arises from the Tyr-Cys· side chain, while the high power component has not yet been assigned. The experimental results show that the EPR spectrum collected at low power includes approximately 7% of the high power component. EPR spectra have been collected for ten different isotope derivatives of GAOX, including ²H-labeled, ¹³C-labeled, 17[superscript]O-labeled, and ³³S-labeled forms. XSophe simulation of the EPR spectra has been performed for the isotopically labeled samples in order to determine the spectroscopic parameters - g-values, hyperfine coupling constants, and linewidths. The g-values and the methylene proton hyperfine coupling constants obtained for the isotopically labeled samples are consistent with the literature values. The magnitude of the hyperfine coupling constants associated with each of the nuclei confirms that significant electron spin density is found on the methylene protons, the alternating carbon atoms within the aromatic π system and the 2p[subscript]z orbital of both sulfur and oxygen. Moreover, the rotation angle of the methylene protons to the phenoxyl ring around the C1-C7 bond has been evaluated based on the experimentally defined hyperfine coupling constants of the two methylene protons.

African sleeping sickness is a parasitic infection spread by the protozoan parasite Trypanosoma brucei, and drugs used today are toxic and painful. Galactose metabolism is essential for the survival of T. brucei and without a functional UDP galactose 4’ epimerase (GalE) galactose starvation occurs and cell death will follow. In this Master thesis project two assays observing binding of small molecules to TbGalE has been investigated in attempt to establish an assay that in the future could be used for screening for drugs. TbGalE was biotinylated through the Pinpoint Xa vector and expressed in E. coli cells. The protein was successfully immobilized to a Streptavidin chip for Surface Plasmon Resonance experiments and the binding of the substrates UDP-galactose and UDP-glucose was observed. Unfortunately, the assay was not optimal for screening due to low signal response. However, the established protocol for expressing biotinylated proteins that bind to Streptavidin surfaces could be used in further experiments with TbGalE and other drug targets for African sleeping sickness. The fluorescent sugar nucleotide analogue UDPAmNS, which is a known inhibitor for E. coli GalE, was synthesised and purified and then used to establish a displacement assay. IC50 of UDPAmNS against TbGalE was determined and a synergic effect in fluorescence between the protein and the inhibitor was proven. Further, evidence for a reduction in fluorescence by displacing UDPAmNS with UDP was obtained. This reduction in fluorescence was also shown by a predicted cofactor inhibitor. The IC50 against TbGalE for this compound was determined before the displacement assay, which showed that the cofactor inhibitor, at least partly, binds to the active site of TbGalE. The UDPAmNS displacement assay could have the potential of becoming a robust screening assay for TbGalE, in the effort to find a better drug for African sleeping sickness.

In this study, the novel idea of in-vivo directed evolution was applied in order to achieve variants of the enzyme galactose oxidase with increased activity. This procedure was done under starvation conditions in Escherichia coli BL21 Star (DE3). Previous studies have been carried out in order to improve the activity of this enzyme using directed evolution methods. In this study, the same idea was used in-vivo, during stationary phase adaptive mutations inside the host organism, hence called in-vivo directed evolution. This method gave variants with improved enzyme activity as compared with the wild-type enzyme, and some variants showed activities that were even higher than the variants of previous directed evolution studies, hence making this method a promising approach for the random mutagenesis of genes of interest. The above mentioned mutations are carried out by a special group of polymerases, the error-prone polymerases. Phylogenetic analysis of these error-prone polymerases was also carried out in order to investigate the relationship between the number of error-prone polymerases and the level of complexity of organisms, and both the number of error-prone polymerases and the ratio of error-prone polymerases to total DNA polymerases of six organisms were studied. It was found that as the organism gets more complex, the number of error-prone polymerases and their ratio to the total polymerases increase.

In this study, self-catalytic maturation of heterologously expressed pro-galactose oxidase was analysed in E.coli by altering some amino acids which were supposed to play a crucial role in pro-peptide removal. Galactose oxidase (GOase / EC 1.1.3.9) from Fusarium graminearum / having a molecular mass of 68kDa, is a monomeric, copper containing enzyme with an unusual thioether bond. The enzyme is produced as a precursor with an additional 8 amino acid pre- and a 17- amino acid pro-sequence at the N terminus. Previous work has shown that the pre-peptide is removed possibly by a protease during secretion, whereas the 17 amino acid pro-peptide is removed autocatalytically by the aerobic addition of Cu2+ to the precursor, preceding the formation of the thioether bond at the active site. The pro-gao gene was on ProGON1 and ProGOMN1 constructs which were previously established on pET101/D/lacZ vector in England by directed evolution. ProGON1 contains silent mutations at the N-terminus different from native galactose oxidase whereas ProGOMN1 has six further mutations within the mature enzyme, providing high expression. The cleavage site mutations R-1P/A1P, R-1X/A1X, S2A, and the H522A mutation just against the cleavage site in the three dimensional configuration, were carried out by site-directed mutagenesis. Those and some extra mutations were confirmed by DNA sequence analysis. Next, mutant galactose oxidases were expressed in E. coli BL21 Star (DE3), and were purified by Strep-Tactin&reg / Sepharose&reg / column, operating on the basis of affinity chromatography. Subsequently, SDS-PAGE was performed to analyze self-processing by detecting molecular mass difference of protein bands resulting from pro-sequence removal or existence. When the bands obtained in SDS-PAGE were compared, it was seen that the products of original recombinant plasmids, i.e. ProGON1, ProGOMN1 / and the mutational variants showed no difference in band size, all slightly above 70kDa / indicating pro-sequence presence on all constructs. Non-mutants and some of the mutants showed galactose oxidase activity, signifying proper active site construction by thioether bond formation. ProGOMN1 was submitted for N-terminal amino acid sequencing to be able to assert that a size above 70kDa is not solely due to the existence of a 1 kDa Strep-tag II at C-terminus. Sequencing data affirmed the presence of both the pre-peptide and the pro-preptide showing that processing has not occurred at the N-terminus. Accordingly, in this study, it was shown for the first time that the existence of a pre-pro-peptide at the N-terminus of galactose oxidase does not prevent thioether bond formation at the active site. Furthermore, since the pro-peptide is cleaved autocatalytically, the lack of removal of the pre-peptide in E.coli in the presence of Cu 2+ and oxygen is very likely to be the cause of lack of pro-peptide cleavage. In future studies the region corresponding to the pre-peptide will be deleted to prove this hypothesis.

Made available in DSpace on 2014-06-11T19:30:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-20Bitstream added on 2014-06-13T20:40:27Z : No. of bitstreams: 1 matos_dc_dr_arafcf.pdf: 766440 bytes, checksum: d75671cd6c4bdad30b7d9a66e5d5621d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / O termo “câncer” corresponde ao conjunto de cerca de 100 doenças que têm em comum uma divisão celular desordenada invadindo os tecidos e órgãos, podendo metastatisar para outras regiões do corpo. Os tumores de mama e de colo do útero são os tipos mais frequentes no sexo feminino, e as estimativas para 2012, no Brasil, são de 52.680 e 17.540 novos casos, respectivamente. Abrina, obtida de sementes de Abrus precatorius é uma proteína inativadora de ribossomos (RIPs) do tipo 2, compostas por duas cadeias peptídicas, uma apresentando atividade enzimática N-glicosidase e a outra atividade lectínica com afinidade a β-D-galactose. Neste estudo, avaliamos o potencial antitumoral contra o câncer de mama de abrina em um modelo de câncer mamário murino já estabelecido em nosso laboratório, concomitante à avaliação dos efeitos imunológicos do tumor e da proteína testada no animal. Para isso avaliamos a partir dos tumores e células do sistema imunológico retirados dos animais do estudo, o percentual de inibição do crescimento e angiogênese tumorais, o percentual de células imunes e de células apoptóticas no peritôneo e baço, a produção de citocinas e mediadores químicos produzidos pelas células imunes. Com base nos resultados obtidos, observou-se que o tratamento com abrina (7,5μg/kg) apresentou eficácia similar ao da droga padrão pois apresentou infiltrado inflamatório misto dentro tumor, percentuais de inibição de crescimento e de angiogênese tumorais, e de células NK e NKT esplênicas similares ao tratamento padrão (doxorrubicina - 6mg/kg); em contrapartida apresentou menor quantidade de macrófagos peritoneais e células dendríticas esplênicas ativadas, células T CD8+ citotóxicas e células T regs (CD25+Foxp3+), e maior percentual de células T CD4+, maior produção de H2O2, IL- 1β, IL-6 e IL-12 por células peritoneais... / The word Cancer corresponds to a group with about 100 diseases which have in common a disordered cell division, resulting in tissues and organs invasion and also metastasis to other body regions. Breast and cervix tumours are the most common type in women and, in Brazil, the estimates for 2012 are of 52,680 and 17,540 new cases respectively. Abrin is obtained from Abrus precatorius seeds being type 2 ribosome inactivating protein (RIP) composed of two peptide chains, one of them possessing N-glycosylase enzyme activity and the other a lectinic activity with affinity for β-D-galactose. In the present work, the antitumour potencial of abrin against breast cancer was evaluated employing a murine breast cancer model already established in our laboratory and concomitantly determining the immunologic effects of the tumour and the protein in mice. From tumours and immune cells isolated from mice belonging to the different experimental groups, the tumour growth inhibition rate and angiogenesis were determined, the percentage of immune and apoptotic cells in the peritoneum and spleen, and also the production of immunologic mediators by immune cells. Based on the results it was found that treatment with abrin (7.5μg/kg) was as effective as the standard drug (doxorubicin - 6 mg/kg) considering that inflammatory tumour infiltrate, tumour growth inhibition and angiogenesis rate, as well as splenic NK and NKT cells percentage were similar. However, abrin-treated animals had a lower percentage of cytotoxic CD8+ and Treg (CD25+Foxp3+) cells as well as activated peritoneal macrophages and splenic dendritic cells accompanied by a higher percentage of CD4+ T cells and increased production of H2O2, IL-1β, IL-6 and Il-12 by peritoneal macrophages and IL-10, IFNγ and TGF-β by splenic lymphocytes. Moreover, abrin-treated animals presented a higher percentage... (Complete abstract click electronic access below)

Altos níveis de galactose circulantes e cerebrais são encontrados em portadores da galactosemia clássica não tratada, cujos pacientes comumente desenvolvem problemas cognitivos e motores ao longo da vida. Entretanto pouco se conhece a respeito dos mecanismos da disfunção celular e molecular responsáveis por estes sintomas. Assim, o objetivo do presente trabalho foi de investigar o efeito da injeção intracerebroventricular de galactose sobre a memória (aversiva e de reconhecimento de objetos) e a coordenação motora em ratos Wistar. Além disso, a atividade, o imunoconteúdo e a expressão gênica da acetilcolinesterase no hipocampo e córtex cerebral foram também avaliados. No cerebelo, foram medidos parâmetros histológicos (contagem de células e imunohistoquímica) e bioquímicos (imunoconteúdo de caspase-3 ativa e BDNF, atividade e imunoconteúdo da acetilcolinesterase, níveis de glutationa e sulfidrilas, bem como o índice de dano ao DNA). Ratos Wistar receberam uma injeção intracerebroventricular de galactose (4 mM) ou salina (controles) sendo esses submetidos às tarefas comportamentais e/ou decapitados em diferentes tempos (1 h, 3 h ou 24 h), logo após, o hipocampo, córtex cerebral e cerebelo foram dissecados. Os resultados mostraram que a galactose prejudicou a memória aversiva e de reconhecimento de objetos, quando injetada antes do treinamento, bem como alterou a atividade e a expressão gênica da acetilcolinesterase em hipocampo. Em relação ao comportamento motor e aos parâmetros histológicos e bioquímicos realizados no cerebelo, a administração intracerebroventricular de galactose prejudicou a coordenação motora e reduziu o número de células e a imunomarcação de neurônios e astrócitos. A galactose, também aumentou o imunoconteúdo de caspase-3 ativa, a atividade da acetilcolinesterase e o índice de dano ao DNA, bem como diminuiu o imunoconteúdo de BDNF e acetilcolinesterase e os níveis de glutationa e sulfidrilas no cerebelo. Tomados em conjunto, nossos resultados mostram que a administração intracerebroventricular de galactose prejudicou a memória e a coordenação motora. Além disso, o modelo experimental utilizado mostrou diversas alterações a nível celular e molecular, os quais podem contribuir pelo menos em parte com o entendimento da fisiopatologia da galactosemia clássica. / Non-treated patients with classical galactosemia present high levels of galactose in the bloodstream and brain. Patients usually develop cognitive and motor impairments during life. However, little is known about the cellular and molecular mechanisms responsible for these symptoms. Thus, the aim of this study was to investigate the effect of intracerebroventricular galactose injection on memory (aversive and object recognition) and motor coordination in Wistar rats. Acetylcholinesterase activity, immunocontent and gene expression were investigated in hippocampus and cerebral cortex. In the cerebellum, we performed histological (cell counting and immunohistochemistry) and biochemical (active caspase-3 immunocontent, BDNF, acetylcholinesterase activity and immunocontent, glutathione and sulfhydryl levels, as well as DNA damage index) parameters. Wistar rats received a single intracerebroventricular injection of galactose (4 mM) or saline (control). The animals performed behavioral tasks and/or were decapitated at different times (1 h, 3 h or 24 h) after injection. The hippocampus, cerebral cortex and cerebellum were dissected. The results showed that injecting galactose before training provokes impairment on aversive and object recognition memories, as well as altered the activity and gene expression of acetylcholinesterase in hippocampus. Regarding to the histological and biochemical parameters measured in the cerebellum, intracerebroventricular galactose injection impaired motor coordination, reduced the number of cells and immunostaining of neurons and astrocytes. In the cerebellum, galactose also increased active capase-3 immunocontent, acetylcholinesterase activity and DNA damage index, as well as decreased BDNF and acetylcholinesterase immunocontent, and glutathione and sulfhydryl levels. Altogether, our results show that intracerebroventricular injection of galactose impaired memory and motor coordination. Moreover, the experimental model used showed several alterations at cellular and molecular levels. These findings may contribute at least in part with the understanding of the physiopathology in classical galactosemia.