Hannover, Universiẗat, Diss., 2000.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Freie Universiẗat, Diss., 2005--Berlin. / Dateiformat: zip, Dateien im PDF-Format.

NADH and NADPH are ubiquitous biological reducing agents essential for both respiration and biosynthesis. The discovery that increased pentose-phosphate pathway activity in cervical cancer cells leads to increased levels of NAD(P)H, emphasises the need for a sensitive detection system as an indication of cellular viability and vitality. The remit of this project was to design and synthesise a novel molecular sensor system whose emissive properties are "switched on" upon reduction by NAD(P)H. Research using the reducible, non-fluorescent dye, resazurin, has shown that, in the presence of a non-enzymic electron transfer agent phenazinium methosulphate (PMS)-NADH can effect reduction to the highly fluorescent dye resorufin. Mechanistic studies have shown that the reduction proceeds via a two-electron hydride transfer to the heterocyclic mediator, followed by a one electron transfer to the dye and disproportionation to furnish the final fluorescent product. It has been shown that direct reduction by NADH does not occur and that the reaction depends upon there being an electron transfer agent present. A new type of reagent for the detection of NAD(P)H has been synthesised, comprising a reducible heterocycle and a masked fluorophore. It has been shown that reduction of the precursor conjugate by NADH results in the release of a detectable fluorescent moiety methylumbelliferone. The synthesis of an analogous conjugate probe containing a known hindered dioxetane moiety is described. Prepared using a previously unreported route, the key vinyl ether intermediate is generated via a Wadsworth-Emmons reductive coupling of an alkoxy phosphonate to 2-adamantanone. Reduction by NADH and subsequent cleavage of a conjugate ether link generates an electron rich phenolate substituted dioxetane which is metastable, resulting in emission from the generated excited product. Work towards a dioxetane containing functionalised alkyl group for conjugation to a fluorophore is also outlined.

Orientador: Lauro Tatsuo Kubota / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T13:04:11Z (GMT). No. of bitstreams: 1 Camargo_MaiuiNagaoLindqquerde_M.pdf: 3614340 bytes, checksum: 6d1263c93417d379a0c3010830acb5e6 (MD5) Previous issue date: 2015 / Resumo: Este trabalho visa demonstrar como o grau de redução do óxido de grafeno eletroquimicamente reduzido (ERGO) pode ser modulado dependendo das condições experimentais utilizadas para se fazer a redução eletroquímica, e como ele influencia nas propriedades eletroquímicas do material final. Esta influência pôde ser constatada por medidas eletroquímicas, de espectroscopia Raman e de fotoelétrons excitados por raios-X (XPS). Através de experimentos eletroquímicos feitos na presença da sonda de ferri/ferrocianeto de potássio, foi possível demonstrar que os eletrodos de ouro modificados com os ERGOs com maiores graus de redução se comportam eletroquimicamente de maneira similar ao não modificado, e portanto, a redução dos grupos oxigenados da superfície do material é importante para que essa similaridade seja atingida. No entanto, essa sonda não permite monitorar o balanço entre grupos oxidados e reduzidos e para fazer isso, foi escolhida uma sonda eletroativa sensível aos grupos oxigenados. Análises feitas na presença de ?-nicotinamida adenina dinucleotídeo (NADH) demonstraram que grupos funcionais oxigenados essenciais para a oxidação dessa espécie estavam diminuindo na superfície do material com o aumento do grau de redução deste. Os espectros de Raman e de XPS também confirmaram essa informação. Além disso, a capacidade adsortiva do ERGO foi testada utilizando o corante Azul de Meldola. Novamente, o grau de redução do ERGO teve papel fundamental, uma vez que interações ?-? ou eletrostáticas podem ser favorecidas entre o ERGO e o corante, dependendo do grau de redução do primeiro, implicando em propriedades distintas dos materiais frente a oxidação de NADH / Abstract: This work aims to demonstrate how the extent of reduction of the electrochemically reduced graphene oxide (ERGO) can be modulated depending on the experimental conditions used for performing the electrochemical reduction, and how it influences on the electrochemical properties of the final material. This influence can be verified by electrochemical, Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements. By means of electrochemical experiments carried out in the presence of the ferro/ferricyanide probe, it was possible to demonstrate that the gold electrodes modified with the ERGOs with higher extents of reduction behave electrochemically in a similar manner to the non-modified, and therefore, the reduction of the oxygenated groups on the surface of the material is important for this similarity to be reached. However, this probe does not permit the monitoring of the balance between oxidized and reduced groups and to do so, an electroactive probe sensitive to the oxygenated groups was chosen. Analyses done in the presence of ?-nicotinamide adenine dinucleotide (NADH) enabled the conclusion that the oxygenated functional groups essential for the oxidation of this species decreased on the surface of the material with the increase of the extent of reduction. The Raman and XPS spectra also confirmed this information. Apart from this, the adsorptive capacity of the ERGO was tested using the dye Meldola's Blue. Once again, the extent of reduction of the ERGO had a fundamental role, since ?-? or electrostatic interactions can be favoured to occur between the ERGO and the dye, depending on the extent of reduction of the former, leading to distinct properties of the materials regarding NADH oxidation / Mestrado / Quimica Analitica / Mestra em Química

No description available.

No description available.

Electron transport systems exist in the plasma membranes of all cells. Although not well characterised they play roles in cell growth and proliferation, hormone responses and other cell signalling events, but perhaps their most important role, especially in erythrocytes, is enabling the cell to respond to changes in both intra- and extracellular redox environments. Human erythrocytes possess a transmembrane electron transport capability that mediates the transfer of reducing equivalents from reduced intracellular species to oxidised extracellular species and is concomitant with proton extrusion. In the work for this thesis I showed that erythrocyte membranes contain a transmembrane WST-1 (water soluble tetrazolium-1) reductase activity that uses reducing equivalents from intracellular NADH to reduce extracellular WST-1. The rate of WST-1 reduction was increased by the presence of phenazine methosulfate and, although of low activity, it showed similar properties to a previously reported transmembrane NADH-oxidase activity. 1H NMR experiments showed that WST-1 was reversibly bound to the membrane and/or proteins in the membrane within the timeframe of the NMR experiment, confirming the location of the WST-1 reduction. Preliminary attempts to purify NADH:WST-1 reductase and NADH:ferricyanide reductase activities from the erythrocyte plasma membrane were inconclusive. The protein(s) responsible for the reduction of these oxidants appear to be of low abundance in the plasma membrane and may be part of a larger protein complex. Further work on the isolation of these redox activities is required before the protein(s) involved can be identified with any confidence. The ability of cells to export electrons suggests that an electron import mechanism might also exist to re-establish the cell�s redox-buffering equilibrium under conditions of oxidative stress. This hypothesis was tested in glucose-deprived erythrocytes using reduced glutathione and NADH as extracellular electron donors. It was shown that neither reduced glutathione nor NADH donated reducing equivalents through a transmembrane redox system. Extracellular NADH was, however, able to produce profound changes in starvation metabolism and methaemoglobin reduction rates. The addition of extracellular NADH caused a six-fold increase in the rate of lactate production above that observed in glucose-starved controls, together with a concomitant decrease in pyruvate production. In erythrocytes containing high levels of methaemoglobin, extracellular NADH increased the rate of methaemoglobin reduction in both the presence and absence of glucose. These results were explained by the leakage of lactate dehydrogenase from erythrocytes due to an admittedly low level of haemolysis. This caused the displacement of the intracellular pseudo-equilibrium of the lactate dehydrogenase reaction via transmembrane exchange of lactate, allowing the conversion of extracellular pyruvate to lactate and resulted in an increase in intracellular NADH concentrations. The latter increased the rate of methaemoglobin reduction. In conclusion, the work described in this thesis showed that erythrocyte membranes do not contain mechanisms for importing electrons or reducing equivalents from extracellular reduced glutathione or NADH. Erythrocytes do, however, contain an electron export system which can reduce extracellular oxidants such as WST-1 and the activity of this system depends on an intricate balance between intracellular antioxidants and enzyme activities. There is much still to be learnt about plasma membrane redox systems, little is known, for example, about the protein composition, mechanism of action, and the in vivo conditions under which these systems are most active.

Many oxidoreductases need nicotinamide cofactors for their reactions. The big obstacle of using these syntheses in industry is the high cost of these nicotinamide cofactors. The work here is about finding novel NADH oxidases from Lactococcus lactis and applying in a cofactor regeneration system with carbonyl reductase or alcohol dehydrogenase. NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of biological redox reactions. The annotated alkyl hydroperoxide reductase (AhpR) and the H2O-forming enzyme (nox-2) genes from Lactococcus lactis (L. lactis, L.lac-Nox2) were cloned and proteins were expressed and characterized. They were compared with the H2O-former from Lactobacillus sanfranciscensis (L. sanfranciscensis, L.san-Nox2). AhpR is composed of H2O2-forming NADH oxidase (nox-1) and peroxidase and the net reaction of AhpR is the same as nox-2 when oxygen is the substrate. Both nox-1 and nox-2 are flavoproteins and turnover-limited. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2. L.san-Nox2 was crystallized and was found to have ADP ligand, but according to the HPLC results, no ADP ligand was found in the L. lac-Nox-2. Enzyme membrane reactor was used for the application of oxidative reaction of cyclohexanol to cyclohexanone, with isolated enzymes horse liver alcohol dehydrogenase and L.lac-Nox2.

Metronidazole is an essential component of the triple therapy regimen against Helicobacter pylori infection. The development of resistance towards metronidazole results in failure to eradicate H. pylori completely. The main aim of the investigation was to understand further the mechanism(s) of resistance in H. pylori. The investigation focussed upon studying the role and function of NADH oxidase in metronidazole resistance. The NADH oxidase levels were shown to be significantly higher in metronidazole susceptible strains than in resistant strains. The purification and characterisation of the enzyme responsible for the oxidation of NADH resulted in isolation of a protein shown to be catalase. The results suggest that NADH oxidase activity in susceptible strains is a function of a bifunctional catalase rather than that of a distinct enzyme. This was confirmed by isolation of catalase from E. coli cells containing cloned H. pylori catalase and demonstration that catalase and NADH activities co-purified. The catalase activity of the purified protein from the bacterial strains used was retained but the oxidation of NADH was not significant in the resistant strain. The base sequence of the catalase gene from the susceptible strain was determined and shown to be 99% identical to that from the cloned gene. The comparison of the derived amino acid sequence of catalase from H. pylori and Proteus mirabilis showed that the heme-binding site is highly conserved. The amino acids in the NAD(P)H binding site are conserved in both strain NCTC 11639 (Mtz s ) and the genomic strain ATCC 26695 (Mtz s) of H. pylori but show significant differences compared with P. mirabilis. A three-dimensional model of catalase from a metronidazole-susceptible H. pylori strain showed stearic hindrance around the NAD(P)H binding site and a substitution of an acidic for a basic residue within the phosphate binding site. Both effects could result in NAD(P)H being less tightly bound and, hence able to leave the catalase in exchange for NADH. Other substitutions may account for the ability of the modified binding site to oxidise NADH. The oxidation of NADH aids in the activation of metronidazole, which damages DNA. The absence of NADH oxidase activity in resistant strains results in the inability of the enzyme to activate metronidazole leading to resistance. The finding that this NADH oxidase activity is a function of a modified catalase in susceptible strains suggests a novel mechanism of metronidazole resistance in H. pylori.