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1

Roeschlaub, Carl Andrew. "The design and synthesis of novel reductively activated molecular sensors." Thesis, University of Surrey, 2000. http://epubs.surrey.ac.uk/843218/.

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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.
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2

Shuler, Elizabeth. "The effects of flavonoids on mitochondrial membrane-associated reduced pyridine nucleotide-utilizing systems of adult Hymenolepis diminuta (cestoda) and Ascaris suum (nematoda)." Bowling Green State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1367950138.

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3

Crowley, Louis J. "Structure-function studies of conserved sequence motifs of cytochrome b5 reductase." [Tampa, Fla] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0001913.

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4

Farooqi, Mohammed Junaid. "METHODS FOR IN SITU PIEZOPHYSIOLOGICAL STUDIES: OPTICAL SECTIONING VIA STRUCTURED ILLUMINATION AND FLUORESCENCE BASED CHARACTERIZATION OF NADH CONFORMATION." Oxford, Ohio : Miami University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1249225952.

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5

Melo, Ana Margarida Nunes Portugal Carvalho. "Characterization of NAD(P)H dehydrogenases from neurospora mitochondria." Doctoral thesis, Porto : Edição do Autor, 2001. http://hdl.handle.net/10216/64566.

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6

Melo, Ana Margarida Nunes Portugal Carvalho. "Characterization of NAD(P)H dehydrogenases from neurospora mitochondria." Tese, Porto : Edição do Autor, 2001. http://catalogo.up.pt/F?func=find-b&local_base=UPB01&find_code=SYS&request=000088166.

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7

Leman, Géraldine. "Régulation de la fonction mitochondriale par le rapport NADH/NAD+ : le rôle clef du complexe I." Thesis, Angers, 2014. http://www.theses.fr/2014ANGE0016/document.

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Le NAD+ apparaît comme un régulateur majeur du fonctionnement mitochondrial. En effet, ce cofacteur régule non seulement l’activité de nombreuses enzymes impliqués dans le métabolisme énergétique (enzymes de la β-oxydation des acides gras, du cycle de Krebs) mais joue également un rôle dans la production d’espèces réactives de l’oxygène (ROS). Le NAD+ est aussi le cofacteur des sirtuines, des enzymes déacétylases régulatrices notamment du métabolisme mitochondrial. De plus, la mitochondrie est l’organite au sein duquel la concentration en NAD+ est la plus élevée (jusqu’à 70% du NAD cellulaire). Le complexe I, qui possède une activité NADH déshydrogénase, pourrait être l’un des régulateurs majeurs du rapport NADH/NAD+ mitochondrial. L’objectif de ce travail de thèse a été d’étudier le rôle du rapport NADH/NAD+ mitochondrial dans le métabolisme énergétique et l’implication du complexe I dans les pathologies mitochondriales. Nous avons mis en évidence qu’une modulation du rapport NADH/NAD+ mitochondrial (augmentation par un activateur pharmacologique ou diminution consécutive à une mutation touchant une sous-unité du complexe I, modifie de manière drastique le métabolisme énergétique notamment en activant ou inhibant la protéine SIRT3, isoforme mitochondriale des sirtuines. Le complexe I semble jouer un rôle majeur dans cette modulation. Le resveratrol, ciblant le complexe I, ainsi que le NMN, un précurseur du NAD+, permettent de restaurer ce rapport et d’améliorer ainsi le métabolisme mitochondrial. Nos résultats suggèrent donc que le rapport NADH/NAD+ pourrait être une cible thérapeutique particulièrement intéressante dans les déficits du complexe I
NAD+ appears as a main regulator of the mitochondrial function. Indeed, this compound not only regulates the enzymatic activity of enzymes involved in energetic metabolism (fatty acid oxidation, tricarboxylic acid cycle) but is also involved in ROS production. NAD+ is also the cofactor of sirtuins, deacetylase enzymes, in particular regulating the mitochondrial function. Moreover, mitochondria sequester most of the cellular NAD+ (up to 70 %). The complex I, which possesses an NADH dehydrogenase activity, is thought to be the most important regualtor of the mitochondrial NADH/NAD+ ratio. The work presented here aimed at studying the role of the mitochondrial NADH/NAD+ ratio in mitochondrial metabolism and to test the involvement of the complex I in mitochondrial disorders. We show that a modulation of the mitochondrial NADH/NAD+ ratio (increase by a pharmacological agent or decrease in complex-I mutated fibroplasts) severely affects the mitochondrial energetic function especially by interacting with SIRT3 a mitochondrial sirtuin isoform. The NADH/NAD+ ratio is highly regulated by complex I activity. Resveratrol, which targets the complex I, as well as NMN, a NAD+ precursor, improves the mitochondrial NADH/NAD+ ratio and consequently increases the mitochondrial metabolism. Our results strongly suggest that the mitochondrial NADH/NAD+ ratio could be an interesting therapeutic target especially in complex I- deficient patients
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8

Marx, Stefanie. "Die Co-Evolution der Cytochrom-c-Reduktase und der mitochondrialen Prozessierungsprotease." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960312099.

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9

Khalily, Mohammad Aref. "Synthesis Of New Mediators For Electrochemical Nad/nadh Recycling." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12612961/index.pdf.

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The synthesis of enantiopure compounds can be achieved by using dehydrogenases as biocatalysts. For instance, reduction reactions of prochiral compounds (ketones, aldehydes and nitriles) into chiral compounds can be achieved by dehydrogenases. These dehydrogenases are cofactor dependent where cofactor is Nicotinamide Adenin Dinucleotite having some restrictions that confines usage of dehydrogenases in organic synthesis including instability of cofactor in water and high cost. Therefore, suitable recycling methods are required and developed which are enzymatic and electrochemical. We will use an electrochemical approach for the regeneration of reduced co-factors. All active compounds
mediator, cofactor and enzyme, will be immobilized on the electrode surface of the constructed reactor surface. Therefore only educts and products will exist in the reactor medium. A gas diffusion electrode will be employed as a counter electrode
which delivers clear protons to the system. Mediator will carry electrons to the cofactor for cofactor regeneration. Then, enzyme will utilize the cofactor and change the substrates to the products in high stereoselectivity. Our aim in this project is the synthesis of mediators and suitable linkers for enzyme, cofactor and mediator immobilization. In the first part of the study, mediators were synthesized which are pentamethylcyclopentadienyl rhodium bipyridine complexes. In the second part of the study, a conductive monomer (SNS) and linker were synthesized for immobilization of the enzyme. In the last part of the study, the reaction of galactitol dehydrogenase with monomer (SNS) was achieved.
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10

Meijers, Rob. "The activation of NADH in liver alcohol dehydrogenase." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2001. http://dare.uva.nl/document/60889.

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11

Barker, C. D. "Studies of NADH oxidation by NADH : ubiquinone oxidoreductase (complex I) from bovine mitochondria." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596366.

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The work described here focuses primarily on the flavoprotein (Fp) subcomplexes of complex I, as a subject for studying NADH oxidation. Fp is the smallest catalytically active subcomplex of complex I. It contains only two subunits and three cofactors – the FMN and a [4Fe-4S] cluster bound by the 51 kDa subunit, and a [2Fe-2S] cluster bound by the 24 kDa subunit. First, the development of a reproducible method for isolating stable and catalytically active Fp from complex I, using chaotropic resolution and ion-exchange chromatography, is described. The subcomplex produced contains the two subunits in equal amounts, and both [Fe-S] clusters and the FMN were present. Fp was capable of NADH oxidation and electron transfer to artificial electron acceptors in standard solution assays. The potential of the FMN was measured and values agree well with those reported from electron paramagnetic resonance (EPR). NADH oxidation was observed and explained by a model which incorporates mass transport of substrate. Michaelis-Menten enzyme kinetics, and interfacial electron transfer. The apparent potential of the active site under non-turnover conditions was higher than the values measured in the absence of substrate, providing insights into the mechanism of NADH oxidation. Unexpectedly, the rate of NAD+ reduction was fastest at intermediate potential – as the driving force for the reaction was increased a sharp maximum in activity was observed, after which there was a marked decrease. Two mechanistic models are proposed and shown to reproduce the experimentally observed voltammograms. In a second investigation, the [Fe-S] clusters in complex I were studied by EPR spectroscopy.
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12

Llanos, Ricardo Pariona. "Caracterização de interações proteína-DNA em tripanossomas." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-22092014-184950/.

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O T. cruzi, é o agente causador da doença de Chagas. O estado redox NAD+/NADH intracelular é fundamental na manutenção do metabolismo celular. A GAPDH apresenta a função de proteção do telômero em mamíferos contra degradação, isto por causa de ligar se ao telômero. Aqui, mostramos que a GAPDH recombinante de T. cruzi (rTcGAPDH) interage com o DNA telomérico. A rTcGAPDH liga ao DNA de simples fita. Mostramos que a GAPDH liga ao DNA telomérico in vivo em células epimastigotas, onde a [NADH] é maior que [NAD+], mas a adição de NAD+ exógeno bloqueia esta interação. Corroborando a hipótese de que o equilíbrio NAD+/NADH determina a interação GAPDH-telômero, vimos que o tripomastigota tem maior [NAD+] intracelular que a [NADH] e a GAPDH não é capaz de ligar se ao DNA telomérico. Além disso, o NADH exógeno resgata a interação GAPDH-telómero nesta fase. É importante o equilíbrio NAD+/NADH desta interação em tripanosomas, sugerindo que a proteção do telômero do parasita pode ser regulada pelo estado metabólico das células.
The T. cruzi, is the causative agent of Chagas disease. The redox state of NAD+/NADH intracellular is critical in the maintenance of cellular metabolism. The GAPDH has the protection function of the telomere in mammals against degradation, because it is connecting to the telomere. Here we show the recombinant GAPDH of T. cruzi (rTcGAPDH) interacts with telomeric DNA. The rTcGAPDH binds to single-stranded DNA. We show GAPDH to bind to telomeric DNA in vivo epimastigotes cells, where [NADH] is greater than [NAD+], but the addition of exogenous NAD+ blocks this interaction. Corroborating the hypothesis that the NAD+/NADH balance determines the GAPDH-telomere interaction, we saw that the trypomastigote has higher [NAD+] that intracellular [NADH] and GAPDH is not able to connect to telomeric DNA. In addition, the exogenous NADH recovers the GAPDH-telomere interaction at this stage. It is important the NAD+/NADH balance this interaction in trypanosomes, suggesting that the protection of the telomere of the parasite can be regulated by the metabolic state of the cells.
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13

Ilic, Stefan. "Utilizing NAD+/NADH Analogs for the Solar Fuel Forming Reductions." Bowling Green State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1499262103862098.

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14

Jiang, Rongrong. "Oxidative Biocatalysis with Novel NADH Oxidases." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/11533.

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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.
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15

Kappes, Kathrin. "Untersuchungen zur Pharmakokinetik von NADH in vivo und in vitro." Berlin : Mensch-und-Buch-Verl, 2005. http://www.diss.fu-berlin.de/2006/281/index.html.

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16

Ekbal, N. J. "NADH monitoring in shock states." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1463985/.

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Shock is defined as inadequate delivery or utilization of oxygen by the body tissues. Currently measured cardiorespiratory variables however, indicate decompensation only when patients become (near) shocked. Belated intervention often fails to reverse injury, leading to organ dysfunction and failure. Precise monitoring of the adequacy of tissue perfusion thus represents a major deficiency in clinical practice, particularly in the critically ill. As mitochondria utilize >90% of the oxygen consumed by the body, predominantly for ATP production, there is an obvious logic in targeting this organelle for monitoring the adequacy of tissue perfusion. Within mitochondria, NADH transfers electrons from the Krebs’ cycle to Complex I of the electron transport chain. In doing so, NADH is oxidized to NAD+. A rise in NADH:NAD+ ratio (redox state) will occur with a downstream block in the chain, e.g. due to lack of oxygen. As NADH (but not NAD+) fluoresces in response to UV light excitation (340nm), with an intensity relating to its concentration, and as most of the NADH signal represents changes in mitochondrial NADH, this property may be utilized for non-invasive assessment of tissue hypoperfusion. I validated the technique in vitro, and investigated its utility in rat shock models. During graded or abrupt decreases in the constituent parts of tissue oxygen delivery (cardiac output, haemoglobin and oxyhaemoglobin saturation), as well as during resuscitation, I assessed the relationship between skeletal muscle NADH fluorescence intensity, organ perfusion and oxygenation. I compared these against measures of global haemodynamics and tissue perfusion routinely measured in critically ill patients. With each graded insult, NADH fluorescence demonstrated increases reflecting severity of the insult, with improvements upon resuscitation. A persisting rise in NADH fluorescence >50% above baseline foretold death within the following 30-45 minutes, in advance of the other monitored variables. My results indicate that NADH fluorescence may be used for monitoring tissue hypoperfusion in shock states, and as a guide to the timing and adequacy of therapeutic interventions.
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17

Ali, Irshad. "Electrochemical investigations of the interactive behavior of Nicotinamide Adenine Dinucleotide (NAD+/NADH) with electrode surfaces: towards direct electrochemical regeneration of enzymatically-active NADH." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117104.

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Nicotinamide adenine dinucleotide NAD(H) is a co-enzyme which participates in a large number of biochemical processes in which it acts as a hydrogen and electron carrier. Hence, NAD(H) is found in two redox forms: oxidized, NAD+, and reduced, 1,4-NADH. Despite its high potential industrial use, due to its very high cost (especially that of 1,4-NADH) and the need to be added in a biochemical reactor in stoichiometric quantities, its current use is very limited. Hence, there is a need to develop in-situ 1,4-NADH regeneration methods. Electrochemical methods are of particular interest because of their potentially low cost and easy product isolation. However, the major problem in the electrochemical regeneration of enzymatically-active 1,4-NADH is the formation of an enzymatically inactive dimer, NAD2. This PhD project focused on (i) the investigation of fundamental aspects of the interaction of NAD+ with a glassy carbon (GC) electrode surface, in terms of the NAD+ reduction kinetics and its adsorption, and on (ii) the development of electrodes for the direct (non-mediated) electrocatalytic regeneration of enzymatically active 1,4-NADH. Potentiodynamic polarization measurements showed that under the experimental conditions employed, the NAD+ reduction reaction is under diffusion control, is irreversible (requires overpotential of more than -550 mV), and is of pseudo-first order with respect to NAD+. The kinetics of reduction of NAD+ on GC at a formal potential of the NAD+/NADH couple (-0.885 V) was found to be rather slow, and only moderately temperature dependent.It was determined that NAD+ is adsorbed on a GC electrode surface. The kinetics of NAD+ adsorption was found to be surface-charge dependent. The adsorption process was described by the Langmuir isotherm. The corresponding apparent Gibbs free energy of adsorption evidenced that the adsorption process is highly spontaneous. The influence of electrode potential and electrode material on the purity of regenerated 1,4-NADH was then investigated. It was found that the regeneration of 1,4 NADH from NAD+ in a batch electrochemical reactor employing non-modified electrodes (GC, Carbon Nanofibers /CNFs/, Ti, Ni, Co and Cd) is feasible. The purity (recovery) of 1,4-NADH regenerated on these electrodes was found to be highly potential- and material-dependant. The origin of the material/potential dependency was related to the strength of the metal-hydrogen (M-Hads) bond, and thus to the potential dependence of the Hads electrode surface coverage and the kinetics of the subsequent NAD-radical protonation by Hads. Among the above outlined non-modified electrodes, only GC and CNF electrodes were capable of producing the highest 1,4-NADH purity (99-100%), but at very high cathodic potentials (–2.3 V).Therefore, to produce high-purity 1,4-NADH at lower cathodic potentials, a GC electrode surface was patterned with electrochemically-deposited platinum and nickel nano-particles (NPs). It was demonstrated that when the GC electrode was patterned with Pt NPs, a 100% pure 1,4-NADH product was achieved at –1.6 V, while the Ni nano patterned GC surface gave 100% pure 1,4-NADH already at –1.5 V. The high purity of 1,4-NADH formed on the two nano-patterned electrodes was prescribed to the formation of Pt-Hads and Ni-Hads at significantly lower potentials than on bare GC and CNFs surfaces. It was found that purity of 1,4-NADH regenerated on the nano-patterned electrodes was dependent on the electrode potential, nano-particles size, and their surface coverage. Considering the energy input, the cost of the electrode, and the percentage of recovery of 1,4-NADH (i.e. its purity), the GC-Ni electrode was suggested as the electrode of choice for 1,4-NADH regeneration among all investigated electrodes (GC, CNF, Ti, Co, Cd, Ni, GC-Pt and GC-Ni).
Nicotinamide-adénine-dinucléotide NAD(H) est une coenzyme qui participe à un grand nombre de processus biochimiques dans lesquels elle agit comme une transporteuse d'électrons et d'atomes d'hydrogène. En dépit de sa forte utilisation potentielle dans l'industrie, son utilisation actuelle est très limitée à cause de son coût très élevé (en particulier celui du 1,4-NADH) et la nécessité d'être ajouté en quantités stœchiométriques dans les réacteurs biochimiques. Par conséquent, il est nécessaire de développer des méthodes de régénération in-situ du 1,4-NADH. Les méthodes électrochimiques sont d'un intérêt particulier en raison de leur coût potentiellement faible et l'isolement facile du produit. Cependant, le problème majeur dans la régénération électrochimique du 1,4-NADH est la formation d'un dimère enzymatiquement inactif, NAD2. Ce projet de doctorat est axé sur (i) l'étude des aspects fondamentaux de l'interaction du NAD+ avec la surface d'un électrode en carbone vitreux (GC), en termes de la cinétique de réduction et l'adsorption du NAD+ sur la surface du GC, et (ii) le développement d'électrodes pour la régénération électrocatalytique directe (non médiatisée) du composé 1,4-NADH, active enzymatiquement actif.Les mesures de polarisation potentiodynamique ont montré que dans les conditions expérimentales utilisées, la réaction de réduction du NAD+ est contrôlée par la diffusion. Cette irréversible (nécessite une surtension de plus de -550 mV) et est de pseudo premier ordre par rapport au NAD+. La cinétique de réduction du NAD+ sur GC, an potentiel formel du couple NAD +/NADH (-0.885 V), est lente, et modérément dépendante de la température. Le NAD+ est adsorbé sur la surface de l'électrode en GC. La cinétique d'adsorption du NAD+ s'est avérée dépendante de la charge surfacique. Le processus d'adsorption a été décrit par l'isotherme de Langmuir. L'énergie de Gibbs d'adsorption correspondante a montré que le processus d'adsorption est très spontané.L'influence du potentiel et du matériel de l'électrode sur la pureté du 1,4-NADH régénéré, a ensuite été étudiée. Il a été constaté que la régénération de 1,4-NADH à partir de NAD+, dans un réacteur électrochimique discontinu, employant des électrodes non modifiés est possible. La pureté (récupération) du 1,4-NADH régénéré sur ces électrodes a été jugée dépendante du potentiel et du matériel de l'électrode. L'origine de cette relationentre la nature elu nature ela matériel et le potentiel été liée à la force de liaison métal-hydrogène (M-Hads), et donc à la couverture du Hads sur la surface de l'électrode, que dépend du potentiel. Seuls les électrodes en GC et CNF ont été capables de produire la plus haute pureté du composé 1,4-NADH (99-100%), mais à des potentiels cathodiques le élevés (-2.3 V). Donc, pour produire 1,4-NADH de haute pureté à faibles potentiels cathodiques, la surface d'une électrode en GC a été modifiée par des nanoparticules (NPs) de platine et nickel, déposées par voie électrochimique. Il a été démontré que lorsque l'électrode en GC a été modifiées avec des NPs de Pt, le produit 1,4-NADH, avec une pureté de 100%, a été obtenu à -1.6 V, tandis que l'électrode en GC modifiée avec les NPs de Ni a produit 1,4-NADH avec une pureté de 100% déjà à -1.5 V. La haute pureté du 1,4-NADH formée sur les deux électrodes nano- modifiée a été prescrite à la formation des liaisons Pt-Hads et Ni-Hads à un potentiel nettement inférieur à celui sur une surface nue en GC. Il a été constaté que la pureté du 1,4-NADH régénérée sur les électrodes nano-modelées est dépendante du potentiel d'électrode, de la taille des nanoparticules et de leur couverture de la surfacique. Compte tenu de l'apport énergétique le coût de l'électrode, et le pourcentage de récupération du 1,4-NADH (i.e. sa pureté), l'électrode GC-Ni a été suggéré l'électrode de choix pour la régénération du 1,4-NADH parmi tous les électrodes étudiés (GC, CNF, Ti, Co, Cd, Ni, GC-Pt et GC-Ni).
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18

Santhiago, Murilo 1984. "Construção de plataformas nanoestruturadas para detecção de Cys e NADH." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248384.

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Orientador: Lauro Tatsuo Kubota
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: Neste trabalho descreve-se o desenvolvimento de dois sensores amperométricos empregando mediadores redox derivados do grupo nitro imobilizados sobre plataformas nanoestruturadas para a detecção da nicotinamida adenina dinucleotídeo (NADH) e L-cisteína. Para a construção da base das plataformas foram empregados nanotubos de carbono funcionalizados com grupos amino. Uma vez funcionalizada, a plataforma possibilitou o ancoramento dos dois mediadores redox. Para a detecção de NADH foi utilizado o ácido 3,5-dinitrobenzoico como mediador redox. Este mediador foi imobilizado na superfície da plataforma utilizando os catalisadores 1-etil-3-(3-dimetilaminopropil) carbodimida (EDC) e N-hidroxi succinimida (NHS). Já para o sensor destinado à detecção de L-cisteína, foi necessário a adsorção de nanopartículas de ouro para imobilizar o ácido 5,5'-ditiobis-2-nitrobenzoico (DTNB) como mediador redox. Para os estudos eletroquímicos, as espécies hidroxilamina foram eletrogeradas in situ a partir do grupo nitro presente em ambos os mediadores. As plataformas nanoestruturadas foram caracterizadas através de voltametria cíclica, cronoamperometria, MEV e eletrodo de disco rotatório. Os valores das constantes heterogênea de transferência de elétrons (ks) e da velocidade da reação mediador-analito foram ~50 s e 10 - 10 L mols, respectivamente. As curvas analíticas para ambos os sensores foram obtidas aplicando baixos sobrepotenciais e resultaram em limites de detecção e quantificação na faixa de 1,2 - 9,1 mmol L. O sensor para L-cisteína foi aplicado em amostras reais e os resultados obtidos foram estatisticamente iguais quando confrontados com um método comparativo
Abstract: This work describes the development of two amperometric sensors using redox mediators derived from the nitro group immobilized on nanostructured platforms for the detection of nicotinamide adenine dinucleotide (NADH) and L-cysteine. Carbon nanotubes functionalized with amino groups were used for the construction of the platforms. Once functionalized, the platform allowed the anchoring of two redox mediators. 3,5-dinitrobenzoic acid was used as redox mediator for NADH detection. This mediator was immobilized on the surface of the platform using the catalysts 1-ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC) and N-hydroxy-succinimide (NHS). For the construction of the sensor for L-cysteine detection, it was necessary the adsorption of gold nanoparticles to receive 5,5-dithio-2-nitrobenzoic acid as redox mediator. For the electrochemical studies, the hydroxylamine species were electrogenerated in situ from the nitro group present in both mediators. Nanostructured platforms were characterized by cyclic voltammetry, chronoamperometry, SEM and by rotating disk electrode experiments. Rate constant values for the heterogeneous electron transfer (ks) and the kinetic constants for the mediator-analyte reaction were ~50 s e 10 - 10 L mols, respectively. The analytical curves for both sensors were obtained applying low overpotentials and resulted in limits of detection and quantification around 1.2 up to 9.1 mmol L. The sensor for L-cysteine was used in pharmaceutical samples and the results were statistically the same to those obtained with a comparative method
Mestrado
Quimica
Mestre em Química
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19

Berkous, Rabiaa. "Greffage de modèles du NADH sur deux nouveaux supports insolubles. Synthèse et réactivité d'un modèle du NADH en série indolo (2,3-b)-pyridine : réduction de substrats non activés." Rouen, 1994. http://www.theses.fr/1994ROUES042.

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Le greffage de réactifs modèles du NADH a été réalisé sur deux nouveaux supports: un matériau mixte silice-polymère et un polymère acrylique à chiralité intrinsèque. Dans les deux cas, la réduction de substrats classiques a été possible. Avec le second support, une induction asymétrique a été observée. Afin d'effectuer la réduction de substrats non activés en présence d'acides de Lewis capables d'activer de tels substrats, un modèle en série indolo (2,3-)pyridinique a été synthétisé. Il est stable et possède une réactivité élevée. Il est le premier modèle du NADH à avoir permis la réduction quasi quantitative de l'acétophénone. Il a également permis de réduire une large gamme de cétones non activées. Le modèle chiral de la même série a réduit l'acétophénone et le phénylglyoxylate de méthyle avec des excès énantiomériques intéressants. Un modèle non chiral en présence d'acides de Lewis chiraux a aussi permis d'observer une induction asymétrique
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20

Vandock, Kurt P. "Mitochondrial Transhydrogenations in Manduca sexta: Relationship between Reversible NADPH → NAD+ Transhydrogenase and Ecdysone 20-Monooxygenase in Fifth Instar Larvae." Bowling Green State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1276033804.

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21

Haramis, Helena. "Developments in flourescence measurements of NADH." Thesis, University of Manchester, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493895.

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The objectives of this research included the development of a novel imaging set-up for fluorescence monitoring of nicotinamide adenine dinucleotide (NADH), in order to investigate the metabolic state of tissues and hence viability in a number of pathological conditions. We validated our system using different concentrations of NADH in the presence and absence of various optical scatterers and absorbers. Fluorescence images of dilute and turbid tissue phantom samples were acquired at an excitation of 366 nm, specific for NADH. These studies were followed by investigations ex vivo and in vivo: (a) to determine if NADH could be used as an indicator of ischaemia in hydrocephalus resulting from an increase in intracranial pressure due to an accumulation of cerebrospinal fluid; (b) monitor the effects of decreased blood flow on metabolic function of xanografted solid tumours in response to an antitumour compound; and (c) if measurement of NADH can be used as an indicator of changes in metabolic activity induced by pentobarbital.
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22

Bružas, Saulius. "NAD(P)H fluorescencijos pokyčiai sąlygoti miogeninių ląstelių diferenciacijos." Master's thesis, Lithuanian Academic Libraries Network (LABT), 2011. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2011~D_20110615_103737-60124.

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Šio darbo tikslas buvo nustatyti ar NAD(P)H fluorescencija, registruojama neinvaziniu fluorescenciniu metodu, gali būti naudojama kaip miogeninių ląstelių diferenciacijos žymuo, ištiriant fluorescencijos pokyčius miogeninių ląstelių diferenciacijos metu bei išaiškinant pagrindines šių pokyčių priežastis. Tyrimai parodė, kad 95 % mioblastų fluorescencijos, išmatuotos prie 460 nm emisijos, priklauso nuo NAD(P)H kiekio. Ląstelėms diferencijuojantis fluorescencija ženkliai didėja, tačiau susiejus fluorescenciją su didėjančiu baltymų kiekiu, ženklus fluorescencijos padidėjimas nustatytas tik po 10 diferenciacijos dienų. Panaudojus ląstelių metabolizmą ir NAD(P)H kiekį veikiančius veiksnius (skyriklį CCCP ir slopiklį KCN) buvo įvertinti santykiniai NADH ir NAD+ kiekiai bei NAD+/NADH santykis, kuris diferenciacijos metu mažėjo. 535 nm fluorescencijos pokyčiai diferenciacijos metu atskleidžia didėjantį FAD kiekį. Tiek NAD+/NADH santykis tiek 535 ir 460 nm fluorescencijos emisijų santykis yra patikimesni diferenciacijos rodikliai nei pati NAD(P)H fluorescencija. Šio darbo rezultatai patvirtina, kad tokie greiti, tikslūs ir neinvaziniai fluorescencijos matavimai gali būti naudingi nustatant mioblastų diferencacijos pradžią ir vyksmą bei taikytini įvairiems bandymams siekiant nustatyti vaistų ir kitų faktorių įtaką ląstelių metabolizmui.
The aim of this study is to check hypothesis that increase of fluorescence at 460 nm emission upon myoblast differentiation display alteration of NADH quantity that change NAD+/NADH ratio and could be a myoblast marker of entering into differentiation process. To evaluate NAD(P)H influence on total cell fluorescence emission intensity at 460 nm wavelenght were used metabolism effectors: carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to obtain minimal NADH fluorescence; and KCN to obtain maximal NADH fluorescence. Assuming, that CCCP caused maximal oxidation and that KCN caused maximum reduction, estimated that NAD+/NADH ratio decreased upon myoblast differentiation. CCCP effect showed that about 95 % of myoblast fluorescence at 460 nm emission is dependent on NAD(P)H. The fluorescence intensity at 460 nm was increasing with myoblast differentiation. Using fluorescence measuring estimated average NADH concentration significantly increased on 10 day of differentiation compared with control cells. Changes of fluorescence intensity at 535 nm reveal accumulation of FAD upon myoblast differentiation. Therefore, the best indicator of myoblast differentiation stage is ratio of fluorescence emission at 535 and 460 nm. Our results indicate that quick, quantitative and real-time fluorescence method may be useful for non-invasive detection and evaluation of myoblast differentiation stage.
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23

Llopart, Sylvie. "Regeneration enzymatique du cofacteur nadh : application a la synthese de l-carnitine." Toulouse, INSA, 1986. http://www.theses.fr/1986ISAT0043.

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L'hydrogenase cytoplasmique d'alcaligenes eutrophus h16 (ec 1. 12. 1. 2) est utilisee pour regenerer le cofacteur nadh. Une etude de la stabilite de cette enzyme met en evidence sa rapide denaturation en presence d'agents reducteurs, ou de nadh. Paradoxalement, l'oxygene agissant comme stabilisateur de l'enzyme, semble etre tres nefaste lors de la reaction en milieu reducteur. On constate une degradation du cofacteur lors de la mise en oeuvre d'un reacteur en absence d'oxygene, en vue de la production de l-lactate avec l'hydrogenase et la l-lactate deshydrogenase (ec 1. 1. 1. 27) coimmobilisees sur des rafles de mais. L'application a la synthese de l-carnitine, a l'aide de la carnitine deshydrogenase (ec 1. 1. 1. 108) est envisagee. Un taux de recyclage de 270 est atteint au bout de 23 h. La degradation du cofacteur dans le milieu reactionnel peut etre ralentie par l'utilisation d'un derive macromoleculaire hydrosoluble, le n**(6)-(6-aminohexyl)-carbamoyl methyl-nad**(+) greffe sur de l'acide poly-l-glutamique. Une decarboxylation spontanee du substrat en milieu basique, la 3-dehydrocarnitine, impose un rendement de bioconversion de 100 %, qui a ete atteint pendant 24 h avec des ajouts reguliers d'hydrogenase.
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24

Tur, Jared. "Cardiovascular regulation by Kvβ1.1 subunit." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6596.

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Heterologous expression systems such as COS-7 cells have demonstrated the profound effects of KCNAB1-3 or Kvβ1-3 proteins on voltage gated potassium channels (Kv) channels. Indeed, in the presence of these β-subunits transiently expressed Kv channels are often modulated in multiple ways. Kv channel membrane expression is often increased in the presence of β-subunits. In addition, non-inactivating Kv currents suddenly become fast-inactivating and fast-inactivating channels become even faster. While much research has demonstrated the profound effects the β-subunits in particular the Kvβ1 subunit have on transiently expressed Kv currents little to date is known of the physiological role it may play. One study demonstrated that by “knocking out” Kvβ1 cardiomyocyte current changes were noted including a decrease in the Ito,f current. While this novel finding demonstrated a key cardiac physiological role of the Kvβ1 subunit it left many unanswered questions as to determine the cardiovascular regulation the Kvβ1 subunit provides. Indeed, cardiac arrhythmias and other electrical abnormalities within the heart such as long QT present patients with many unfortunate unknowns. Many of these incidences occur often abruptly with cardiac electrical abnormalities. Genetic research has begun to shine light on key cardiovascular genes in particular those coding for ion channels and auxiliary subunits or β-subunits. Kv channels and their β-subunits have gained particular notoriety in their key responsibility in restoring the resting membrane potential known as the repolarization phase. Indeed genetic manipulation and physiological examination of Kv channels and recently their β-subunits has demonstrated profound physiological results including prolonged QT durations within mice altered functional activity during physiological cycles such as estrus. While initial findings of Kvβ1 have demonstrated profound cellular and cardiomyocyte current alterations much still remains unknown. Therefore, this work hypothesizes that the Kvβ1 subunit provides a profound cardiovascular role in regulation and redox sensing at the physiological and pathophysiological level in both males and females. This work identifies a sex-based difference in cardiovascular regulation by Kvβ1 as well as demonstrated a profound redox sensing ability during altered metabolic states seen in pathophysiological conditions.
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25

Kadowaki, Jonathan. "Electrochemical Regeneration of Cofactors Using a Novel Cuprous Oxide Derived Cathode." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555578179760958.

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26

Camargo, Maiuí Nagao Lindqquer de 1990. "Influência do grau de redução do óxido de grafeno eletroquimicamente reduzido nas suas propriedades eletroquímicas." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248391.

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Orientador: Lauro Tatsuo Kubota
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química
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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
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27

Nhamposse, Catarina Tivane. "Avaliação morfoquantitativa nos músculos estriados esqueléticos de ratos wistar (Rattus norvegicus) dos efeitos da dieta utilizada na alimentação de crianças das zonas rurais de Moçambique." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/10/10132/tde-18042013-154159/.

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Moçambique é um País da África Austral onde cerca de 55% da população vive abaixo da linha de pobreza absoluta com menos de uma refeição por dia sobrevivendo a muito custo com base em donativos. A insegurança alimentar e a nutrição extremamente precária, principalmente nas crianças, são fatores que induzem a níveis de Desnutrição crônica (DC) infantil em torno de 44%. Esta DC é responsável por um terço de mortes em menores de cinco anos. O objetivo deste estudo foi avaliar os efeitos que uma ração preparada com alimentos produzidos e consumidos pela população das zonas rurais de Moçambique exerceu no músculo gastrocnêmio de ratos wistar (Rattus norvegicus) alimentados com esta ração. Foram usados 75 ratos Wistar pesando aproximadamente 300 g divididos em três grupos: Nutrido ou controle (N), Desnutrido (D) e Moçambique ou grupo experimental (M), avaliados ao nascimento e ao desmame. Os animais foram mantidos sob as mesmas condições de alojamento, temperatura umidade e luz, porém com alimentação diferente consoante o grupo; Grupo N com ração normoproteica (20% de caseína), Grupo D com ração desnutrida (5% de caseína) e grupo M com ração de Moçambique. Em todos os grupos foi feita avaliação da massa corporal ao nascimento e desmame e coletado o músculo gastrocnêmio direito dos filhotes machos ao desmame para processamento. Foram realizadas cortes seriados de 10 µm de espessura em criostato e, as secções foram submetidas às técnicas da hematoxilina e eosina, picro-sirius, NADH-tr e análise em microscopio eletrônico de transmissão. A avaliação estatística das diferenças inter-grupos foi determinada pelos testes análise de variância, (ANOVA) e Tukey. Foram encontradas diferenças significativas entre os grupos N, D e M. Observou-se nos animais do grupo M uma grande variação no peso e tamanho que foi similar ao do grupo D; os mesmos apresentaram também alterações no formato das fibras musculares que exibiram contornos arredondados e, ainda, predominância de fibras colágenas tipo III tal como os desnutridos. Ultra estruturalmente os animais de Moçambique apresentaram um desalinhamento das linhas Z dos sarcômeros e rompimento das miofibrilas, diminuição na área de seção transversa e menor proporção de fibras glicolíticas e glicolítico-oxidativas, e maior porcentagem e área de seção transversa igual ao grupo D no respeitante ás fibras oxidativas.
Mozambique is a country of Southern Africa where about 55% of the population lives below the absolute poverty line with less than one meal per day, hardly surviving based on donations. Food insecurity and precarious nutrition, especially in children, are factors that induce to levels of 44% chronic infant malnutrition (DC). DC is responsible for one third of deaths in children under five years. The aim of this study was to evaluate morphoquantitative effects in gastrocnemius muscle of Wistar rats (Rattus norvegicus) fed with a diet utilized by people of rural areas of Mozambique. We used 75 Wistar rats weighing approximately 300 g divided in three groups: Nourished or control (N), Malnourished (D) and Mozambique or experimental group (M), measured at birth and weaning. The animals were kept under the same housing conditions, temperature, humidity and light, but fed with different diet depending on the group; Group N with normal protein diet (20% casein), Group D with hypo-proteic diet (5% casein) and group M with Mozambique diet. Body mass at birth and weaning was evaluated the right gastrocnemius muscle of male pups at weaning was colletcted for processing. Serial sections of 10 µm were performed in a cryostat prior to histology techniques of hematoxylin and eosin, picro-sirius, NADH-tr and analysis in transmission electron microscope. Statistical evaluation was determined by analysis of variance tests (ANOVA) and Tukey. Significant differences were found between groups N, D and M. Group M exhibit a great variation of body mass that was similar to group D; these animals (group M) also showed structural changes in muscle fiber which exhibited round-shaped contours, and a predominance of type III collagen similarly to malnourished group. Ultra structurally animals from Mozambique showed a disorganization of Z lines of sarcomeres and myofibrils disruption, decreased cross-sectional area and a smaller proportion of glycolytic and glycolytic-oxidative fibers, and higher percentage and cross-sectional area identical to group D with respect to oxidative fibers.
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28

Courtois, Philippe. "Peroxydases buccales et NADH-hypothiocyanite-oxydoréductase bactérienne." Doctoral thesis, Universite Libre de Bruxelles, 1996. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/212313.

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29

Marques, Isabel Maria Medeiros. "Molecular biology charactersation of mitochondrial NADH dehydrogenase." Doctoral thesis, Instituto de Ciências Biomédicas Abel Salazar, 2007. http://hdl.handle.net/10216/7281.

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30

Binay, Patrice. "Nouveaux modèles du NADH : réactivité et énantiosélectivité." Rouen, 1986. http://www.theses.fr/1986ROUES001.

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Dans une première partie, synthèse d'alkyl-4 dihydro-1,4 benzyl-1 et -phényl-1p éthyl-1 (diméthyl-4,4 oxazoline-2yl-2)-3 pyridines et étude de leur activité réductrice vis-a-vis de p-nitrobenzaldéhyde et de benzenéglyoxylate de méthyle en présence de mg**(2+) ; dans la seconde partie, étude de modèles plus énantiosélectifs : méthyl-1 dihydro-1,4 n-(hydroxyméthyl-1 propyl) nicotinamide (=méthyl-1 a), o-, m- et p- xylylene-1, 1' bis-a, dihydro-1,4 methyl-1 nicotinate de (dihydro-1,4 methyl-1 nicotinoylamino)-2 butyle et le cyclophane correspondant à ce dernier composé (pont xylylene entre les azotes des pyridines) ; interaction entre les noyaux pyridines
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31

Binay, Patrice. "Nouveaux modèles du NADH réactivité et énantiosélectivité." Grenoble 2 : ANRT, 1986. http://catalogue.bnf.fr/ark:/12148/cb37596051d.

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32

Marques, Isabel Maria Medeiros. "Molecular biology charactersation of mitochondrial NADH dehydrogenase." Tese, Instituto de Ciências Biomédicas Abel Salazar, 2007. http://hdl.handle.net/10216/7281.

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33

Allali, Naoual. "Covalent funtionalization of carbon nanomaterials for bioelectrochemical applications." Thesis, Université de Lorraine, 2019. http://www.theses.fr/2019LORR0021/document.

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Les dispositifs bioélectrochimiques utilisent souvent le co-facteur NADH (nicotinamide adénine dinucléotide) comme biomolécule impliquée dans les réactions d’oxydo-réduction avec des enzymes de grand intérêt biochimique, comme par exemple les glucose oxydases ou les déshydrogénases. Il est nécessaire d’utiliser de nouveaux matériaux d’électrode afin de diminuer les sur-potentiels nécessaires au transfert d’électrons avec le système NADH/NAD+ et éviter l’adsorption des produits de la réaction à la surface de l’électrode (biofouling). Les nanotubes de carbone (NTCs) constituent un matériau conducteur de grande aire spécifique qui semble prometteur pour modifier ainsi la surface des électrodes. Ce travail de thèse a consisté à développer de nouvelles méthodes de greffage covalent de groupements fonctionnels électro-actifs vis-à-vis du système NADH/NAD+ en contrôlant les différentes étapes du procédé avec un protocole particulièrement poussé d’analyses physico-chimiques impliquant les spectroscopies de diffusion Raman, d’absorption infrarouge, de photo-électrons X, les microscopies électroniques à transmission, l’ellipsométrie spectroscopique et les analyses thermogravimétriques et de volumétrie d’adsorption. Nous avons développé un procédé reposant sur une première étape d’oxydation des NTCs par assistance micro-ondes dans des milieux acides dilués. Ceci permet de transformer les défauts existant à la paroi des nanotubes (atomes de carbone en hybridation sp3) pour les convertir en fonction acides carboxyliques, qui serviront dans les étapes ultérieures du procédé au greffage covalent des groupements électro-actifs. Ainsi l’intégrité structurale des NTCs, et donc leurs excellentes propriétés électroniques et mécaniques, sont préservées. Le succès de cette approche est pleinement démontré dans ce travail aussi bien en utilisant des nanotubes monoparois purifiés que des nanotubes multiparois. Un net effet électrocatalytique est obtenu avec les groupes fonctionnels dérivés du ferrocène. On montre également le rôle crucial de la nature du bras espaceur reliant les groupes électro-actifs à la paroi des NTCs. Ce travail a permis de mettre au point une méthode générale de greffage covalent des NTCs et son contrôle étape par étape. On montre enfin en perspective de ce travail qu’il est possible de greffer directement la molécule de NAD+ à la surface des NTCs
Bioelectrochemical devices often use the NADH co-factor (nicotinamide adenine dinucleotide) as a biomolecule involved in oxidation-reduction reactions with enzymes of high biochemical interest, such as glucose oxidases or dehydrogenases. It is necessary to use new electrode materials to reduce the over-potentials required for electron transfer with the NADH/NAD+ system and avoid adsorption of the reaction products to the electrode surface (biofouling). Carbon nanotubes (CNTs) are a conductive material with a large specific surface area that seems promising for modifying the surface of electrodes. This thesis work consisted in developing new methods for covalent grafting of electro-active functional groups with respect to the NADH/NAD+ system by controlling the various stages of the process with a particularly advanced physico-chemical analysis protocol involving Raman scattering spectroscopy, infrared absorption, X-ray photoelectron spectroscopy, transmission electron microscopy, spectroscopic ellipsometry and thermogravimetric and volumetric adsorption analyses. We have developed a process based on a first step of oxidation of the CNTs by microwave assistance in diluted acid media. This makes it possible to transform existing defects in the wall of the nanotubes (carbon atoms in sp3 hybridization) into carboxylic acid functions, which will be used in the subsequent steps of the process for covalent grafting of electro-active groups. Thus, the structural integrity of the CNTs, and therefore their excellent electronic and mechanical properties, are preserved. The success of this approach is fully demonstrated in this work both by using purified single-walled nanotubes and multi-walled nanotubes. A clear electrocatalytic effect is obtained with the functional groups derived from ferrocene. The crucial role of the nature of the spacer arm connecting the electro-active units to the wall of the CNTs is also shown. This work made it possible to develop a general method for covalent grafting of CNTs and its step-by-step control. Finally, we show in perspective of this work that it is possible to directly graft the NAD+ molecule onto the surface of the CNTs
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34

Ito, Takeshi. "Mode of action study of inhibitors of energy converting NADH-quinone oxidoreductases." Kyoto University, 2018. http://hdl.handle.net/2433/232338.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21137号
農博第2263号
新制||農||1057(附属図書館)
学位論文||H30||N5111(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 三芳 秀人, 教授 宮川 恒, 教授 加納 健司
学位規則第4条第1項該当
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35

Cheng, Jun. "MONITORING METABOLIC RESPONSES IN SACCHAROMYCES CEREVISIAE USING FLUORESCENCE-BASED DETECTION OF NADH CONFORMATION." Miami University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=miami1313788354.

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36

Alquist, Erik James. "The Effects of High Hydrostatic Pressures on NADH Conformation." Miami University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1281640692.

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37

Åström, Nina. "NADH/NAD⁺ analogues and cyclodextrins in enzyme mimicking systems an experimental and computational investigation /." Lund : Organic Chemistry 1, Lund University, 1995. http://catalog.hathitrust.org/api/volumes/oclc/39781586.html.

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38

Policarpo, Anna Carolina de Freitas. "Vias alternativas mitocondriais: clonagem e caracterização bioquímica do gene NADH desidrogenase alternativa de \'A. fumigatus\'." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-17122008-111144/.

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Aspergillus fumigatus é um fungo filamentoso e saprofítico encontrado em todas as regiões do mundo. A principal forma de infecção ocorre através da inalação de conídios do fungo com predominância de infecções no trato respiratório, principalmente em pacientes imunocomprometidos. Foi caracterizada a função mitocondrial de A. fumigatus e sugeriu-se a presença de vias alternativas, dentre elas, a presença da NADH desidrogenase alternativa. A fim de colaborar com estudos para elucidação do papel desta enzima, foi realizada a clonagem dos genes das NADH desidrogenase alternativa interna e externa de A. fumigatus. A análise da seqüência de aminoácidos revelou um perfil de hidropaticidade com a presença de quatro regiões hidrofóbicas, semelhante às outras seqüências já descritas. Além disso, foram identificados dois motivos (GXGXXG) altamente conservados para ligações a nucleotídeos, dentro de um domínio os quais estão relacionados com a estrutura e atividade da enzima. A seqüência de cDNA do gene ndhiAf foi clonada em plasmídeo pYES2 e expressa em S. cerevisiae cepa CEN.PK873-2B; a expressão da NDHIAf foi verificada por técnica de Western-blot. A proteína foi expressa de forma ativa, conferindo à levedura uma respiração e a formação de um potencial de membrana resultantes da oxidação de substratos clássicos do complexo I, sugerindo sua localização na membrana mitocondrial interna. Além disso, as cepas expressando essa proteína foram capazes de crescer em meio contendo apenas lactato como fonte de carbono, uma característica atribuída à presença da NADH desidrogenase alternativa interna (NDHi). Considerando que a atividade das NDHIAf e NDHEAf estão sob controle metabólico, nos avaliamos o efeito de diferentes concentrações de glucose como única fonte de carbono no meio de cultura. Durante a germinação conidial, não foi observada expressão da NDHIAf e NDHEAf na presença de baixas concentrações de glucose. Entretanto, durante a fase de hifas foi observado um aumento na expressão de NDHIAf e NDHEAf. Por outro lado, em alta concentrações de glucose, durante a germinação conidial, foi observado expressão da NDHIAf mas nenhuma expressão da NDHEAf. Na fase de hifas observou-se um aumento da expressão da NDHIAf e uma diminuição da NDHEAf.
Aspergillus fumigatus is a filamentous and saprophytic fungus found in all regions of the world. The main form of infection occurs through inhalation of fungi conidial, with predominance of infections in the respiratory treat, mainly in immunocompromised host. It was characterized the mitochondrial function of A. fumigatus and suggested the presence of alternative pathways, including the alternative NADH dehydrogenase. In order to elucidate its role, genes of the external and internal enzymes were cloned. Analysis of the amino acid sequence and hydropathy plot revealed a profile with four hydrophobic regions, similar to other already described sequences. Moreover, two highly conserved motifs (GXGXXG) for the nucleotides interaction, related with the structure and activity of the enzyme, were identified inside the a domain. The sequence of DNA of the ndhiAf gene was cloned in an expression plasmid pYES2 and expressed in CEN.PK873-2B S. cerevisiae strain; the expression was confirmed by Western-blot analysis. The protein was expressed in an active form, providing to the yeast an oxygen consumption and a potential membrane formation due to oxidation of the complex I substract, suggesting its localization in the inner mitochondrial membrane. Moreover, strains expressing this protein were capable of growing in medium with only lactate as a carbon source, a characteristic associated with the presence of internal alternative NADH dehydrogenase (NDHI). Considering that NDHI and NDHE activities are under metabolic control, we evaluated the effect of different concentrations of glucose as the only carbon source in the medium of culture. During conidia germination, it was not observed neither an expression of NDHI nor NDHE in the presence of low concentrations of glucose. However, in hyphae phase was observed an increase an expression of NDHI and NDHE. On the other hand, in high concentration of glucose, during conidia germination was observed an expression of NDHI and not expression of NDHE. In hyphae phase was observed a decrease an expression of NDHI and an increase of NDHE.
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39

Silva, Robson Pinho da. "Aplicações analíticas de eletrodos quimicamente modificados por espécies de interesse biológico." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46133/tde-18102007-164848/.

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O trabalho apresentado nesta Dissertação de Mestrado descreve o desenvolvimento e aplicação de eletrodos de pasta de carbono modificados eletroquimicamente em soluções de guanina e de eletrodos de grafite pirolítico modificados em soluções de dopamina. Estes eletrodos foram empregados na detecção e, a quantificação, por voltametria de pulso diferencial (VPD), de alguns compostos de importância biológicas tais como NADH, NADPH, 8-oxo-guanina, ácido úrico (AU), ácido ascórbico (AA), dopamina (DA) e xantina (XA). No primeiro caso, os eletrodos de pasta de carbono foram modificados em solução de guanina por aplicação de um potencial de 1,1 V (vs Ag/AgCl, KClsat) ao eletrodo de trabalho por 12 minutos sob sat agitação constante. Com estes eletrodos detectaram-se NADH, NADPH, 8-oxo-guanina e AU, com limites de detecção de 3,3, 3,7, 2,0 e 6,6 x 10-6 mol L-1 respectivamente, na faixa de concentração de 7,5 x 10-6 a 8,1 x 10-4 mol L-1 . No segundo caso, eletrodos de grafite pirolítico, previamente tratados em solução de NaOH, foram modificados eletroquimicamente em solução de DA por aplicação de um potencial 1,5 V (vs Ag/AgCl, KClsat ) ao eletrodo de trabalho durante 2 minutos. Com estes eletrodos foi possível a determinação simultânea de AA, AU e DA. Para obtenção das curvas analíticas variou- se a concentração do analito de interesse, mantendo-se constante a concentração dos possíveis interferentes nos valores de 1,0 x 10-4 mol L-1 (DA), 5,0 x 10-5 mol L-1 (AU) e 1,0 x 10-3 mol L-1 (AA). Os limites de detecção calculados para AU, AA e DA foram respectivamente de 1,4 x 10-6 mol l-1 , 2,5 x 10-5 mol l-1 e 1,1 x 10-7 mol l-1 . Ácido úrico foi determinado em amostras de urina, sangue e soro humano com 92 a 103 % de recuperação, sem a necessidade de tratamento prévio das amostras.
Chemically Modified Carbon Paste and Pyrolitic Graphite Electrodes were prepared via electrochemical deposition from guanine and dopamine solutions. Carbon paste electrodes were modified in guanine solutions under an applied potential of 1.1 V (vs Ag/AgCl, KClsat ) during 12 minutes under constant stirring. They were used for sat electrochemical detection of NADH, NADPH, uric acid and 8-oxoguanine. Detection limits were 3.3, 3.7, 6.6 and 2.0 10-6 mol L-1 respectively, with sensitivity of 0.13, 0.10, 0.26 and 0.40 A mol-1 L cm-2 , respectively. The electrodes showed high reproducibility and absence of surface poisoning effects. Good analytical performance was attributed to the formation of superficial dimer or trimers species of guanine during the modification process. Pyrolitic graphite electrodes, previously submitted an electrochemical treatment in NaOH solution, were modified in dopamine solution (phosphate buffer, pH 10) under an applied potential of 1.5 V (vs Ag/AgCl, KClsat ) during 2 minutes under constant sat stirring and, further used for the simultaneous determination of ascorbic acid (AA), uric acid (AU) and dopamine (DA). The analytical curves were obtained changing the concentration of the wished analyte, at constant concentration levels of the interferences: 1.0 x 10-4 mol L-1 (DA), 5.0 x 10-5 mol L-1 (UA) and 1.0 x 10-3 mol L-1 (AA). Detection limits were 1.4 x 10-6 mol L-1 , 2.5 x 10-5 mol L-1 and 1.1 x 10-7 mol L-1 for UA, AA and DA, respectively. Uric acid was determined in human urine, blood and serum samples without any previous treatment. Recovering percentages of 92 to 103 % were obtained.
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40

Retamal, Morales María Fernanda. "Generación de electricidad en mutantes de Escherichia coli de la producción de NADH y NADPH." Tesis, Universidad de Chile, 2018. http://repositorio.uchile.cl/handle/2250/151296.

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Tesis presentada a la Universidad de Chile para optar al grado de Magíster en Bioquímica área de Especialización Bioquímica Ambiental y Memoria para optar al Título de Bioquímico
1.- RESUMEN Durante la generación de electricidad en sistemas de Celda de Combustible Microbiológicas (MFC, por su sigla en inglés), se han manipulado distintas vías metabólicas para generar un aumento en la bioelectricidad, debido a que los electrones necesarios para este proceso provienen del poder reductor generado dentro de la célula. Esto no solo se ha estudiado en cepas electrogénicas como lo son Geobacter o Shewanella, sino que también se han realizado estudios en Escherichia coli, la cual puede transferir electrones a un electrodo a través de un mediador. Estos últimos años, la investigación se ha centrado específicamente en la manipulación cofactores, principalmente el NADH. En estos casos, alteran tanto vías de consumo como de generación de este cofactor, pero evalúan principalmente la eficiencia de esta modificación en el voltaje, la corriente o en la potencia producida. Sin embargo, pocas veces consideran observar los factores intrínsecos de la bacteria que están produciendo estas mejoras. Debido a esto, en este trabajo se utilizó una MFC con distintas cepas de E. coli con el mediador rojo neutro para observar no solo los cambios en la generación de corriente, sino que también, el consumo de la fuente de carbono (glucosa en este caso), rendimiento, y flujo de electrones en estos cultivos electrogénicos. Esto ayudó a comprender en parte lo que puede estar pasando en el metabolismo bacteriano durante el proceso de generación de corriente. Las cepas utilizadas fueron la cepa de E.coli K-12 MG1655, la cepa NAD-G6PDH la cual presenta un cambio en la especificidad de cofactor de la enzima G6PDH (en la cepa silvestre utiliza NADP+ , en este caso utiliza NAD+), la cepa Δpgi (con la deleción del gen que codifica la enzima fosfogluco isomerasa) y la cepa Δpgi-NAD-G6PDH que posee las dos mutaciones mencionadas. Finalmente, el trabajo demostró que las cepas MG1655 y NAD-G6PDH, que presentan más NADH, generan mayor electricidad en el tiempo que las cepas que tienen la mutación Δpgi. No obstante, el flujo de los electrones de todas las cepas fue similar, ya que no se observaron diferencias significativas. Además, se observó que la cepa Δpgi podría poseer un mejor rendimiento de mmoles de electrones por mmoles de glucosa consumida que las otras cepas, ya que produce una corriente relativamente alta para la poca biomasa y el bajo consumo de glucosa.
During the electricity generation in Microbiological Fuel Cell (MFC) systems, different metabolic pathways have been manipulated to generate an increase in the bioelectricity since the electrons needed for this process come from the reducing power generated inside the cell. This has not only been studied in electrogenic strains such as Geobacter or Shewanella, but also studies have been carried out in Escherichia coli, which can transfer electrons to an electrode through a mediator. Over the last few years, the research has focused specifically on the manipulation of cofactors, mainly in NADH. In these cases, they alter both consumption and generation pathways of this cofactor, but they mainly evaluate the efficiency of this modification in the voltage, the current or the power produced. However, they rarely consider observing the intrinsic factors of the bacteria that are producing these improvements. Due to this, in this work we used an MFC with different strains of E. coli with the Neutral Red mediator to observe not only the changes in the current generation, but also the consumption of carbon source (glucose in this case), yields, and electron flux in these electrogenic cultures. This helped to partially understand what may be happening in the bacterial metabolism during the current generation process. The strains used in this work were E. coli strain K-12 MG1655, strain NAD-G6PDH, which shows a change in the specificity cofactor of the G6PDH enzyme (in the wild type strain uses NADP+, in this case uses NAD+), strain Δpgi (with a deletion of the gene encoding the phosphoglucose isomerase enzyme) and strain Δpgi-NAD-G6PDH that possesses the two mentioned mutations. Finally, the work demonstrated that the strains MG1655 and NAD-G6PDH that present more NADH generate higher current per time than the strains that have the Δpgi mutation. Nevertheless, the electrons flux of all the strains was similar, since no significant differences were observed. In addition, it was observed that the strain Δpgi could have a better yield of mmol of electrons per mmol of consumed glucose than the other strains, since it produces a relatively high current for the low biomass and the low glucose consumption
PAIFAC 2016. Fac Cs. (RC) y Proyecto ENL012/16.
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41

Kennett, Eleanor. "Transmembrane Electron Transport Systems in Erythrocyte Plasma Membranes." University of Sydney. School of Molecular and Microbial Biosciences, 2005. http://hdl.handle.net/2123/620.

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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.
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42

Sprules, Steven David. "Investigations into novel screen-printed electrodes for biosensor applications." Thesis, University of the West of England, Bristol, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306892.

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43

Wallace, Emma Naomi Kathleen. "Poly(aniline) composites as bioelectrochemical sensors." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242655.

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44

Osborn, Helen. "Undesirable pinking in meat and meat model systems." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343321.

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45

Kennett, Eleanor. "Transmembrane Electron Transport Systems in Erythrocyte Plasma Membranes." Thesis, The University of Sydney, 2004. http://hdl.handle.net/2123/620.

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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.
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46

Pilkington, Stephanie Joan. "Nuclear encoded subunits of mammalian NADH-ubiquinone reductase." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385445.

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47

Mercier, Claire. "Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10025.

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48

Boussad, Nadjib. "Sulfoxydes pyridiniques précurseurs de modèles chiraux du NADH. Synthèse et étude de modèles stabilisés du NADH en série benzo(b)thiénopyridinique." Rouen, 1991. http://www.theses.fr/1991ROUE5019.

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Des modèles simples portant un groupe sulfoxyde chiral en position de la pyridine ont été synthétisés et utilisés dans la réduction de la α, α', α'' acétophénone. Les rendements sont moyens car il se forme une réaction de désulfénylation pendant la réduction. Les produits secondaires ont été isolés et identifiés. Un modèle analogue en série quinoléinique a été synthétisé. Il a conduit à la réduction totale du même substrat. Les excès énantiomériques sont variables. D'autres modèles du NADH portant un groupement carbonyle en position de la dihydropyridine ont été synthétisés en série benzo[b]thiéno3,2-b ou 3,2-[b]pyridiniques. Les résultats les plus importants sont les suivants: la 1-méthyl 3-carbamoyl-1,4 benzo[b]thieno2,3-b pyridine conduit à un complexe binaire avec le perchlorate de magnésium en présence de substrat; le même modèle reéduit l'acétophénone en présence d'acide de Lewis
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49

Strain-Damerell, Claire Michelle. "Functional analysis of Rex, a sensor of the NADH/NAD+ redox poise in Streptomyces coelicolor." Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/6308/.

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Maintenance of the intracellular NADH/NAD+ redox poise is vital for energy generation in cells. Gram-positive bacteria, including the antibiotic-producing organism, Streptomyces coelicolor, have evolved a regulatory protein Rex that both senses this ratio and mediates an adaptive response to changes in it. Rex is a dimeric redox-sensitive transcriptional repressor. It is capable of binding to both NAD+ and NADH, although only NADH is an effector, causing dissociation of the protein from operator (ROP) sites. As NADH levels rise during oxygen limitation Rex dissociates from its target genes allowing expression, which helps to restore the NADH/NAD+ ratio. Microarray-based expression studies had suggested that Rex regulated only a small number of genes. In this work, however, ChIP-on-chip analyses revealed 38 genes that are potential regulon members. Analysis of the Rex binding sites in S. coelicolor revealed new insights into the mode of binding and show that Rex can bind with low affinity to incomplete half sites. This work also focused on characterising two key Rex targets, ndh and nuoA-N, that encode non-proton-translocating and proton translocating NADH dehydrogenases, respectively. Whereas nuoAN is not essential and was not expressed in liquid media, ndh was essential for growth. Depletion of NDH from growing cells led to the induction of Rex target genes confirming that ndh and Rex play key roles in maintaining redox homeostasis. Structure-based dissection of Rex, via a close homologue in Thermus aquaticus, identified a key interaction between the NADH- and DNAbinding domains of Rex. An R29-D203' salt-bridge, that traverses the NADH binding and DNA binding domains of Rex, appeared to stabilise the DNA-bound form of Rex, but is ‘broken' in the presence of NADH. In the NADH-bound form of Rex, D203 alternatively interacts with Y111, which in turn interacts with the nicotinamide ring of NADH. In order to assess the importance of individual subunits in the dimeric Rex, a single-chain derivative was constructed and the NADH binding and DNA binding domains individually disrupted.
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Holowiecki, Andrew. "Catalysis of Mitochondrial NADH→NAD+ Transhydrogenation in Adult Ascaris suum (Nematoda)." Bowling Green State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1256953439.

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