Rozprawy doktorskie na temat „Nicotinamide adenine dinucleotide”

Introduction: Synovial fibroblasts (SF) display a ‘hyperactive’ phenotype in patients with rheumatoid arthritis (RA). Nicotinamide adenine dinucleotide (NAD+) plays a role in cell metabolism, but may also be a key molecule in maintaining this ‘activated’ phenotype. NAD+ can be synthesised from precursor vitamin molecules, nicotinamide (Nam), nicotinic acid (NA) and Tryptophan (TRP); with their respective phosphoribosyl transferases (NAMPT, NAPRT, QAPRT) and Indoleamine (IDO) being the rate limiting enzymes involved in these pathways. NAMPT and IDO are known to be elevated in RA synovial tissue (ST). However, the expression and regulation of other NAD+ biosynthesis enzymes are unknown. Methods: RA, OA and normal ST were obtained from joints of patients undergoing surgery and expression of NAD+ biosynthesis enzymes were quantified using qPCR. Synovial fibroblasts were cultured and stimulated with 10ng/ml of TNF-α, IL-1β, OSM & IFN- and the expression of NAD+ biosynthesis enzymes were quantified using qPCR. Results: qPCR analyses showed that all NAD+ biosynthesis enzymes tested were constitutively expressed in synovial tissue ex vivo and in vitro, with the exception of NMN adenyltransferase (NMNAT)-3. NAMPT, IDO, QAPRT, NADSYN and NMNAT-2 were all upregulated in RA ST compared to normal tissue, however only NAMPT was significantly upregulated in RA compared to OA and normal, (NAMPT reached statistical significance when patients on anti-TNF therapy were excluded). Moreover, NAMPT was found to be upregulated in ST of young actively developing individuals, decreasing with age. Expression of NAD salvage enzymes, NAMPT and NMNAT-2 in ST correlated with each other and de novo NAD enzymes, IDO, QAPRT, NADSYN and NMNAT-2 were also correlated with each other in ST. NAMPT and IDO were both significantly upregulated in vitro following stimulation with OSM & IFN- but only NAMPT and NMNAT-2 were upregulated following stimulation with TNF-α & IL-1β. NAPRT expression was found to be low in RA ST and there was no upregulation following stimulation by OSM, IFN-, TNF-α & IL-1β. Conclusion: The data presented in this thesis emphasises NAMPT and IDO as a potential therapeutic target in rheumatoid arthritis.

Nicotinamide adenine dinucleotide (NAD+) is a cofactor in hydride transfer reactions and consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD+, its biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome) is altered in many metabolic disorders, such as aging and obesity. Supplementation with the novel NAD+ precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations of the NAD metabolome are both a symptom and cause of metabolic disease, accurate assessment of the abundance of these metabolites is expected to provide insight into the biology of diseases and the mechanism of action of NR in promoting metabolic health. Current quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid chromatography mass spectrometry methodologies to quantify the NAD metabolome and applied these methods to determine the effects of disease states and NR supplementation on NAD+ metabolism. My investigations indicate that NR robustly increases the NAD metabolome, especially NAD+ in a manner kinetically different than any other NAD+ precursor. I provide the first evidence of effective NAD+ supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD+, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD+ supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus.

Septic shock is a leading cause of death in intensive care units. As part of the septic process, the endothelium becomes activated and propagates the septic condition. It has become evident that reactive oxygen species (ROS) are involved in the signaling of mediators of sepsis, such as tumor necrosis factor-alpha (TNF-alpha) and the lipopolysaccharide coating of gram-negative bacteria (LPS). An important source of these ROS is NADPH oxidase, which is a ubiquitously expressed enzyme complex that also exists in endothelial cells (EC). We showed that O2- from NADPH oxidase was important for LPS, as well as TNF-alpha, induction of two markers of an activated endothelium, interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC).
Expression of a gene can be increased by a rise in transcription as well as post-transcriptional changes, such as mRNA stability modifications. We assessed the role of NADPH oxidase in this process and found a complex interaction. Although LPS increases IL-8 transcription, it also destabilizes IL-8 mRNA in a p38 and extracellular signal-regulated kinase (ERK) MAPK dependent manner, which was only evident after blocking NADPH oxidase. This regulation involved the mRNA de-stabilizing factor tristetraprolin (TTP). In contrast, TNF-alpha enhanced the stability of IL-8, IL-6 and ICAM-1 mRNA in a p38 MAPK dependent, but NADPH oxidase independent manner. Furthermore, LPS did not have an effect on mRNA stability of IL-6 or ICAM-1 in our system. Thus, we conclude from our studies that the NAPDH oxidase is important for the induction of inflammatory molecules in LPS and TNF-alpha treated EC and is also involved in mRNA stability regulation of these molecules in a signal and gene specific fashion.

Nicotinamide adenine dinucleotide (β-NAD+) is a primary metabolite involved in fundamental biological processes. Its molecular structure with characteristic functional groups, such as the quaternary nitrogen of the nicotinamide ring, and the two high-energy pyrophosphate and nicotinamide N-glycosidic bonds, allows it to undergo different reactions depending on the reactive moiety. Well known as a redox substrate owing to the redox properties of the nicotinamide ring, β-NAD+ is also fundamental as a substrate of NAD+-consuming enzymes that cleave either high-energy bonds to catalyse their reactions. In this study, a panel of novel adenine-modified NAD+ derivatives was synthesized and biologically evaluated against different NAD+-consuming enzymes. The synthesis of NAD+ derivatives, modified in position 2, 6 or 8 of the adenine ring with aryl/heteroaryl groups, was accomplished by Suzuki-Miyaura cross-couplings. Their biological activity as inhibitors and/or non-natural substrates was assessed against a selected range of NAD+-consuming enzymes. The fluorescence of 8-aryl/heteroaryl NAD+ derivatives allowed their use as biochemical probes for the development of continuous biochemical assays to monitor NAD+-consuming enzyme activities. The introduction of different substituents in position 8 on the adenine ring allowed the modulation of their fluorescence, resulting in the development of more sensitive and alternative probes compared to the known fluorophore ε-NAD+. The different substitutions introduced on the adenine ring also allowed us to probe the active site of an NAD+-dependent bacterial DNA ligase. The selective activity of 8-aryl/heteroaryl NAD+ derivatives against different NAD+-consuming enzymes offers excellent opportunities for their application as tool compounds in in-vitro/in-vivo studies, and as inhibitor templates for drug discovery.

O A. fumigatus é um fungo saprofítico e tornou-se um dos principais agente patogênico oportunista em pacientes imunossuprimidos. Estudos prévios em nosso laboratório foi demonstrado que em mitocôndrias de P. brasiliensis e de A. fumigatus, o NAD+ era capaz de induzir a formação de potencial de membrana mitocondrial, o qual podia ser dissipado por FCCP, sugerindo a presença de um transportador de NAD+/NADH, conforme havia sido descrito em S. cerevisiae. Através de ferramentas de bioinformática, foi identificado no Aspergillus Gene Database, uma sequência com 32% de identidade com o gene ndt1p de S. cerevisiae. A sequência de cDNA, contendo 1.194 pb foi obtida usando PCR-Overlaping e clonada em vetor pGEM®-T Easy. Em seguida, a sequência foi subclonada em vetor de expressão pET28-a(+) e expressa em E. coli BL21(DE3). A proteína recombinante foi purificada a partir dos corpos de inclusão e sua identidade confirmada por espectrometria de massas e por Western Blotting usando anticorpo anti-His-tag. A proteína recombinante foi utilizada para produção de anticorpo policlonal anti-Ndt1 em coelho. Para expressão em levedura, o cDNA do gene ndt1 de A. fumigatus foi subclonado em vetor pYES2 e as leveduras S. cerevisiae ?ndt1?ndt2 foram transformadas. Foi realizada a curva de crescimento e indução da expressão da proteína recombinante Ndt1, a presença da proteína foi detectada utilizando anticorpo policlonal anti-Ndt1 após 16 horas de expressão. Nesse período foi verificado que as leveduras estavam em fase de crescimento exponencial. A cepa duplo mutante apresenta uma taxa de crescimento menor quando comparada com a cepa expressando a proteína recombinante quando crescidas em meio fermentável. As mitocôndrias isoladas de ambas as cepas foram submetidas à medida do potencial de membrana onde apresentavam acoplamento entre a oxidação de substratos e a fosforilação oxidativa. Além disso, ficou evidenciado que na cepa expressando a proteína recombinante, NAD+ induziu a formação de um potencial de membrana maior que na cepa controle. O transporte de NAD+ foi realizado e demonstrou que a cepa expressando a proteína Ndt1 tinha um aumento na fluorescência de NADH, mostrando que NAD+ foi capaz de entrar na matriz mitocondrial e posteriormente ser reduzido a NADH por enzimas da matriz mitocondrial. A determinação da produção de espécies reativas de oxigênio foi realizada utilizando as sondas fluorescentes CM-H2DCFDA e MitoSox Red em esferoplastos da levedura S. cerevisiae. Ambos experimentos não houve diferença significativa entre a cepa expressando a proteína Ndt1 e a cepa controle. As proteínas carboniladas foram determinadas utilizando anticorpo anti-DNP, após a reação com dinitrofenilhidrazona e não há diferença significativa entre as cepas. Finalmente, para confirmação da localização celular da proteína Ndt1, os esferoplastos de S. cerevisiae foram submetidos à microscopia confocal, onde ficou evidenciado a co-localização da proteína Ndt1 com as mitocôndrias na cepa de S. cerevisiae transformada com a construção pYES/ndt1, o mesmo perfil não foi observado na cepa ?ndt1?ndt2.
The A. fumigatus is a saprophytic fungus and is a major opportunistic pathogen in immunosuppressed patients. Previous studies in our lab has showed that mitochondrias of the P. brasiliensis and A. fumigatus, NAD+ is able to induce the formation of mitochondrial membrane potential, which could be dissipated by FCCP, suggesting the presence of a NAD+/NADH carrier as described in S. cerevisiae. Using bioinformatics tools, it was identified in Aspergillus Gene database a sequence containing 32% of identity with the gene ndt1p of S. cerevisiae. A fragment of cDNA was obtained from the ndt1p mRNA sequence, containing 1194 bp by using PCR-overlaping and cloned in pGEM®-T Easy vector. Then, the sequence was subcloned in pET28-a(+) vector and expressed in E. coli BL21 (DE3). The recombinant protein was purified from inclusion bodies and the identity confirmed by mass spectrometry and by Western Blotting using anti-His-tag antibody. The recombinant protein was used to produce polyclonal antibody anti-Ndt1 in rabbit. For expression in yeast, the ndt1 cDNA of A. fumigatus was subcloned into pYES2 vector and the yeast S. cerevisiae ?ndt1?ndt2 were transformed. Growth curve and induction of the recombinant protein expression Ndt1 was performed and the protein was detected using a polyclonal anti-Ndt1 antibody after 16 hours of expression. During this period it was found that the yeast were in exponential growth phase. The double mutant strain shows a slower growth rate compared to the strain expressing the recombinant protein growth rate when grown in fermentable medium. The isolated mitochondria from both strains were subjected to measurement of the membrane potential which showed coupling between substrate oxidation and oxidative phosphorylation. Furthermore, these measurements evidenced that the strain expressing the recombinant protein NAD+ induced the formation of a membrane potential larger than the control strain. The transport NAD+ was evaluated and showed that the strain expressing the protein Ndt1 has an increase in NADH fluorescence, indicating that NAD+ was able to enter into the mitochondrial matrix and then reduced to NADH by enzymes from the matrix. The determination of the production of reactive oxygen species was performed using the fluorescent probe CM-H2DCFDA and MitoSox Red in spheroplasts of the yeast S. cerevisiae. Both experiments showed no significant difference between the strain expressing Ndt1 protein and strain control. The carbonylated proteins levels were checked using anti-DNP, after the reaction with dinitrophenylhydrazone and there is no significant difference between the strains. Finally, to confirm the cellular localization of the protein Ndt1, spheroplasts of S. cerevisiae were subjected to confocal microscopy, which evidenced the co-location of Ndt1 protein with mitochondria in S. cerevisiae strain transformed with the construction pYES/ndt1. This same profile was not observed in strain ?ndt1?ndt2.

Nicotinamide adenine dinucleotide (NAD+) represents a long-known key molecule in cellular metabolism. It was initially identified for its ability to convey electrons and protons between redox partners in multiple bioenergetic and biosynthetic reactions. In addition, NAD+ also serves as a substrate for NAD+-consuming enzymes such as sirtuins and poly ADP-ribose polymerases (PARPs). These latter enzymes catalyze dynamic post-translational modifications that control virtually every signaling pathway orchestrating cell fate. The aim of this work was to analyze the role of NAD+ in the context of programmed cell death mechanisms.

Our findings indicate that NAD+ is protective against DNA damage-induced cell death and FAS-induced apoptosis, while, unexpectedly, it promotes TNF-induced necroptosis, a regulated form of necrosis. Indeed raising NAD+ cellular levels sensitized culture cells to necroptosis, while NAD+ depletion protected cells from this form of cell death. Furthermore, specific silencing of NAD+-dependent sirtuins was also found to be protective against TNF-induced necroptosis. Consistently, a pharmacological pan-sirtuin inhibitor called cambinol protected cells from necroptosis. Then, as necroptosis represents a back-up mechanism that may have evolved in response to viral pathogens expressing anti-apoptotic proteins, we demonstrated in an in vitro model mimicking viral infection that pharmacological sirtuin inhibition protected cells from poly I:C-induced necroptotic cell death. In vivo, we demonstrated that cambinol partially protected kidney from necrosis after ischemia/reperfusion. We have also shown that enhancing liver NAD+ concentration via isonicotinamide increases the susceptibility of mice to systemic inflammatory response syndrome (SIRS). Moreover, our preliminary data show that isonicotinamide substantially improves the ability of cyclophosphamide to trigger the rejection of the murine mastocytoma P815 tumor cell line.

Collectively, our observations point to a role for NAD+ in the control of necroptosis in a sirtuin-dependent manner. These observations may bear relevance to the better understanding of the pathophysiological consequences of excessive production of the pro-inflammatory cytokine TNF and the control of viral infections and tumor progression/immunotherapy. &
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

The F420-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F420 as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum of 7.5. The NADP reductase had an apparent Km of 42 uM for reduced F420, and an apparent Km of 4l uM for NADP. The enzyme was stable in 0.05 M sodium phosphate buffer (plus 10 mM cysteine) at pH 7.0, when gassed with nitrogen or hydrogen and stored at 4°C.

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).

Ageing is associated with ultrastructural changes in the liver, including a reduction in hepatocyte mitochondrial number density and porosity of the liver sinusoidal cells. Histologically-verifiable changes include lipid and collagen accumulation, and increased perisinusoidal von Willebrand factor (VWF) expression. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is renowned for its range of anti-ageing actions. This study evaluated whether the anti-ageing effects of SIRT1 would extend to these liver ageing characteristics. Here the effects of whole-body SIRT1 overexpression on the livers of young (3-5 month) and old (13-20 month) SIRT1 transgenic mice were evaluated and compared to their wildtype controls. In addition, because NAD+ levels diminish with age, some old animals were supplemented with the NAD+ precursor, nicotinamide mononucleotide (NMN), to remove any limitation of naturally available NAD+ on SIRT1 overexpression. NAD+ is a SIRT1 cofactor. Old age is orthodoxly associated with reduced mitochondrial function and number in many tissues. However, transmission electron microscopy determination of liver mitochondrial numerical density (mitochondrial particles per cytosolic µm2) showed that there was no change with age. Additionally, there was no intervention effect of SIRT1 overexpression or NMN supplementation. Old age is also conventionally associated with reduced number and size of pores in the liver sinusoidal endothelial cell called fenestrae. However, analysis of the liver blood vessels with scanning electron microscopy was unable to confirm an effect of age on the number and size of fenestrae. Analysis did, however, reveal that SIRT1 overexpression was associated with increased sinusoidal porosity (%) in older but not younger mice (F(1,8) = 14.9, p < 0.01). Furthermore, although SIRT1-mediated increases in fenestrae diameter were not statistically verifiable in a 2-way ANOVA analysis, when plotting maximal fenestrae diameter size against groups, there was a trend suggesting a shift in the size distribution towards larger fenestrae for SIRT1-overexpressing animals. Histological examination of the mice livers showed that age was associated with an increase in lipid and collagen levels. However, there was no effect of age on the presence of activated stellate cells or perisinusoidal expression of VWF. Mice that were treated with NMN were 0.11 times less likely to have elevated α-SMA expression than controls, yet a combination of SIRT1 overexpression and NMN supplementation led to a 20.43 times greater likelihood of elevated α-SMA expression. The lack of canonical ageing-associated changes in a number of regards raises the question of whether the older mice were sufficiently old for such effects to emerge. Future studies should examine mice of at least 24 months of age. In summary, although SIRT1-overexpression shows promise in promoting liver sinusoidal endothelial porosity, the attendant risk related to stellate cell activation may be prohibitive in the use of SIRT1-elevating strategies with respect to the liver. The possibility for tissue- specific SIRT1-elevating strategies remains for other tissue types which do not incur this extra risk. Finally, NMN might reduce stellate cell activation by a SIRT1-independent pathway. Collectively, the results raise the possibility of a novel pathway leading to stellate cell activation and a new therapeutic approach to reducing stellate cell activation – both possibilities have important implications for the treatment of hepatic fibrosis. Keywords: sirtuin 1 (SIRT1), nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD), ageing, anti-ageing, liver, gastrocnemius muscle, liver sinusoidal endothelial cells (LSECs), fenestrae, defenestration, pseudocapillarization, mitochondria, mitochondrial numerical density, fibrosis, steatosis, hepatosteatosis, lipid, collagen, α-smooth muscle actin (α-SMA), von Willebrand factor (VWF), scanning electron microscopy, transmission electron microscopy, histology, haematoxylin and eosin, Sirius Red, immunohistochemistry.

Poly(ADP-ribosyl)ation is a reversible post-translational modification of histones and nuclear proteins rapidly stimulated by DNA damage. Its homeostasis is a dynamic process regulated by the synthesizing enzymes poly(ADP-ribose) polymerases (PARPs) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). PARP-1, the first-discovered and major PARP, has been the focus of many studies aimed at clarifying the biological function of poly(ADP-ribose) (PAR). This abundant nuclear enzyme plays key roles in a variety of cellular processes, including the regulation of chromatin structure, transcription and genomic integrity. Its multifunctionality has made it an attractive and potential target for therapy, as evidenced by the numerous PARP-1 inhibitors currently undergoing clinical trials. The transient nature of PAR, explained by the close coordination between PARP-1 and PARG, has also highlighted the potential of targeting PARG for diseases of inappropriate cell death. A number of obstacles, however, have prevented PARG from being studied as extensively as PARP-1. The extreme sensitivity of PARG to proteases and its insolubility at high concentrations have limited structure-activity relationship analyses and structural studies of PARG, and the unavailability of high-throughput activity assays has stalled the discovery and development of specific and cell permeable PARG inhibitors, subsequently slowing down the validation of PARG as a therapeutic target. The work presented in this dissertation describes in detail strategies devised to overcome these difficulties. First, a novel colorimetric high-throughput assay for PARG was evaluated and its sensitivity and precision were compared to a widely-used radiolabelling assay. Second, several expression and purification systems were constructed in order to obtain high quantities of soluble human PARG protein adequate for in vitrostructural studies. The efficacy of these strategies was demonstrated in structure-activity analyses of PARG which led to the identification of a regulatory segment far removed linearly from the catalytic site of PARG. This region, necessary for catalytic activity, corresponds with a recently identified mitochondrial targeting sequence (MTS) and was thus named the ‘regulatory segment/MTS’ (REG/MTS). Finally, based on structural data obtained, secondary structure predictions were made to provide insight into the molecular composition of the different domains of PARG, whose structures still remain to be determined.

The mechanism by which nicotinamide adenine dinucleotide (NAD) stimulates the activity of adenylate cyclase (AC) in canine plasma membrane has been studied. Using [3 2P]-NAD, the activation by NAD was correlated with the radiolabeling of the stimulatory guanosine triphosphate (GTP) binding protein Gsa. Further characterization demonstrated that the modification occurred only in the presence of G-protein activators and that arginine residue(s) were modified by ADP-ribose by the action of a mono-ADP-ribosyltransferase. Inhibitors of the transferase blocked both the modification of Gsa and the activation of AC. Collectively, these studies suggest that ADP-ribosylation of Gsa by an endogenous mono-ADP-ribosyltransferase may regulate cardiac AC.

The nicotinamide riboside (NR) form of vitamin B3and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B3are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD+decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD+levels. BHB is naturally produced by the body when individuals fast or consume a ketogenic (KD) or calorically restricted (CR) diet. These diets have been shown to extend lifespan in mice, while they are also protective in many disease models. Caenorhabditis elegans, a roundworm with a short mean lifespan of roughly 2 to 3 weeks depending upon the temperature, is used as a model system to study aging. BHB has been previously shown to increase lifespan by roughly 20% when administered to C. elegans.We administered NR and BHB individually and together to C. elegans starting at two different developmental stages (larval stages 1 and 4) and measured lifespan. We found that administration of 20 mM DL-BHB decreased lifespan when first given at the L1 stage, while it robustly increased lifespan when first given at the L4 stage. Administration of 0.5 mM NR increased lifespan when first given at L1, with only a very slight increase when first given at L4. When initiating administration at L1, NR greatly mitigated the BHB-mediated decline in longevity, however, NR did not increase BHB-mediated lifespan extension when first administered at L4.

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environmental Engineering
Full Text

Les cellules dendritiques (DC) sont des cellules présentatrices de l’antigène. Quand la DC présente un antigène exogène sur les molécules du CMH-I et active un lymphocyte T CD8+, on parle de cross présentation de l’antigène. Nous savons que seules les DC CD8+ (CD8+) cross présentent de manière efficace les antigènes. Les DC ont une adaptation de leur voie de phagocytose leur permettant une dégradation partielle des antigènes par les protéases lysosomales. Ainsi la NADPH oxydase est recrutée à la membrane des phagosomes précoces de DC ne produisant que de faible quantité de dérivés oxygénés responsables de l’alcalinisation du phagosome. Les DC issues de souris déficientes pour l’activité de la NADPH oxydase présentent une acidification plus élevée du phagosome responsable d’une dégradation plus importante de l’antigène et par conséquent un défaut majeur de cross présentation de l’antigène. J’ai montré que les DC CD8+ ont un pH phagosomal quasi neutre alors que la sous population CD8- a un pH phagosomal acide. Le pH neutre du phagosome des DC CD8+ est à l’origine de leur capacité à cross présenter les antigènes en opposition avec les DC CD8-. On a montré que la NADPH oxydase est localisée au phagosome des DC CD8. Le recrutement différentiel de la NADPH oxydase au phagosome des DC CD8+ est dépendante de l’activité de Rac2 alors que la localisation membranaire dans les CD8- est dépendante de Rac1.

Thesis (Ph.D.)--Case Western Reserve University, 2008
Department of Chemical Engineering Abstract Title from OhioLINK abstract screen (viewed on 10 April 2009) Available online via the OhioLINK ETD Center

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Nicotinamida adenina dinucleotídeo (NAD + ) e seu derivado NADP + são compostos essenciais em todos os organismos vivos e que estão envolvidos em transações energéticas, na sinalização celular e em diversas reações na forma de coenzimas. Alterações no catabolismo ou no anabolismo do NAD + podem modificar não apenas a sua concentração, mas também o poder redutor celular, impactando aspectos importantes do crescimento e desenvolvimento. Uma vez que as etapas finais da biossíntese de NAD + ocorrem no citosol, o mesmo precisa ultrapassar membranas biológicas para exercer suas funções dentro de organelas celulares. Neste trabalho, avaliou-se em Arabidopsis thaliana o papel dos transportadores cloroplastídico e mitocondrial de NAD + , denominados AtNDT1 e AtNDT2, respectivamente. Para tal, foram utilizadas linhagens mutantes obtidas por inserção do T-DNA e foram geradas plantas transgênicas contendo uma construção antisenso para os transportadores NDT1 e NDT2, sob o controle do promotor 35S, das quais, três linhagens com reduzida expressão de cada transportador foram selecionadas. Estas plantas foram caracterizadas extensivamente a nível fisiológico e bioquímico. Verificou-se que plantas com expressão reduzida de NDT1 apresentam maior número de folhas e área foliar específica em relação às plantas não transformadas (WT). Observaram-se também maiores taxas de fotossíntese por unidade de massa e, consequentemente, maior acúmulo de sacarose e amido em comparação com o WT. Entretanto, estas plantas apresentaram menor viabilidade do grão de pólen, redução no comprimento das síliquas e sementes abortadas. Já em plantas com reduzida expressão do transportador NDT2 descobriu-se que apesar de possuírem menor número de folhas, a área foliar aumentou. A fotossíntese nestas plantas diminuiu consideravelmente em decorrência de uma menor densidade estomática e condutância estomática. Concomitantemente, plantas com reduzida expressão de NDT2 apresentaram menor acúmulo de amido. A germinação das sementes e formação de plântulas foram menores e mais lentas em comparação com o WT. Além disso, houve aumento na concentração relativa de alguns ácidos graxos durante o estabelecimento das plântulas. Diante dos resultados obtidos, sugere-se que os transportadores de NAD + estão envolvidos na determinação do número de folhas e área foliar, na fotossíntese, na condutância estomática e nas concentrações de amido. Além disso, tecidos heterotróficos também são fortemente influenciados, especialmente aqueles envolvidos em processos reprodutivos. Para compreender a função deste transportador é necessário analisar não apenas a concentração celular dos nucleotídeos de pirimidina, mas também como alterações no status redox celular afetam o metabolismo vegetal. Assim, os dados obtidos neste trabalho sugerem que alterações no estado redox em cloroplastos e mitocôndrias causadas pela diminuição da expressão de NDT1 ou NDT2 afetam processos em outras organelas que não aquela em que ocorre a redução da expressão. Estes resultados abrem possibilidades para aplicações biotecnológicas, uma vez que a regulação da biossíntese e transporte de cofatores pode ser usada como uma ferramenta para estratégias de aumento da assimilação de carbono em plantas. Para tal, é necessário, no entanto compreender mais o papel destes transportadores em outros tecidos vegetais e condições adversas bem como o papel de enzimas chave que atuam no processo.
Nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) are indispensable redox-active components of all living organisms. These nucleotides play essential roles in signaling and in many reactions as they act as co-enzymes. NAD catabolism or anabolism changes impact not only the concentration of these coenzymes but also the cellular poise, modifying plant growth and development. Since the final steps of NAD + biosynthesis occurs in cytosol, this nucleotide needs to be imported by organelles to play its functions inside cellular organelles. This work analyzed the function of a plastid and a mitochondrial Arabidopsis thaliana NAD + transporter, named as AtNDT1 e AtNDT2 respectively. We have isolated T-DNA insertion mutants and three antisense lines, controlled by the 35S promoter, for NDT1 and NDT2. Plants with reduced expression of NDT1 and NDT2 were individually selected and characterized at physiological and biochemical level. It was verified that NDT1 low expression lines displayed higher leaf number and specific leaf area compared to wild type plants (WT). Interestingly, higher photosynthesis per mass unit and sucrose and starch concentration was observed. On the other hand, NDT1 low expression lines show less pollen viability and reduced silique length and increased the number of aborted seeds. The low expression NDT2 lines displayed reduced number of leaves and increased leaf area. Despite these phenotypes, the net photosynthetic rate decreased considerably due to lower stomatal conductance, possible as a consequence of lower stomatal density. Concomitantly, these plants showed lower starch accumulation during the day. The germination and seedling growth rate were reduced as compared to WT. Furthermore, the oil relative concentration increased during seedling establishment. Based on these results, we conclude that NAD + transporters have important function in determining leaf number, area, stomatal conductance, photosynthetic rate and starch accumulation. Moreover, heterotrophic tissues are strongly affected by the reduction in the expression of these transporters, especially those tissues involved in reproductive process. For a better understanding of the role played by these transporters, it is necessary to analyze not only nucleotide cellular concentration but also the redox poise in different cell compartments. Thus, the results presented in this thesis suggest that lower expression of NAD transporters alters redox poise in different organelles affecting important physiological processes. These results open possibilities for biotechnology application, once the regulation of biosynthesis and transport of cofactors might be used as a tool for increasing carbon assimilation in plants. Nonetheless, it is necessary understanding the role of these transporters in other plant tissues and plant cultivated under stress conditions involved in these processes.

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Background: Nitric oxide (NO) donors can act in the control and treatment of various cardiovascular complications, such as hypertension. In particular, an organic nitrate, 2-nitrate-1,3-dibutoxipropane (NDBP) developed in recent years by UFPB has shown great therapeutic potential. However, its mechanisms are not fully clarified. Aim: To investigate the effects and mechanisms of NDBP, elucidating their therapeutic potential in angiotensin II-mediated hypertension. Methods: In the first stage of the study, were performed in vitro experiments in normotensives Wistar rats (n=11) tissues for characterization of compound about ability to release NO by direct measurement of NO; and modulation of NADPH oxidase activity, by chemiluminescence technique. In the second stage of the study, in vivo experiments in C57BL / 6J mice (n=24), divided in four groups (control, NDBP, Ang II and Ang II + NDBP) were performed to investigate the therapeutic potential of NDBP in angiotensin II-mediated hypertension. The animals had implantation of osmotic minipumps containing Ang II (400 mg / kg / 24h) and after 9 days were treated with NDBP (40 mg / kg / day; ip). Blood pressure (BP) was measured by tail-cuff. At the end of the experiments, the animals were euthanized and blood and tissues were collected for analysis. Histopathological analyzes were performed using staining picrosirius. Superoxide levels were measured by NADPH oxidase activity via chemiluminescence and DHE fluorescence (5 mmol • L-1). Levels of serum nitrate/nitrite and cGMP were performed using specific kits. NADPH oxidase subunit expression were analyzed by real time qPCR. Putative antioxidant activity was determined by autoxidation with pyrogallol. Results: In vitro experiments revealed a dose-dependent increase in NO production mediated by NDBP in liver and kidney tissues, which was attenuated by the xanthine oxidase inhibitor febuxostat. In addition, the NDBP reduced NADPH oxidase activity and prevented its activation induced by ANG II. In vivo experiments showed that NDBP prevents the increase in hypertension in mice with chronic infusion of ANG II. This results were associated to the attenuation of cardiac hypertrophy, renal fibrosis, and reduced NADPH oxidase-derived oxidative stress in the kidneys, the heart, and liver. Furthermore, NDBP also increased plasma levels of nitrite and nitrate. On the other hand, the autoxidation with pyrogallol revealed that NDBP does not act as a direct antioxidant, justifying its involvement in the modulation of NADPH oxidase. Conclusions: The organic nitrate NDBP prevents the progression of angiotensin II-mediated hypertension. Regarding the mechanisms, our findings suggest that NDBP treatment is associated with sustained NO release and attenuated activity of NADPH oxidase, which requires functional xanthine oxidase.
Introdução: Doadores de óxido nítrico (NO) podem atuar no controle e tratamento de diversas complicações cardiovasculares, como por exemplo, a hipertensão arterial. Em particular, um nitrato orgânico, 2-nitrato-1,3-dibutoxipropano (NDBP) desenvolvido nos últimos anos pela UFPB vem apresentando grande potencial terapêutico. No entanto, seus mecanismos não estão totalmente esclarecidos. Objetivo: Investigar os efeitos e os mecanismos de ação do NDBP, elucidando seu potencial terapêutico na hipertensão arterial induzida por Angiotensina II (ANG II). Métodos: Na primeira etapa do estudo foram realizados experimentos in vitro em tecidos de ratos Wistar normotensos (n=11) para caracterização do composto, sobre sua capacidade de liberação de NO, por meio da medida direta de NO; e modulação da atividade da enzima NADPH oxidase, pela técnica de quimioluminescência. Na segunda etapa do estudo, experimentos in vivo em camundongos C57BL/6J (n=24), classificados em quatro grupos (Controle, NDBP, ANG II e ANG II + NDBP), foram realizados para investigar o potencial terapêutico do NDBP na hipertensão induzida por ANG II. Os animais tiveram implante de minibombas osmóticas contendo ANG II (400 μg/kg/24h) e após 9 dias receberam tratamento com NDBP (40 mg/kg/dia; i.p.). A pressão arterial (PA) foi verificada via pletismografia de cauda. Ao término dos experimentos, os animais foram eutanasiados e tecidos e sangue foram coletadas para análise posterior. Análises histopatológicas foram realizadas utilizando a coloração com Picro-sirius. Os níveis de superóxido foram mensurados por meio da atividade da enzima NADPH oxidase, via quimioluminescência e fluorescência com DHE (5 mmol·L−1). Além disso, os níveis séricos de nitrato/nitrito e cGMP foram realizados, utilizando kits específicos. Foi investigada também a expressão de subunidades da enzima NADPH oxidase pela técnica de qPCR em tempo real e a possível atividade antioxidante por meio da autoxidação com pyrogallol. Resultados: os experimentos in vitro utilizando tecidos de fígado e rim revelaram um aumento dose-dependente na produção de NO, mediado pelo NDBP, que foi atenuado pelo inibidor da enxima xantina oxidase, o febuxostato. Além disso, o NDBP reduziu a atividade da NADPH oxidase e preveniu sua ativação induzida pela ANG II. Os experimentos in vivo mostraram que o NDBP previne o aumento da hipertensão arterial em camundongos com infusão crônica de ANG II. Esses resultados foram associados à atenuação da hipertrofia cardíaca, fibrose renal, e redução do estresse oxidativo mediado pela redução da atividade da enzima NADPH oxidase nos rins, coração e fígado. Além disso, o NDBP aumentou os níveis plasmáticos de nitrito e nitrato. Por outro lado, a autooxidação com pyrogalol revelou que o NDBP não atua como um antioxidante direto, justificando então sua participação na modulação da NADPH oxidase. Conclusão: o nitrato orgânico NDBP impede a progressão da hipertensão arterial mediada pela angiotensina II. Em relação aos seus mecanismos, os achados sugerem que o tratamento com NDBP está associado a uma contínua liberação de NO e redução da atividade da enzima NADPH oxidase, que parece ser dependente da xantina oxidase funcional.

La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico.
In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1.
També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut.
Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation.
We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.

Social-cognitive difficulties in Multiple Sclerosis (MS) have devastating psychosocial impacts, yet assessment is challenging and treatments scarce. This pilot longitudinal study examined the potential role of inflammatory cytokines and nicotinamide adenine dinucleotide (NAD+) as predictive biomarkers of social-cognitive abilities in people with MS (pwMS). Seventeen pwMS (13 female, 18-62 years old) provided blood samples and were assessed for the social-cognitive domains of emotion recognition and sarcasm detection tapping into Theory of Mind, using The Awareness of Social Inference Test-Short (TASIT-S) at baseline and after 5-to-6 years. As hypothesised, higher levels of anti-inflammatory cytokine IL-4 related to better sarcasm detection. However, relationships between pro-inflammatory cytokines and TASIT-S scores were unexpected, with lower levels of cytokines relating to better emotion recognition, but higher levels relating to better sarcasm detection at baseline and follow-up. TASIT-S scores and cytokine levels did not change over time. Select cytokines and NAD+ may be predictive of long-term social-cognitive performance. These pilot results suggest a larger, fully powered study to further examine these relationships is needed. The identification of predictive biomarkers could both facilitate timely assessment and intervention for social-cognitive difficulties, and the development of potential pharmacotherapeutic targets, which would improve health-related quality of life in pwMS.

碩士
中州科技大學
保健食品系
100
Caloric restriction (CR) can extend the lifespan of organisms. Nicotinamide adenine dinucleotide (NAD+) is one of bioactive from of vitamin B3. Sirtuins is a protein family called NAD+-dependent deacetylase, which is considered as a longevity protein. FK866 is an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway of NAD+ synthesis from nicotinamide and has ability to decrease intracellular NAD+ level. In this study, we examined whether CR can increase intracellular NAD+ levels, and then activate SIRT1 and extend replicative lifespan of human Hs68 cells. Using cells cultured at lower levels of glucose than normal level of glucose in medium (glucose restriction; GR) to mimic CR, we compared the effects of GR, NAD+ and FK866 on cell lifespan, intracellular NAD+ levels, SIRT1 activity and senescence associated -galactosidase (SA-G) activity. We also examined whether the effects of FK866 on Hs68 cells can be antagonized by GR and adding NAD+ into the culture medium. The lifespan of cells was measured as the cumulated growth curve of population doubling levels. The biomarker of cell senescence was detected by determination of senescence associated -galactosidase (SA-G) activity using X-Gal staining. Intracellular NAD+ was determined by an acid extraction method and followed by an enzymatic cycling method. SIRT1 activities were determined by SIRT1 Direct Fluorescent Screening Assay Kit or used p53 acetylation to represent cellular activities of SIRT1. The results showed that GR (>5day) could significantly increase NAD+ levels in cells, activated SIRT1 activity, and extended the lifespan of Hs68 cells. Add NAD+ in to the medium showed with similar effects as GR. Oppositely, FK866 has abilities to reduce intracellular NAD+ levels, shorten the cells lifespan, decrease SIRT1 activity, and increase SA-βG activity. Importantly, all of these effects of FK866 on Hs68 cells can be effectively blocked by GR or adding NAD+ into the medium. The results demonstrated that increase of intracellular NAD+ level plays a crucial role in the mechanism of CR-extended lifespan of human Hs68 cells.

Thesis (M.S.)--State University of New York at Buffalo, 2008.
Title from PDF title page (viewed on Feb. 12, 2009) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Bhattacharjee, Arin, Slaughter, Malcolm Includes bibliographical references.

碩士
國立陽明大學
生醫光電工程研究所
96
Stem cells have been demonstrated having potential applications in tissue engineering and regenerative medicine. One of the known metabolic properties of stem cell is the change of metabolic switch from stem cells to differential state. The metabolic shift is form anaerobic glycolysis to the more efficient mitochondrial oxidative metabolism. Base on this property of stem cell, we observed the lifetime of reduced nicotinamide adenine dinucleotide (NADH) via two-photon Fluorescence lifetime imaging microscopy (FLIM) during osteogenic differentiation of human mesenchymal stem cells (hMSCs), to replace many of these methods are invasive that damage cell or need pre-labeling. In our study, we investigated that NADH lifetime increased from ~1000 ps to ~1200 ps and the free-/bound- protein ratio of NADH decreased from~3.0 to ~2.1 during the osteogenic differentiation of hMSCs. Then we treated different metabolic inhibitors into fiberblasts(CCD) and measured NADH lifetime. After treating different inhibitors 24 hr, NADH lifetime only increased to ~1200 ps in the treatment of oligomycin A. In the short terms (2 hr), NADH lifetime after treating KCN decreased to ~700 ps initially, and then increased to ~1000 ps after 1 hr, which is almost the same with control. Other inhibitors didn’t influence the change of NADH lifetime in the short terms. These result is very different from our hypothesis. In MSCs, we considered the increase of NADH lifetime resulted from Complex number of OXPHOS system increased. However, in treatment of metabolic inhibitors, cells not only changed in metabolism but also the cellular condition became very complicated. Our results suggest that detecting NADH lifetime by FLIM has potential to observe the metabolic change during the osteogenic differentiation of hMSCs. But we need more researches to understand the complicated condition resulted from the treatment of metabolic inhibitors in the measurement of NADH lifetime.