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.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Extremophiles are the organisms that survive in environments which are inhospitable to other creatures. This thesis made an attempt to understand the enviromental effects, in particular saline, on enzyme structure by using three dimensional NMR. The enzyme DHFR1 from halophile Haloferaxi volcanii is complexed with its coactor NADPH, and the saline effects on the complex are studied by comparison with its apoenzyme, hvDHFR1. Backbone chemical shift assignments of the hvDHFR1:NADPH complex were attained which can be functional (along with future work) in understanding the effect of salts on enzyme structure, function, and flexibility. A total of 27 amino acids were found to show a significant change upon binding of NADPH and their positions were identified on enzyme complex. The secondary structure of hvDHFR1:NADPH is also predicted and overall global structure is found to be similar with the crystal structure of hvDHFR1 with few changes.

N-hydroxylating monooxygenases (NMOs) are Class B flavin-dependent monooxygenases found only in fungi and bacteria. These enzymes catalyze the hydroxylation of nucleophilic primary amines, such as those found in histamine, L-ornithine, L-lysine, and small aliphatic diamines. The hydroxamate moiety produced by this reaction is key for the production of siderophores, small chelating compounds that allow survival in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. Herein we present the characterization of TheA, an NMO from Thermocrispum agreste. The enzyme mechanism was studied using steady state kinetic measurements, thermostability, and stopped flow spectrophotometry assays. Using these techniques, the catalytic rates, substrate binding affinities, thermal stability, and coenzyme specificities were determined. Additionally, NADPH analogues were produced to use as tools to study FAD reduction in NMOs. An unspecific reduction reaction of NADP+ using NaB2H4 yielded [6-2H]-NADPH, [2-2H]-NADPH, and [4-2H]-NADPH. Compound identity was confirmed by mass spectrometry and unidimensional proton nuclear magnetic resonance (NMR). Results presented in this thesis lay the foundation for future studies of NMOs using NADPH analogues. In conjunction, these results will improve the general knowledge and understanding of flavoenzymes, ornithine monooxygenases, and their associated mechanisms. / Master of Science in Life Sciences

Orientador: Cristina Antoniali Silva / Coorientadora: Lusiane Maria Bendhack / Banca: Graziela Scalianti Ceravolo / Banca: Michele Mazzaron de Castro / Resumo: A enzima NAD(P)H oxidase (NOX) é a principal fonte de espécies reativas de oxigênio (ERO) no sistema cardiovascular e sua atividade e expressão podem ser regulada pela angiotensina (Ang) II. Demonstramos previamente que o tratamento crônico com apocinina, um inibidor de NOX, reduziu a pressão arterial e preveniu o desenvolvimento da disfunção endotelial em SHR. Estes efeitos da apocinina foram associados a redução de geração de ERO e ao aumento da biodisponibilidade de óxido nítrico em células endoteliais de SHR. Dados de nosso laboratório mostraram que o tratamento com apocinina, também reduziu o efeito pressor da Ang II em SHR. A associação entre Ang II, via receptores AT1, e o estresse oxidativo tem sido implicada na patogênese da hipertensão. Levantamos a hipótese que a apocinina, ao alterar a sinalização redox, reduz a expressão de receptores AT1 e a resposta vasoconstritora da Ang II em SHR. Neste estudo, avaliamos o efeito do tratamento crônico com apocinina sobre as respostas contráteis à Ang II em vasos sanguíneos de SHR e os mecanismos envolvidos nestes efeitos, utilizando ensaios bioquímicos, biomoleculares e funcionais. SHR foram tratados com apocinina (30 mg/Kg, v.o.) da 4ª a 10ª semana de vida e ratos Wistar foram utilizados como controle normotenso. Analisamos os efeitos da apocinina na capacidade antioxidante plasmática, expressão de NOX, geração de ERO, níveis de nitrato/ nitrito, expressão de receptores AT1 e AT2, e respostas vasoconstritor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: NAD(P)H oxidase (NOX) is the major source of reactive oxygen species (ROS) in the cardiovascular system and its activity and expression could be regulated by angiotensin (Ang) II. We previously demonstrated that chronic treatment with apocynin, a NOX inhibitor, reduced blood pressure and prevented the development of endothelial dysfunction in SHR. These effects of apocynin have been associated with reduced generation of ROS and increased bioavailability of nitric oxide in endothelial cells of SHR. Data from our laboratory showed that treatment with apocynin also reduced the pressor effect of Ang II on SHR. The association between Ang II, via AT1 receptors, and oxidative stress has been implicated in the pathogenesis of hypertension. We hypothesized that apocynin, altering redox signaling, could reduce expression of AT1 receptors and Ang II vasoconstrictor response in SHR. In this study, we evaluated the effect of chronic treatment with apocynin on the contractile responses to Ang II in blood vessels of SHR and the mechanisms involved in the effects of these on biochemical, biomolecular and functional assays. SHR were treated with apocynin (30 mg/kg, p.o.) from the 4 th to the 10th week of life and Wistar rats were used as normotensive control. Analysis of the effects of apocynin on plasma antioxidant capacity, NOX expression, ROS generation, nitrate/ nitrite levels, expression of AT1 and AT2 receptors, and vasoconstrictor responses to Ang II on mesenteric and aortic arteries.... (Complete abstract click electronic access below) / Mestre

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-30T12:19:46Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Georgia Maria Ricardo Felix dos Santos_Mestrado.pdf: 1018948 bytes, checksum: eaff2e3a96759aec9a4685114c9635cb (MD5) / Made available in DSpace on 2016-06-30T12:19:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Georgia Maria Ricardo Felix dos Santos_Mestrado.pdf: 1018948 bytes, checksum: eaff2e3a96759aec9a4685114c9635cb (MD5) Previous issue date: 2013-08-15 / CAPEs / Elevados níveis de hiperglicemia durante a gestação pode ter consequências deletérias para prole que inclui doenças cardiovasculares (DCV) e renais. As complicações cardiovasculares do diabetes resultam de um conjunto de fatores que envolvem a hiperglicemia crônica, a formação excessiva dos produtos finais de glicação avançada (AGEs) e de oxidação proteica (AOPP) e a ativação do fator de transcrição nuclear kappaB (NF-κB), associada à liberação de citocinas pró-inflamatórias. Portanto, é de grande relevância a elucidação de mecanismos envolvidos na gênese e/ou manutenção do processo oxidativo que pode ser perpetuada na prole exposta à hiperglicemia. O objetivo deste estudo foi investigar os efeitos da exposição intra-uterina a hiperglicemia (> 200 mg/dL) sobre AGEs, AOPP, bem como a expressão proteica do NF-κB, a expressão gênica da interleucina-1 beta (IL-1ß) e Nox 4 (homólogo da subunidade da NADPH oxidase) na prole adulta. Para isto, o DG foi induzido no sétimo dia de gestação com dose única de estreptozotocina (STZ, 42mg/kg, i.p.) dissolvida em tampão citrato (veículo). Fêmeas controles receberam apenas veículo. A prole foi dividida em dois grupos de acordo com o tratamento da mãe: controle não diabético (ND) e diabetes gestacional (DG). Na idade adulta, foram realizadas as dosagens de glicose, triglicerídeos, colesterol total e HDL. A concentração plasmática de AGEs foi determinada por fluorescência e a de AOPP por espectrofotometria, os níveis de peroxidação lipídica pelas substâncias reativas ao ácido tiobarbitúrico (TBARS). A expressão proteica do NF-κB foi analisada em aortas através de Western Blot. Foi utilizada reação em cadeia de polimerase quantitativa em tempo real (RTq-PCR) para avaliar a expressão gênica da IL-1ß e Nox 4. Os níveis de glicose, triglicerídeos, colesterol total foram significativamente (p < 0,05) maiores na prole DG quando comparadas a prole ND. A prole DG apresentaram níveis elevados de AGEs (p < 0,01), AOPP (p < 0,05) e TBARS (p < 0,05). Em aorta, a expressão proteica do NF-κB foi mais elevada (p < 0,05) no grupo DG do que no ND. Adicionalmente, foi observado um aumento da expressão gênica da IL-1ß e da Nox4. O número de néfrons desses animais foi significativamente reduzido (p < 0,001). A associação de níveis elevados de estresse oxidativo e interleucina-1 beta podem ser responsáveis pela estimulação da expressão proteica do NF-κB. Juntas, essas alterações em um ambiente hiperglicêmico, com indícios de comprometimento renal, poderiam desencadear alterações vasculares. / Experimental evidences support the hypothesis that diseases can be programmed during the period intrauterine. This concept of programming fetal has been used as basis for knowledge of some chronic diseases, such the cardiovascular. Cardiovascular disease has been associated with hyperglycemia, increased level of advanced glycation end products (AGE) and of advanced oxidation products (AOPP) activate the transcription factor nuclear factor-kappaB (NF-κB) associated with inflammatory cytokine liberation. Therefore, the present study was designed to examine the alterations produced by maternal diabetes over the AGEs, AOPP, NF-κB protein expression, and mRNA expressions of IL-1β and Nox4 in adult offspring rats. Maternal diabetes was induced in female Wistar rats with a single dose of streptozotocin (STZ; 42 mg/kg, i.p.) on the 7th day of pregnancy. Control animals only received STZ vehicle. The offspring were divided into two groups: control non-diabetic (ND) and gestational diabetes (DG). Plasma levels of glucose, total cholesterol, HDL cholesterol and triglycerides were analyzed through biochemical measurements. Plasma levels of AGEs and AOPPs were detected using spectrofluorimetry and spectrophotometry, respectively. Lipid peroxidation was assessed in liver tissue by measuring thiobarbituric acid reactive substances (TBARS). The NF-κB protein expression was evaluated by Western blot. The mRNA levels of IL-1β and Nox4 were detected quantitatively by real time polymerase chain reaction (real-time PCR). In the DG offspring, it was observed that plasma levels of glucose, total cholesterol, HDL cholesterol and triglycerides were higher when compared to the control group. AGEs, AOPP and TBARS concentration were significantly higher in animals exposed to the DG compared with ND. The NF-κB protein expression was higher (p < 0,05) in aorta from DG than in ND. In addition, the expression of IL-1β and Nox4 mRNA was elevated. The number of nephron in DG was significantly reduced (p < 0,001). These results also support the involvement of oxidative stress species in IL-1β induced NF-κB expression. Together, these data suggest that hyperglycemia is a risk factor for vascular alterations.

Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmique. / The NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane.

No description available.

No description available.

Kaposi's sarcoma (KS), caused by the oncogenic Kaposi's sarcoma herpesvirus (KSHV), is an angiogenic tumor characterized by intense angiogenesis, inflammation and proliferation of KSHV-infected spindle cells. We describe the characterization of a mouse model of KS by transfection of a KSHV bacterial artificial chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells which generated a cell (mECK36) that forms KS-like tumors in mice. Our results define mECK36 as a biologically sensitive animal model of KSHV-dependent KS with the following characteristics: (1) the pathological phenotype is a consequence of KSHV gene expression in normal progenitor cells subjected to in vivo growth conditions, (2) the histopathologic phenotype of the tumors resembles KS lesions, and (3) the model is suitable for analysis of vGPCR-driven tumorigenesis in the context of the whole KSHV genome. The mechanism by which vGPCR promotes tumorigenesis is not fully understood. The characterization of a Rac1 transgenic mouse model that produces KS-like lesions that highly resemble human KS has helped us to identify the potential role of Rac1, which is activated by vGPCR, in the pathogenesis of KS. The results from the RacCA transgenic mouse suggest that viral and host genes triggering Rac1 and ROS production may play an important role in KS tumorigenesis. We set out to determine how vGPCR physiologically activates Rac1 in KSHV-infected cells in the KS model mECK36. We found that KSHV oncogenesis in mECK36 is promoted by vGPCR activation of a paracrine oncogenic mechanism through PDGF-BB, which requires a Rac1- and ROS-mediated loop, leading to STAT3 transcriptional activation of c-Myc, VEGF and KSHV latent viral gene expression. We also found that the latency-associated nuclear antigen (LANA) upregulates the PDGFR in vivo, priming latently-infected cells to the PDGF signaling pathway. This oncogenic mechanism can be targeted with the antioxidant N-acetylcysteine (NAC) and FDA-approved PDGF receptor inhibitors to control KSHV-induced tumorigenesis. Our results highlight a ROS-dependent axis whereby Rac1 activating oncogenes and inflammatory signaling drive paracrine stimulation of neoplastic growth and angiogenesis in neighboring cells, defining this axis and its components as attractive anti-tumor targets in KS pathogenesis.