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It is known that African American ethnicity is an independent risk factor for exaggerated oxidative stress which is intricately intertwined with inflammation, hypertension (HT), and cardiovascular disease (CVD). The purpose of this dissertation study was to examine the racial differences that exist between African Americans and Caucasians in oxidative stress levels at the molecular level using an in vitro model of Human Umbilical Vein Endothelial Cells (HUVECs). African American HUVECs were found to have significantly higher baseline levels of oxidative stress in vitro compared to Caucasian HUVECs. In order to establish proof of concept, three preliminary studies were conducted. The first preliminary study, an acute exercise protocol was conducted in young healthy adults in order to measure plasma oxidative stress markers in response to a single moderate intensity treadmill exercise bout. In this study, it was found that the treadmill exercise did not elicit a race-dependent responses, but that African American adults had higher level of oxidative stress at all sample times when compared to the Caucasians. A second preliminary study was conducted using a parallel cell culture design to measure basal oxidative stress levels in African American and Caucasian HUVECs without stimulation. These data were shown in relation to the plasma levels of oxidative stress in resting African American and Caucasian adults. This was done in order to show that the common oxidative stress markers measured in human plasma can also be measured in cell culture supernatant and lysate. It was found that both African American adults and HUVECs had heightened oxidative stress and inflammatory markers when compared to their Caucasian counterparts. The third preliminary study was conducted using tumor Necrosis Factor-#945; (TNF-#945;) as an inflammatory stimulant and measuring the oxidative stress response in both African American and Caucasian HUVECs. This was done in order to show that cells of different race respond differently to stimuli. It was found that the response to TNF-α was blunted in African American HUVECs. The final step was to use laminar shear stress (LSS) as an exercise mimetic in order to examine whether HUVECs from different race respond differently. HUVECs from both race were harvested under static condition (no LSS), with low LSS at 5 dyne/cm2, and with a moderate level of LSS at 20 dyne/cm2. It was found that despite the fact that African American HUVECs had higher levels of oxidative stress under static conditions, when LSS was applied, protein expressions and oxidative stress biomarkers adjusted to levels that were similar to the Caucasian HUVEC adaptations to LSS. From this, it appears that African American HUVECs have a larger response to LSS stimulus indicating that aerobic exercise prescriptions may be valuable for this population since the potential exists for large improvements in oxidative stress levels for this population. / Kinesiology

The parkinsonian inducing drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is bioactivated in a reaction catalyzed by the flavoenzyme monoamine oxidase B (MAO-B) to form the corresponding dihydropyridinium (MPDP+) subsequently pyridinium (MPP+) metabolites. As part of our ongoing studies to characterize the structural features responsible for this unexpected biotransformation, we have synthesized and examined the MAO-B substrate properties of a variety of MPTP analogs bearing various heteroaryl groups at the 4-position of the tetrahydropyridinyl ring. The results of these SAR studies indicate that electronic features, steric features and polar interactions can contribute to the substrate activities. Additionally, isotope effects have been examined to investigate the mechanism and stereoselectivity of the MAO-B catalytic pathway. The synthesis and characterization of regio and stereoselectively deuterated MPTP analogs have been achieved. The results indicate that the catalytic step occurs exclusively at the allylic C-6 position and is rate-determining for both good and poor substrates. The two enantiomers of MPTP bearing a deuterium atom at C-6 have been prepared via chiral aminooxazolinyl derivatives and have been characterized by 2H NMR in a chiral liquid crystal matrix. These enantiomers were used to determine the selectivity of the MAO-B catalyzed a C-H bond cleavage reaction leading to the dihydropyridinium metabolite MPDP+. Some of the cyclopropyl analogs of MPTP have also been synthesized as the potential inhibitors. / Ph. D.

The neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) protects against the neurotoxicity of MPTP in a mouse model of neurodegeneration. Since 7-NI also inhibits the monoamine oxidase-B (MAO-B) catalyzed bioactivation of MPTP, the role of nNOS inhibition as a mediator of 7-NI's neuroprotective properties have been challenged. In order to examine in greater detail the neuroprotective effects of indazolyl derivatives, the synthesis of water soluble indazolyltetrahydropyridinyl derivatives as potential "prodrugs" that may undergo MAO bioactivation in the brain was undertaken. During the course of the studies on the synthesis of indazolylpyridinium derivatives, precursors to these "prodrugs", an interesting reaction involving the rearrangement of 4-(2H-indazolyl)-1-methylpyridinium iodide to the corresponding 1H-isomer was encountered. A detailed investigation of this rearrangement reaction is reported in this thesis. The syntheses and interaction of nitroindazolyltetrahydropyridinyl "prodrugs" with MAO-B have been investigated previously. Molecular docking studies that attempt to explain the MAO-B substrate and inhibitor properties of members of this series of compounds are described. Finally, the MAO-A substrate properties of nitroindazolyltetrahydropyridinyl derivatives are reported. / Ph. D.

The flavin-containing monoamine oxidases (MAO) A and B catalyze the oxidative deamination of primary and secondary amines. The overall process involves a two electron oxidation of the amine to the iminium with concomitantreduction of the flavin. Based on extensive studies with a variety of chemical probes, Silverman and colleagues have proposed a catalytic pathway for the processing of amine substrates and inactivators by MAO-B that is initiated by a single electron transfer (SET) step from the nitrogen lone pair to the oxidized flavin followed by α-proton loss from the resulting amine radical cation that leads to a carbon radical. Subsequent transfer of the second electron leads to the reduced flavin and the iminium product. In the case of N-cyclopropylamines, the initially formed amine radical cation is proposed to undergo rapid ring opening to form a highly reactive primary carbon centered radical that is thought to be responsible for inactivation of the enzyme. In this thesis we have exploited the unique substrate and inactivator properties of 1,4-disubstituted tetrahydropyridine derivatives to probe the mechanism of MAO-B catalysis. Reports of the parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a structurally unique substrate of MAO-B initiated these studies. Consistent with the SET pathway, the N-cyclopropyl analog of MPTP proved to be an efficient time and concentration dependent inactivator but not a substrate of MAO-B. On the other hand, the 4-benzyl-1-cyclopropyl analog is both a substrate and inactivator of MAO-B. These properties may not be consistent with the obligatory formation of a cyclopropylaminyl radicalcation intermediate. In an attempt to gain further insight into the mechanism associated with the MAO catalyzed oxidation of 1,4-disubstituted tetrahydropyridines, deuterium isotope effects studies on both the substrate and inactivation properties of the 4-benzyl-1- cyclopropyl derivative were undertaken. A series of 1-methyl- and 1-cyclopropyltetrahydropyridine derivatives bearing various heteroaro-matic groups at C-4 also have been examined. The MAO-B substrate properties, inactivator properties and partition ratios for these compounds together with preliminary results from chemical model studies are discussed in terms of the MAO-B catalytic pathway. / Ph. D.

Smoking is one of the leading causes of death in the world. About 80% of smokers start smoking before the age of 18. In the Appalachian area and the South in the United States, smoking percentages among adults and adolescents are higher than in other regions. Female smoking shows a variety of different trends from male smoking, and smoking brings particular health problems related to production to female smokers. These findings highlighted the importance of studying female teenage smokers in southwest Virginia. The initial project aimed to identify risk factors that might prevent smoking in an early stage. Dr. Helen Crawford led the Cognitive Neuroscience Lab at Virginia Tech in discovering the psychophysiological vulnerabilities of female teenage smokers. Toward this end, event-related potential (ERP), personality, and behavioral data were collected in teenage female smokers and non-smokers. These data were analyzed to examine possible psychophysiological vulnerabilities in female teenage smokers such as deficits in brain and cognitive function, personality traits, and environment influences. The purpose of this dissertation is to further analyze these data to elaborate and clarify the relationships among these vulnerabilities toward understanding teenage smoking behavior. Participants were 49 teenage girls (smokers and non-smokers) with age from 14 to 18. The measures included sensory gating, platelet MAO-B activity, attention, memory, temperament, schizotypal personality, recognition of facial expressions, taste and smell. The initial set of analyses compared smokers and non-smokers, including those classified as high and low dependent, on all dependent measures. The results suggested some psychophysiological vulnerabilities in female teenage smokers, which have been used as support for the self-medication and the orbito-frontal dysfunction models of why teenagers smoke (Crawford et al., 2004). Further examination of these factors may help teenagers to reduce the smoking dependency and possibly improve cognitive function. Specifically, this dissertation focused on the role of the variable of monoamine oxidase-B (MAO-B) in the correlations among sensory gating, MAO and other cognitive and personality measures. All smokers were divided into high and low MAO groups first. Comparison analyses were conducted between them. The high MAO group showed better sensory gating function than the low MAO group. Correlation analyses were conducted among all of the measures. The significant linear relationships between MAO and sensory gating, MAO and CO level and MAO and temperament were demonstrated. MAO activity positively correlated with the sensory gating function and negatively correlated with CO level and temperament characteristics. Finally, to explore the mechanisms of the relationship between MAO and sensory gating, the neurotransmitter systems related to MAO and sensory gating were discussed. / Ph. D.

Dissertation (MSD) --Boston University, Goldman School of Dental Medicine, 2012 (Department of Oral Biology). / Includes bibliographic references: leaves 71-77. / Lysyl oxidases constitute a family of enzymes responsible for the formation of crosslinks in collagen and elastin. These enzymes have also been linked to pathological fibrosis. The importance of collagen in the structural and mechanical properties of bone led us to investigate the hypothesis that the absence of one or more of these enzymes could lead to a significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate it could facilitate the development of drugs that control its activity in pathological fibrosis. [TRUNCATED]

Yes / Quercetin is reported to reduce blood pressure in hypertensive but not normotensive humans, but the role of endothelial redox signaling in this phenomenon has not been assessed. This study investigated the effects of physiologically obtainable quercetin concentrations in a human primary cell model of endothelial dysfunction in order to elucidate the mechanism of action of its antihypertensive effects. Angiotensin II (100 nM, 8 h) induced dysfunction, characterized by suppressed nitric oxide availability (85 ± 4% p<0.05) and increased superoxide production (136 ± 5 %, p<0.001). These effects were ablated by an NADPH oxidase inhibitor. Quercetin (3 μM, 8 h) prevented angiotensin II induced changes in nitric oxide and superoxide levels, but no effect upon nitric oxide or superoxide in control cells. The NADPH oxidase subunit p47(phox) was increased at the mRNA and protein levels in angiotensin II-treated cells (130 ± 14% of control, p<0.05), which was ablated by quercetin co-treatment. Protein kinase C activity was increased after angiotensin II treatment (136 ± 51%), however this was unaffected by quercetin co-treatment. Physiologically obtainable quercetin concentrations are capable of ameliorating angiotensin II-induced endothelial nitric oxide and superoxide imbalance via protein kinase C-independent restoration of p47(phox) gene and protein expression. / Innovate UK and Boots Pharmaceuticals

The unexpected monoamine oxidase (MAO) substrate properties of 1-cyclopropyl-4-substituted-1 ,2,3,6-tetrahydropyridines have been interpreted in terms of partitioning of these tertiary cyclic allylamines between substrate turnover and ring opening inactivation processes. To evaluate further this proposal, we examined the bioactivities of the related saturated analogs. Several 1,4-disubstituted piperidine derivatives were synthesized and their interactions with MAO-A and MAO-B were characterized. These compounds displayed poor substrate properties toward MAO-A and MAO-B and led to the expected α-carbon oxidized metabolites which were fully characterized. Both the N-methyl and N-cyclopropyl derivatives were good inactivators of MAO-B, suggesting that some species other than the radical resulting from cyclopropyl ring opening is responsible for the inactivation. Both the N-methyl and N-cyclopropyl derivatives also inactivated MAO-A. In this instance, the N-cyclopropyl analogs were much more potent inactivators than the N-methyl analogs. These results suggest that the radical derived from cyclopropyl ring opening may be involved in this inactivation process. The MAO substrate/inactivator properties of these piperidine analogs are discussed in terms of current proposed mechanisms for the MAO catalyzed oxidation of amines. / Master of Science

La bactérie Erwinia amylovora est responsable de la maladie du feu bactérien des Maleae (pommier, poirier…). Le pouvoir pathogène de cette bactérie dépend d'une seringue moléculaire appelé système de sécrétion de type 3 (SSTT). Ce SSTT permet à la bactérie d’injecter des effecteurs dans les cellules de la plante. Parmi les effecteurs injectés, DspA/E est l'effecteur indispensable au pouvoir pathogène d’E. amylovora. Cet effecteur est à lui seul capable de provoquer la mort des cellules chez le pommier et le tabac et permet à la bactérie de se multiplier de manière transitoire chez A. thaliana. L’objectif de ce travail de thèse était de comprendre la fonction de DspA/E dans la cellule végétale et d’identifier des facteurs végétaux impliqués dans la toxicité de DspA/E. Afin de répondre à cette question, des plantes transgéniques exprimant DspA/E sous contrôle d’un promoteur inductible à l’estradiol ont été construites.Dans un premier temps, la caractérisation phénotypique des lignées exprimant DspA/E a été effectuée. Les résultats obtenus montrent que DspA/E est toxique lorsqu'il est exprimé in planta (il provoque la mort des cellules, inhibe la germination, la croissance racinaire et la traduction) et permet la multiplication in planta d’un mutant dspA/E. Un crible de mutants suppresseurs de la toxicité de l'effecteur DspA/E a été effectué sur une lignée transgénique exprimant DspA/E dans le but d'identifier un ou plusieurs gènes impliqués dans la toxicité de DspA/E. Ce crible suppresseur a permis d'identifier un candidat potentiel impliqué dans la photo-respiration, la glycolate oxydase 2 (GOX2). L’analyse fonctionnelle réalisée sur le mutant gox2-2 a permis de montrer que le gène GOX2 est un régulateur positif des réponses de défense d’A. thaliana en réponse à l’infection par E. amylovora.Enfin, la caractérisation du stress oxydant a permis de montrer que plusieurs formes actives de l’oxygène (H2O2 et O2.-) s’accumulent au cours de l’interaction entre A. thaliana et E. amylovora. Ceci a permis également de comprendre le rôle de DspA/E sur ce stress oxydant. Nos résultats suggèrent que la glycolate oxydase 2 participerait à l’induction du stress oxydant en perturbant le métabolisme des sucres. / The bacterium E. amylovora is responsible for the fire blight disease of Maleae (apple, pear…). The pathogenicity of this bacterium relies on a molecular syringe, the type three secretion system (TTSS). This TTSS allows the bacterium to inject effector proteins into the plant cell. Among these effectors, DspA/E is essential for the pathogenicity of E. amylovora. This effector can provoke cell death on apple and tobacco and allows the bacterium to multiply transiently in A. thaliana.The purpose of the thesis was to understand the function of DspA/E in the plant cell and to identify plant factors involved in the toxicity of DspA/E. To answer this question, transgenic plants which express DspA/E under an estradiol-inducible promoter were built.At first, phenotypical characterization of DspA/E transgenic lines was performed. Our results showed that DspA/E is toxic when expressed in planta (it provokes cell death, inhibits germination, root growth and translation) and allows in planta multiplication of dspA/E bacterial mutant. A screening for suppressor mutants of DspA/E toxicity was performed on a DspA/E transgenic line in order to identify one or several genes involved in the toxicity of DspA/E. This screening allowed us to identify a potential candidate involved in photorespiration, the glycolate oxidase 2 (GOX2). Functional analysis performed on the gox2-2 mutant allowed us to show that the GOX2 gene is a positive regulator of A. thaliana responses against E. amylovora.Finally, characterization of oxidative stress allowed us to show that several reactive oxygen species (H2O2 et O2.-) accumulate during A. thaliana and E. amylovora interaction. This allowed us to understand the role of DspA/E in the oxidative stress. Our results suggest that the glycolate oxidase 2 could be involved in the induction of the oxidative stress by disrupting sugar metabolism.