Introduction and aim: Increases in the bioavailability of nitric oxide have been shown to reduce the oxygen (O2) cost of exercise, improve exercise performance, alter gastric blood flow and mediate glucose uptake in healthy individuals. Aim: Does dietary nitrate reduce the O2 cost of exercise, improve walking performance in individuals with type 2 diabetes mellitus (T2DM) and chronic obstructive pulmonary disease (COPD) or alter hepatic diffusion and positively affect glucose homeostasis in healthy adults? Methods: Experimental chapters utilised a double-blind, placebo-controlled, randomised, experimental design. Breath by breath pulmonary gas analysis was utilised to assess the O2 cost of exercise in 48 individuals with T2DM and 13 with COPD. Walking performance was assessed via the six minute walk test (6MWT) in cohorts 1 and 2. Magnetic resonance imaging was used to assess portal vein flux, velocity and the apparent diffusion coefficient, in order to assess hepatic microvascular diffusion (apparent diffusion coefficient (ADC)). Blood pressure (BP) was measured in all trials. Results: Relative to placebo, beetroot juice resulted in a significant increase in plasma nitrate and nitrite. There were no differences between placebo vs. beetroot juice for the O2 cost of walking (T2DM: placebo; 946 ± 221 vs. beetroot juice; 939 ± 223 ml.min-1; P=0.59) or cycling (COPD: placebo; 933 ± 323 vs. beetroot juice; 939 ± 302 ml: min-1; P=0.88), distance covered in the 6MWT (T2DM: placebo; 550 ± 83 vs. beetroot juice; 554 ± 90m; P=0.17 or COPD: placebo; 456 ± 86 vs. beetroot juice; 449 ± 79 m; P=0.37) or BP (T2DM: systolic: placebo; 134 ± 10 vs. beetroot juice; 132 ± 12 mmHg, P=0.17; diastolic: placebo; 77 ± 7: vs. beetroot juice; 76 ± 11 mmHg, P=0.27. COPD: systolic: placebo; 123 ± 14 vs. beetroot juice; 123 ± 14 mmHg; P=0.91; diastolic: placebo; 77 ± 9 vs. beetroot juice; 79 ± 9 mmHg; P=0.27). No differences were seen between placebo and beetroot juice for ADC (young adults: F(3, 45) = 0.25, P = 0.74; older adults; F(3, 42) = 1.3, P = 0.28), portal vein flux (young adults: F(3, 45) = 0.339, P = 0.79; older adults; F(3, 42) = 1.65, P = 0.19) however, there was an interaction effect in the young adults: (F(3, 45) = 2.9, P = 0.04) but not in the older adults; F(3, 42) = 1.8, P = 0.16) between visits for portal vein velocity. Nitrate supplementation did not reduce plasma glucose concentrations (young adults: F(3, 45) = 0.96, P = 0.42; older adults; F(3, 42) = 0.04, P = 0.99). Nitrate supplementation did not reduce systolic blood pressure (young adults: F(3, 45) = 0.20, P = 0.89; older adults; F(3, 42) = 1.7, P = 0.18) or diastolic blood pressure (young adults: F(3, 45) = 0.25, P = 0.86; older adults; F(3, 42) = 0.45, P = 0.72). Conclusion Dietary nitrate supplementation does not alter the O2 cost of exercise, improve walking performance or reduce BP in individuals with T2DM or COPD. Nitrate supplementation does not alter hepatic diffusion, glucose homeostasis or BP.

The need to inject treated seawater to enhance reservoir pressure and secondary oil recovery is increasing in the oil field, so also is the reservoir souring potential caused by the activities of Sulphate Reducing Bacteria (SRB) generating H2S in the reservoir. The total cost of SRB mediated corrosion in the United States alone is estimated to be 1-2 billion US dollars per year. In the last few years, a number of potential souring mitigation and prevention tools have been studied. These include: sulphate-reduction using membranes, biocide injection and nitrate injection. Out of all the various methods used for the mitigation and prevention of reservoir souring, the use of nitrate injection in conjunction with waterflood projects is becoming more popular because of its economic benefits and least environmental impact. However, nitrate injection is still widely considered as an emergent technology because there are still many unknowns. One of the major unknowns, of great concern is the susceptibility of subsea hardware components to nitrite, which is a by-product of nitrate anti-souring treatment. Any detrimental effect can compromise the technical integrity of subsea installations. The objective of this research is to study the corrosion susceptibility of CRA (13Cr- Martensitic, 22Cr, and 25Cr super duplex stainless steel) to pitting and stress corrosion cracking in the presence of nitrite. Research hitherto, has investigated corrosion susceptibility of carbon steel to nitrite and found out that nitrite causes pitting in carbon steel. This research work built on previous studies and extensively investigated the effect of nitrite on CRA materials in terms of pitting and stress corrosion cracking. Using electrochemistry techniques in conjunction with C-ring test and slow strain rate test, with variables such as temperature, and nitrite concentration all under anaerobic conditions. Metallographic examination and further evaluation using scanning electron microscopy confirmed pitting and intergranular stress corrosion cracking of 13Cr-L80 and 25Cr due to presence of nitrite.Test data confirmed that sodium nitrite is an anodic inhibitor; it shifts the corrosion potentials to more noble potential and also shifts the anodic curve to lower current, given a net reduction in corrosion rate. A critical concentration of 400ppm is required for inhibition to be effective on 13Cr-L80 and 25Cr. However, below the critical concentration, nitrite significantly increases the corrosion rate. The experimental data generated from this research work provides very valuable information that will tremendously assist the materials selection process for subsea and subsurface hardware components and also serve as a guide in the corrosion management process in existing systems.

Membrane proteins play an integral role in the control of ion transport across the cell membrane in biological systems. However, due to experimental constraints, structural and functional data available for these proteins is limited, especially considering their importance. In this study, two membrane proteins which transport nitrate and nitrate into the model filamentous ascomycete Aspergillus nidulans were investigated. Work on the twelve trans-membrane domain nitrate transport protein NrtA is well established. As a member of the major facilitator super family (MFS) the role of signature sequences characteristic of this family have previously been studied. Here, a series of point mutations were made to facilitate an understanding of key residues in the nitrate binding domain, the first nitrate signature motif and residues of the unique fungal central-loop domain. Using an expanded alignment package, the proposed secondary structure of NrtA was enhanced and used as a starting point for mutagenesis. Alanine scanning mutagenesis showed that glycine residues in the conserved nitrate nitrite porter (NNP) motif were critical for NrtA function. Two asparagines in the NNP were investigated; N160 and N168. N168 was found to be critical for NrtA function as all mutants were devoid of growth on nitrate solid agar medium though they expressed in the membrane to varying degrees. The nitrate binding site has been studied previously, revealing the interaction of conserved arginine residues with the anion as it traverses the bilayer. Though it was thought that mutations of residue T83 to a small, charge neutral, amino acid would substitute for no alteration to enzyme kinetics in mutant T83S was found when using ¹³NO₃⁻. Another major part of this thesis examined NitA which is part of a distinct nitrite transport family to NrtA (the Formate Nitrite Transporters, FNT). A mutagenesis approach targeted NitA residues conserved amongst homologous proteins. Residues in position D88 in an alignment of homologues were conserved in terms of charge. Mutagenesis of D88 revealed that maintaining charge at this position was essential for NitA function, likely due to a role in salt-bridge formation during conformational changes. Mutations to asparagine, glutamine, serine and valine showed reduced growth on agar though the protein was expressed to approximately wild-type levels. Nitrite uptake assays using a ¹³NO₂⁻ tracer were performed on D88N, D88E and D88Q and all showed wild-type Km and Vmax. Finally, the role of conserved asparagine residues found throughout NitA was investigated by mutagenesis. Expression studies revealed that mutants created in N122 and N246, changed to aspartic acid, lysine, glutamine and serine were generally not present in the membrane and thus did not grow on nitrite agar. However, mutations in N173 (in Tm 4) and N214 (in Tm 5), which are conserved in > 95 % of NitA homologues, showed varying degrees of growth and expression. Both of these residues are located in FNT signature motifs, so it is likely that they are involved with conformational changes or protein dynamics.

Abstract Aim: The purpose of the study is to show the influence of Proton Pump Inhibitors (PPI in form of esomeprazole) on the bioactivation of dietary nitrate (sodium-nitrate solution) in submaximal exercise, through affecting the gastric pH. Method: Randomized, doubled-blinded, placebo-controlled and crossover study with six subjects (mean ± SD, age 29 ± 5years, height 170 ± 5 centimeters, weight 70 ± 5 Kg, BMI 24,36 ± 1,75 Kg/m2 blood pressure 119/ 77 ± 6 mmHg, 3 male and 3 female). They were tested in two different trials. Every trial consisted of two parts. One part was cycling on 4 different submaximal stages (80W/60RPM, 80W/90RPM, 120W/60RPM, 120W/90RPM) for 5 minutes each, with 90 minutes rest in between. The same protocol was repeated. In the beginning of the resting time a sodium nitrate solution (NaNO3-, 10mg/kg body weight) was ingested. VO2, VCO2, RER, VE, Lactate, Glucose, heart rate and blood pressure were recorded. Venous blood samples were taken. Whether esomeprazole (10mg) or a placebo were taken 24h, 12h and directly before being tested in both trials. Subjects were pleased to have a nitrate poor diet starting when taking the pills. An information sheet was provided. Results: No significant differences were found between the post values and the treatment. Tendencies of a higher oxygen consumption when taking esomeprazole (2,62%) in comparison to placebo (0,11%) were observed. Systolic BP decreased by 3,91% with the placebo while it decreased just 2,04% with esomeprazole after intake. Sex-specific differences occurred in the metabolism of esomeprazole and dietary nitrate. RER showed a significant post nitrate difference between the female and male participants with t=.006 and a significance in predietary nitrate intake. VE in female (40,79 ± 7,20 L/min) and (50,03 ± 10,09 L/min) in male were as well significant (t=.017). Conclusion: Tendencies of effects of PPI are seen in the post-values of VO2 and BP after intake of dietary nitrate. Gender-differences are shown in RER and VE. More research is needed to see the impact of dietary nitrate on the human body under submaximal load.

Made available in DSpace on 2016-12-08T16:24:10Z (GMT). No. of bitstreams: 1 PGCA10MA084.pdf: 10543370 bytes, checksum: c4278e36095798e986451ebb91edcf8f (MD5) Previous issue date: 2010-11-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Epidemiological data, clinical and pathological findings of the spontaneous and experimental poisoning by nitrate and nitrite in cattle fed by oats (Avena sativa) and ryegrass (Lolium spp), diphenylamine test, and the nitrate content in the samples of the pasture where the outbreaks occurred are described. The disease occurs in different regions of the state of Santa Catarina, in which pastures have exuberant growth, after receiving excessive amounts of chemical and/or organic fertilizer, mainly when raining occurs after a period of dry wheather. The animals grazing these pastures quickly developed brown mucosa, tachypnea, staggering gait, frequent urination, bloating, lateral recumbency and death in few minutes or hours. At necropsy of four animals that died spontaneously, the main lesions found were brown mucosa, dark color of the blood (chocolate), intense red color of the skeletal muscles and of the left part of the myocardium. The experimental reproduction of the disease was performed in seven cattles, with pastures of four farms where the disease occurred. The animals were fed with fresh and hay oats and ryegrass. Four animals died, two became ill and recovered, and one was treated with 2 mg / kg per body weight methylene blue 1%, and one cattle did not show changes. Clinical signs and lesions of the sickened and died animals were similar to natural cases. Microscopic changes were not observed in spontaneous and experimental poisoning. The diphenylamine test was positive in all the farms where the outbreaks occurred. The chemical analysis performed on samples of pasture from several farms in which outbreaks of the disease occured ranged from 0.30% to 3.36% of nitrate in the dry matter. The disease is associated with the ingestion of oats and ryegrass pastures heavily fertilizer, which accumulates high levels of nitrate after a period of rain and is characterized by rapid breathing, dark-colored blood, brown mucous and rapid death / Descrevem-se os dados epidemiológicos, sinais clínicos e lesões da intoxicação espontânea e experimental por nitrato e nitrito em bovinos que pastoreiam em pastagens de aveia (Avena sativa) e azevém (Lolium spp), o teste da Difenilamina e os teores de nitrato nas amostras de pastagens onde ocorreram os surtos. A enfermidade ocorre em diferentes regiões do estado de Santa Catarina, quando as pastagens tem crescimento exuberante, após receberem quantidades excessivas de adubo químico e/ou orgânico, principalmente quando ocorrem condições climáticas de seca e posteriormente chuva. Os animais em contato com essas pastagens desenvolveram rapidamente mucosas de coloração marrom, taquipnéia, andar cambaleante, micção frequente, timpanismo, decúbito lateral e morte em poucos minutos, ou recuperação algumas horas após. Na necropsia de quatro animais que adoeceram espontaneamente, as principais lesões encontradas foram a coloração marrom das mucosas, a cor escura do sangue (cor de chocolate) e a coloração vermelho intensa da musculatura esquelética e do miocárdio esquerdo. A reprodução experimental da doença foi realizada em sete bovinos, com pastagens de quatro propriedades onde ocorreu a doença. Aveia e azevém verdes e sob a forma de feno foram administradas aos bovinos, destes quatro morreram, dois adoeceram e recuperam-se, um naturalmente e outro com a aplicação de azul de metileno a 1%, na dose de 2 mg/kg/peso vivo, e um bovino não apresentou alterações. Os sinais clínicos observados e lesões dos animais que adoeceram e morreram foram idênticos aos casos naturais. Alterações microscópicas não foram observadas nos casos naturais e experimentais. O teste da difenilamina resultou positivo em todas as propriedades em que ocorreram os surtos. A análise bromatológica realizada em amostras coletadas de várias propriedades em que ocorreram surtos da enfermidade variaram de 0,30% a 3,36% de nitrato na matéria seca. A enfermidade caracterizou-se principalmente por respiração ofegante, sangue de coloração escura, mucosas de coloração marrom e morte rápida de bovinos e está relacionada á ingestão de pastagens de aveia e azevém superadubadas, que acumularam alto teor de nitrato, após um período de chuvas precedido de seca