published_or_final_version / abstract / toc / Physiology / Master / Master of Philosophy

Nitric oxide (NO) has become a molecule of interest in biological research. NO is generated via the oxidation of L-arginine, by NO synthase (NOS), and plays a key role in many bioregulatory systems, including smooth muscle relaxation, platelet inhibition, neurotransmission, and immune stimulation, primarily through the formation of cGMP. N-Diazeniumdiolates (NONOates) are an interesting class of compound that can deliver NO specifically to a target site, with potential biological or therapeutic value and minimal side effects. The versatility of NONOates makes them ideal for studying NO in many different scenarios. Primary amine diazeniumdiolates such as isopropyl amine (IPA/NO) can release HNO under physiological conditions.\\Quantitative Structure Activity/Property Relationships (QSAR/QSPR) relate the structure of a compound, to a property/activity of interest ( biological activity). QSAR/QSPR studies are of great importance in drug design. Model that predict the half-lives of NONOates was built and were studied the influence of each variable on decomposition rate. External validation of this model will be made using new set of NONOates to test the Model.

Nitric Oxide (NO) has been detected in the expiratory air of normal animals and human subjects. Recent experiments revealed that expiratory NO production rises during exercise and correlates well with O$ sb2$ consumption and heart rate. Whether or not physical conditioning influences expiratory NO output production remains unclear. In this study, NO concentration in expired gas was measured in 18 healthy male volunteers subdivided into 3 groups (sedentary, intermediate, athletes) based on their state of physical conditioning. Measurements were taken at rest and during two steady-state exercise bouts on a bicycle ergometer designed to elicit VO$ sb2$ of 1 and 2 1/min with the athletes performing an additional bout at VO$ sb2$ of 4 1/min. In the sedentary and intermediate groups, expired NO concentrations declined significantly with increasing VO$ sb2.$ In contrast, expired NO levels declined only slightly with increasing VO$ sb2$ in athletes. At a VO$ sb2$ of 2 1/min, expired NO concentrations were significantly higher in athletes compared with the other groups. When correlated with V$ rm sb{E},$ expired NO concentrations declined linearly with the increase in $ rm V sb{E}$ in sedentary and intermediate groups but not in the athletes. Only the athletes had a significant linear increase in NO output (expired NO x V$ rm sb{E})$ with increasing VO$ sb2$ (p $<$ 0.001). These results support the notion that physical conditioning increases expiratory NO output during exercise. We speculate that the rise in expiratory NO output in athletes might be due to increased vascular and/or epithelial production of NO. Enhanced vascular NO production may be the result of increased shear stress and/or upregulation of endothelial NO synthase gene expression.

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Field and laboratory studies were performed to estimate fluxes of the a-ace gases nitrogen monoxide (NO), nitrous oxide (N2O), and methane (CH4) in an organic soil, to determine the microbial processes involved, and to assess how soil water and nitrogen controlled flux rates. Metabolic inhibitors showed microbial nitrification to be the major NO source, regardless of the soil moisture content. Nitrification also produced N 2O, but denitrification losses of this gas from flooded, anaerobic soil were much higher. Up to 26% of nitrified N was converted to NO, but most of this (95%) was consumed microbially before it could escape across the soil surface. The NO-consuming process appeared to be co-oxidation by soil heterotrophs, not coupled to energy production. Organic matter content and CO2 production were therefore good predictors of NO oxidation rates across soil types, and NO oxidation was stimulated by manure addition. / Soil water and nitrogen had nonlinear effects on trace gas fluxes, acting on both production and consumption. Kinetic analysis showed that nitrate was a weak noncompetitive inhibitor, but ammonium a strong competitive inhibitor of soil CH4 oxidation at field fertilization rates. However, spatial and temporal factors complicated fertilization effects on CH4 oxidation in situ. Ammonium was immobilized in surface soil and rapidly nitrified, limiting its inhibitory effect on CH 4 oxidation. Fertilizer N stimulated nitrification and denitrification and therefore gaseous N-oxide production, but other, unexpected fertilizer effects were also observed. Ammonium fertilizer decreased NO oxidation rates. Nitrate and other salts stimulated NO and N2O losses during nitrification, an effect apparently related to soil nitrite accumulation. / The controls exerted on trace gas fluxes by soil water were mediated primarily through diffusion rates. Oxygen diffusion controlled the balance of anaerobic (methanogenesis and denitrification) versus aerobic (CH 4 oxidation and nitrification) processes. Soil moisture content also controlled the diffusion rate of atmospheric CH4 to soil methanotrophs, and the escape of gaseous N-oxides from production sites across the soil surface.

Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

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In vitro analysis of osteoblasts deficient in endothelial NO synthase (eNOS) or inducible NOS (iNOS) enzymes, indicated that NO produced via eNOS is required for normal osteoblast growth and differentiation. The transcriptional regulation of two genes, the extracellular matrix protein Tenascin-Cytotactin (Tn-C) and eNOS, which contain strain-related regulatory sites and are known to respond to mechanical stimuli, was investigated in osteoblasts and osteocytes (ob-mix). Stretch was applied via 2 different systems and the transcription level of the gene promoters assayed by dual luciferase reporter assay. It was demonstrated that transcription of Tn-C and eNOS is upregulated in response to physiological levels of tensile and/or shear stress. This signal is mediated via the putative StRE and SSRE in Tn-C promoter and at the proximal end of the eNOS promoter is indicated. A system was built to study calcium (Ca²⁺) fluxes in response to shear stress in primary ob-mix and an immortalised simian virus human foetal osteoblast cell-line (SV-HFO). Studies have shown that pulsating fluid flow (PFF) stimulates a rapid [Ca²⁺]_i increase in primary ob-mix. Rapid transient increases in [Ca²⁺]_i were also recorded in a synchronised culture of SV-HFO when subjected to PFF. Both these observations are consistent with the hypothesis that Ca²⁺ increases contribute to the osteoblastic NO response to shear stress. Taken together, these results confirm the important role NO plays in bone physiology and elucidate aspects of transcriptional regulation and activation of the eNOS enzyme.