Cardiovascular disease (CVD) such as hypertension and stroke are serious illnesses that impact on the lives of millions all over the world, with 972 million (26% of the world’s population) suffering from hypertension in year 2000, and an estimated 1.56 billion to be affected by 2025. Hypertension, being one of the most common CVD is associated with the development of stroke, peripheral vascular diseases, myocardial infarction, renal failure as well as cardiac failure. Several studies have shown a seasonal correlation for both the systolic and diastolic blood pressure in mankind. A hypertension trial done by the Medical Research Council in the 1980s showed the average blood pressure being lower in summer than winter, and this difference was more significant in the elderly than the younger population. Other than seasonal variation, blood pressure (including hypertension prevalence) is also noted to correlate with latitude, being higher at places further away from the equator. Other cardiovascular related diseases such as stroke and acute coronary syndrome are also shown occur more frequently in winter. The morbidity and mortality of CVD could be due to various factors including diet, culture, race and social status, but within the United Kingdom, all cause mortality (with cerebral-vascular disease being the major one) correlates with latitude even after accounting for all known risk factors, with CVD risks highest in the north. We propose that this difference in cardiovascular mortality is caused by variations in ultraviolet exposure other than temperature. Known mechanisms of sunlight exposure that affect cardiovascular health include temperature and the ultraviolet B (UVB) mediated photolysis of 7-dehydrocholesterol in the skin to produce 1,25 dihydroxycholecalciferol (Vitamin D). UVB is however a potent skin carcinogen, and calculating risk-benefit ratios for exposure will be important. We believe that independently of vitamin D, nitric oxide plays an important role in blood pressure regulation and cardiovascular health, accounting for seasonal and latitude variation. In 1961, Furchgott demonstrated relaxation of rabbit aorta by irradiating them with ultraviolet light, and in later research he noted this effect is most significant in the ultraviolet A (UVA) spectrum. Recently, Mowbray showed a rich store of various nitro-species within human skin and Oplander showed a reduction of blood pressure in human after giving whole body UVA irradiation. We therefore hypothesize that independently of vitamin D, NO mediates the UVA induced beneficial effects on cardiovascular health. To support our hypothesis, in vivo as well as in vitro studies were conducted. We recruited a total of 63 healthy volunteers and monitored blood pressure, forearm blood flow as well as other cardiovascular parameters before and after UVA irradiation. Blood samples were also taken for the measurement of circulatory nitro-species. We have noted a significant reduction of blood pressure (from 84.5±1.76 to 81.33±1.37 mmHg) and increased forearm blood flow (1.95±0.28 to 2.94±0.47 mL/100mL of tissue/min) after UVA irradiation of human skin; simultaneously, we also noted a rise in circulatory nitrite (0.5±0.04 μM before irradiation to 0.72±0.04 μM) and a drop in circulatory nitrate (11.79±0.64 μM before irradiation and 8.99±0.4 μM). For us to further clarify the role of nitric oxide in different latitude, a monochromator machine that generates specific wavelength of light was been used to irradiate aqueous nitrite solution, and the total amount of nitric oxide release at different latitude was then calculated according to the irradiance of various wavelength across the globe. The results of our studies provide evidence suggesting that nitric oxide release induced by UVA irradiation of the skin can account for the difference in cardiovascular mortality and morbidity by latitude. The current public health advice of avoiding sun exposure to reduce the risk of developing skin cancer may need to be modified.

Tumour hypoxia has been demonstrated to cause development of an aggressive tumour phenotype and is associated with increased patient mortality and poorer response to treatments such as chemotherapy and radiotherapy. Previous studies have established that hypoxia exists within a nidus of prostate cancer. Based on the importance of the tumour microenvironment, especially hypoxia, in prostate cancer, the major aims of this thesis were to establish: (a) the role of a novel putative survival factor, dermcidin, in prostate cancer survival under hypoxia/oxidative stress; and (b) the effect of nitric oxide-donating non-steroidal anti-inflammatory drugs (NO-NSAIDS), a new class of drugs, on the killing of prostate cancer cells subjected to hypoxia. A wide-range of confirmatory, cellular and molecular biology techniques were employed in this thesis. The PC-3 hormone-insensitive prostate cancer cell line was used for the majority of studies as this cell line represents hormone-independent prostate cancer, treatment of which is currently palliative. Cell incubation at 0.2% oxygen for 48 hours was established as suitable conditions to stimulate the development of the hypoxia response. Upregulation of nuclear hypoxia-inducible factor-1α protein was the main marker used to assess the hypoxia response. Dermcidin messenger RNA production was found to occur in a range of prostate cancer cell lines; was upregulated in cell lines by both hypoxic and oxidative stress; and found to act as a proliferation, survival and pro-invasion factor under hypoxia and oxidative stress in immortalised prostate cancer cell lines. Furthermore, the portion of the dermcidin molecule responsible for the survival advantage was localised to the proteolysis-inducing factor core peptide subunit. However, subsequent analysis of primary cancer samples from prostate cancer patients revealed that dermcidin was not expressed in these tumours, although dermcidin mRNA was identified in analysis of other primary tumours. As such, the role of dermcidin in prostate cancer was not evaluated further in this thesis. Investigation of NO-sulindac (a NO-NSAID drug) in hypoxic PC-3 cells showed that these agents were significantly more pro-necrotic, pro-apoptotic and anti-invasive than traditional, unnitrated sulindac. NO-sulindac was found to downregulate the hypoxia response mounted by PC-3 cells under hypoxia via the Akt signalling pathway. Finally, analysis of the role of NO-sulindac in radiosensitising hypoxic PC-3 cells showed that NO-sulindac caused significant radiosensitisation under normoxia, but particularly in hypoxic conditions. As such, NO-NSAIDs show great promise as neoadjuvant, concurrent and adjuvant treatments for patients with hypoxic prostate cancer. The findings of this thesis illustrate several potential novel strategies for treatment of hormone-independent prostate cancer.

Approved for public release; distribution is unlimited / The production of nitric oxide by soft solar X-rays in the E-region of the ionosphere is investigated. An empirical expression for the variation in X-ray flux as a function of F10.7 is determined. This expression is incorporated into a one-dimensional diffusive photochemical model to compute nitric oxide densities. No results of these calculation are compared with NO observation from the Solar Mesosphere Explorer satellite. Variations of X-ray flux by a factor of 30 over the solar cycle can explain the observed variation in nitric oxide densities. / http://archive.org/details/investigationofp00duma / Lieutenant, United States Navy

A novel in vitro membrane oxygenator circuit was developed to test gas exchange where particular elements could be examined whilst keeping other variables constant. The circuit comprises two membrane oxygenators connected to form a continuous blood circuit resembling venous and arterial blood conditions. The effects of Isoflurane, a volatile anaesthetic, on oxygen transfer were investigated. RBC resistance to nitric oxide diffusion (DNO) was tested in this circuit by haemolysis and addition of the haemoglobin-based-oxygen-carrier (HBOC) Oxyglobin. The circuit was primed with equine blood flowing at 2.5 l/min. The oxygenator was ventilated with 5 l/min air/oxygen/N2 mix providing a range of FiO2. The deoxygenator received 5 l/min 5% CO2 in N2 with 0.2-0.3 l/min CO2. Isoflurane 1%, NO 4000-16000 ppb and CO 0.03% were added to the oxygenator gas. Uptake of O2, CO2, CO and NO were calculated by gas inlet and outlet concentrations and flow rates. Arterial and venous oxygen dissociation curve (aODC and vODC) comparisons were made. Isoflurane uptake by the circuit blood was evident and 1% Isoflurane did not affect oxygen uptake (p=0.981), aODC or vODC (p=0.311 and p=0.751). Haemolysis did not affect O2 or CO2 transfer but increased DNO (p<0.001). 250ml free Hb solution addition to the circuit increased DNO by 91% (p<0.0001). Addition of 250ml Oxyglobin increased DNO by 143% from 7.41±2.77 to 17.97±1.83 ml/min/mmHg. Oxyglobin caused a right shift of aODC and vODC (p<0.0001) but NO-bound Oxyglobin caused a left vODC shift (p<0.0001). Conclusion: Isoflurane administered via a membrane oxygenator does not affect O2 uptake or carriage in the blood. RBC surroundings provide significant resistance to DNO in circuit tests. Significant uptake of NO by Oxyglobin supports the potential of HBOCs to scavenge endothelial NO in vivo, causing vasoconstriction.

Following pathogen recognition, nitric oxide (NO) is rapidly produced in plants, this small molecule has emerged as a key signal in plant defence responses. S-nitrosylation is the major route of NO signal transduction in plants, a redox-based modification by addition of an NO moiety on cysteine thiol to form an S-nitrosothiol (SNO). S-nitrosoglutathione reductase (GSNOR) regulates cellular levels of S-nitrosylation and displays a key role in regulating the plant defence response. In this context, NO is important to orchestrate both defence gene expression and the hypersensitive response (HR) during attempted microbial infection. However, how the plant immune system recognizes NO and how NO level could elicit plant defence responses are poorly understood. The Arabidopsis thaliana (Arabidopsis) mutant NO overproducing 1 (nox1) was employed to characterize how NO level elicits defence dynamics. In response to microbial infection, resistance (R) gene-mediated defence and basal resistance were found to be compromised in the nox1 mutant relative to wild type Col-0 plants. Interestingly, nox1 mutant exhibit similar levels of HR and pathogen susceptibility to the GSNOR loss-of-function mutant atgsnor1-3. This phenomenon suggests that NO might regulate defence responses via GSNOR-mediated S-nitrosylation. Therefore, the nox1 atgsnor1-3 double mutant was generated and characterized to clarify this hypothesis. Accelerated HR and increased pathogen susceptibility are shown in the double mutant, which implies that increased NO mediated by nox1 and elevated SNOs resulting from atgsnor1-3, are additive with respect to the plant defence response. To identify genes responsible for NO perception, forward genetic screens were developed to identify Arabidopsis mutants with abnormal NO recognition. NO marker genes for genetic screens were identified from both lab and open source microarray data. Two genes, At3g28740 and At1g76600 were selected and experimentally confirmed to be strongly induced by NO. Transgenic Arabidopsis plants were generated carrying a NO reporter cassette, which consist of a luciferase reporter gene (LUC) driven by the promoter of NO marker gene. This forward genetic approach might be a powerful tool to identify genes integral to NO signal transduction. Three C2H2 zinc finger transcription factors (ZnTFs) ZAT7, ZAT8 and ZAT12 were identified as being rapidly and strongly induced by NO donors, which could be modulators of redox/NO-dependent signalling pathway. T-DNA insertion mutants within these ZnTFs have been identified. Basal resistance against Pseudomonas syringae pv tomato (Pst) DC3000 is compromised in all single knockout lines. Therefore, the full characterisation of defence phenotype of these mutants would be necessary to explore the role of these TFs in the plant defence. Furthermore, zat8 mutant is more sensitive to nitrosative stress when compared to wild type Col-0. This suggests that ZAT8 may be involved in protecting plants against nitrosative stress. However, the molecular mechanisms that underpin this function remain to be determined. In conclusion, NO and SNOs might regulate plant disease resistance via distinct pathways. Our work has also established NO-reporter lines to identify genes responsible for NO perception. In addition, three NO-induced ZnTFs have been identified that participate in regulation of basal resistance, which might unveil aspects of NO signalling related to the regulation of transcription.

Name of the thesis Effect of nitric oxide on cerebral blood flow during neuronal activity Aim of the thesis The aim of this thesis is to determine whether the application of 7-nitroindazole, relatively specific inhibitor of neuronal nitric oxide synthase, affects the baseline blood pressure. Furthermore, to determine whether the application of the substance affects the baseline cerebral blood flow and whether it influences blood flow in brain during transcallosal stimulation with increasing frequency. Research method The research took place at the premises of the Institute of Physiology, Academy of Sciences of the Czech Republic. Experiments were carried out on laboratory albino Wistar rats. The group contained both experimental and control sample. General anesthesia was performed to rats, stimulating and sensing electrodes were implanted in epidural area of sensorimotor cortex and Laser Doppler flow probe was implanted into the contralateral hemisphere. A plastic catheter was applied in the carotid artery for measuring systemic blood pressure. In the first part of the experiment, we tested the effects of 7-nitroindazole on the systemic blood pressure. In the second part of the experiment, we investigated the effects of 7-nitroindazole on baseline cerebral blood flow. The third part of the...

Title: The role of nitric oxide in the pathophysiology of neurodegenerative diseases Objectives: The main objective of this thesis is to evaluate the effect of nitric oxide on the formation and development of neurodegenerative diseases. Another objective was to determinate, whether NO affects by its impact processes involved in apoptosis in the CNS. Methods: The thesis is prepared in the form of research, drawing from available relevant resources. Results: Nitric oxide is widely applied in the pathophysiology of selected neurodegenerative diseases, either directly or through other reactive nitrogen and oxygen. It also affects other factors that are involved in apoptosis in the CNS. Keywords: Nitric oxide, NMDA receptors, neurodegenerative diseases, excitotoxicity, apoptosis

Pulmonary diffusing capacity for nitric oxide (DLNO) is a relatively new pulmonary function test to assess gas transfer in the lung. To date, there are no prediction equations made for healthy adult African-American (black) subjects. Thus, the purpose of this study was to create prediction equations for DLNO in this ethnic/racial group. A total of 59 healthy subjects (27 males and 32 females) were recruited to perform pulmonary function testing at Georgia State University. They were diverse in age (18-67 yr), height (140-189 cm), and body mass index (17.2-32.3 kg/m2). All subjects completed single-breath maneuvers at rest inhaling 43 ± 4 ppm NO with a standard diffusion mixture. The breath-hold duration was 5.6 ± 0.6 s. Multiple linear regression predicted DLNO based on the subject’s age, height, and sex. The prediction equation for DLNO (mL/min/mmHg) = 0.92·(height in cm) +38.8·(sex) – 0.012·(age2) – 25, where 1 = male, 0 = female for sex. About 77% of the variance in DLNO was accounted for by sex (67%), age2 (7%), and height (4%). The standard error of the estimate in predicting DLNO was 16.3 mL/min/mmHg. Those with higher resting heart rates had a lower DLNO (r =-0.28, p = 0.03) but it was not included in the regression model as it did not enhance the fit. Black males had a 7-10% lower DLNO and black females had a 12-15% lower DLNO compared to matched white subjects. Black males of the same age and height had a 10% smaller alveolar volume, while black females had a 15% lower alveolar volume compared to matched white subjects. In conclusion, DLNO values and alveolar volumes are reduced in blacks compared to matched whites. The regression model presented best predicts DLNO in African-Americans below 40 years of age.

Master of Science / Department of Kinesiology / David C. Poole / Introduction: Dietary nitrate(NO[subscript]3[superscript]-) supplementation, via its reduction to nitrite (NO [subscript] 2 [superscript]-) and subsequent conversion to nitric oxide (NO) and other reactive nitrogen intermediates, reduces blood pressure and the O[subscript]2 cost of submaximal exercise in humans. Despite these observations, the effects of dietary NO [subscript]3 [superscript]- supplementation on skeletal muscle vascular control during locomotory exercise remain unknown. We tested the hypotheses that dietary NO [subscript]3 [superscript]- supplementation via beetroot juice (BR) would reduce mean arterial pressure (MAP) and increase hindlimb muscle blood flow in the exercising rat. Methods: Male Sprague-Dawley rats (3-6 months) were administered either NO [subscript]3 [superscript]- (via beetroot juice; 1 mmol · kg[superscript]-[superscript]1 · day[superscript]-[superscript]1, BR n=8) or untreated (control, n=11) tap water for 5 days. MAP and hindlimb skeletal muscle blood flow and vascular conductance (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m · min[superscript]-[superscript]1, 5% grade). Results: BR resulted in significantly lower exercising MAP (control: 137 ± 3, BR: 127 ± 4 mmHg, P<0.05) and blood [lactate] (control: 2.6 ± 0.3, BR: 1.9 ± 0.2 mM, P<0.05) compared to control. Total exercising hindlimb skeletal muscle blood flow (control: 108 ± 8, BR: 150 ± 11 ml · min[superscript]-[superscript]1 · 100 g[superscript]-[superscript]1, P<0.05) and vascular conductance (control: 0.78 ± 0.05, BR: 1.16 ± 0.10 ml · min[superscript]-[superscript]1 · 100 g[superscript]-[superscript]1 · mmHg[superscript]-[superscript]1, P<0.05) were greater in rats that received beetroot juice compared to control. The relative differences in blood flow and vascular conductance for the 28 individual hindlimb muscles and muscle parts correlated positively with their percent type IIb + d/x muscle fibers (blood flow: r=0.74, vascular conductance: r=0.71, P<0.01 for both). Conclusion: These data support the hypothesis that NO [subscript]3 [superscript]- supplementation improves vascular control and elevates skeletal muscle O [subscript]2 delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO [subscript]3 [superscript]- supplementation improves metabolic control.

Doctor of Philosophy / Department of Anatomy and Physiology / Timothy I. Musch / Nitric oxide (NO) is synthesized via distinct NO synthase (NOS) enzymes and constitutes an essential cardiovascular signaling molecule. Whereas important vasomotor contributions of endothelial NOS (eNOS) have been well-described, the specific vasomotor contributions of nNOS-derived NO in healthy subjects during exercise are unknown. The purpose of this dissertation is to test the global hypothesis that nNOS-derived NO is a critical regulator of exercising skeletal muscle vascular control. Specifically, we utilized the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) to investigate the effects of nNOS-derived NO on skeletal muscle vascular function within established rodent models of exercise performance. The first investigation (Chapter 2) identifies that nNOS inhibition with SMTC increases mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle blood flow at rest whereas there are no effects during low-speed (20 m/min) treadmill running. In Chapter 3 it is reported that nNOS inhibition with SMTC reduces blood flow during high-speed treadmill running (>50 m/min) with the greatest relative effects found in highly glycolytic fast-twitch muscles and muscle parts. Chapter 4 demonstrates that nNOS-derived NO modulates contracting skeletal muscle blood flow (increases), O2 consumption (VO2, increases), and force production (decreases) in the rat spinotrapezius muscle and thus impacts the microvascular O2 delivery-VO2 ratio (which sets the microvascular partial pressure of O2, PO2mv, and represents the pressure head that drives capillary-myocyte O2 diffusion). In Chapter 5 we report that systemic administration of the selective nNOS inhibitor SMTC does not impact lumbar sympathetic nerve discharge. This reveals that the SMTC-induced peripheral vascular effects described herein reflect peripheral nNOS-derived NO signaling as opposed to centrally-derived regulation. In conclusion, nNOS-derived NO exerts exercise-intensity and muscle fiber-type selective peripheral vascular effects during whole-body locomotor exercise. In addition, nNOS-derived NO modulates skeletal muscle contractile and metabolic function and, therefore, impacts the skeletal muscle PO2mv. These data identify novel integrated roles for nNOS-derived NO within healthy skeletal muscle and have important implications for populations associated with reduced NO bioavailability and/or impaired nNOS structure and/or function specifically (e.g., muscular dystrophy, chronic heart failure, advanced age, etc.).