<p>Nitric oxide (NO) is an important chemical factor that controls vascular tone in the cardiovascular system. NO is a vasodilatory molecule that plays a role in blood pressure and blood flow regulation as well as vessel formation and tissue cell proliferation. NO influences the flow by which nutrients and other metabolites required for growth are transported to the tissues. The aim of this study was to investigate if NO, through mediation by the NO donors Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) affect growth and oxygen consumption of prenatal broiler chicken. The results indicate that, although the treatments did not have clear significant effects on the embryos or the organs examined, a slight delay in development can be observed in the GSNO treatment embryos. The study could not conclude, however, if this was due to effects of NO donors</p>

<p>The primary aim of this thesis was to modify saphenous vein (SV) harvesting technique and evaluate its clinical importance. A new "no touch" (NT) technique of SV preparation was developed where the vein is harvested with a pedicle of surrounding tissue, which protects the vein from spasm therefore obviating the need for distension.</p><p>Firstly, a prospective randomised study in 156 patients who underwent coronary artery bypass grafting was done to compare this new "no touch" technique to two others, the conventional (C) and the intermediate (I) techniques. A morphological study of the endothelium showed an endothelial integrity of 97% in NT vessels while about half of endothelial surface of veins harvested by the other two techniques was devoid of endothelium. At angiographic follow up, the patency for NT was 95.4%, 88.9% for grafts in group C and 86.2% for grafts in group I. A statistically significant difference in patency rate was found between the NT group and group C (p=0.025) and the poorest result was observed in group I.</p><p>Secondly, the immunohistochemistry assessment using CD31-antibody confirmed a better-preserved endothelium for NT vessels. Putative NOS was identified by NADPH-diaphorase histochemistry and autoradiographic localization of [3H] L-nitroarginine (NOARG) binding. NADPH staining was almost continuous on the luminal aspect and was also present in the intact adventitia of NT vessels, which was markedly reduced in conventionally harvested veins. Autoradiographic analysis of specific NOARG binding showed greater binding in the no-touch vessels, confirming the histochemistry results. All three NOS isoforms were identified in the media of SV grafts. In NT, NOS I was abundant in adventitial nerves; NOS II was found in adventitial vasa vasorum and NOS III was associated with endothelial cells lining both the vessel lumen and microvessels within the adventitia.</p><p>In conclusion, this study demonstrated that the endothelial integrity and NOS activity are better maintained when using the no-touch technique for vein graft harvesting. The vasorelaxant and thromboresistent activities of NO may be responsible for the reduced venospasm and improved early patency rates observed. Furthermore, the mechanical properties provided by the cushion of surrounding tissue in grafts harvested by NT technique may contribute to the observed high patency rate.</p>

<p>Nitric oxide (NO) is a mediator of vascular smooth muscle tension that metabolises rapidly in blood. NO delivered by inhalation can therefore be used as a selective pulmonary vasodilator to relieve pulmonary hypertension or to improve oxygenation with no systemic effects. In artificial ventilation nitric oxide has been administered in inspiration gas as a continuous gas flow or to form constant inspired concentration. Homogeneous inspired gas mixture has been regarded essential for successful therapy and the therapy has been characterized by the mixture NO concentration. The response in oxygenation on NO therapy has, however, been variable. Administration of NO as a short pulse synchronously with inspiration has been suggested to improve the response. In this study the NO administration was examined theoretically and experimentally with the aim to relieve pulmonary hypertension and improve oxygenation during artificial ventilation. For the experimental study a system for the synchronized administration was developed.</p><p>The effect on oxygenation was studied during equine anaesthesia where hypoxemia develops regularly secondary to left-to-right shunt caused by atelectasis. By administering the NO as a short pulse in early inspiration to well ventilated lung areas the oxygenation could be effectively improved. Delayed administration to low ventilated lung areas was found possible for a negative contribution on oxygenation, which reduces the improvement gained in the well-ventilated lung areas. When NO is delivered into the whole inspiration, the net effect on oxygenation is the sum of these negative and positive contributions, whereas with pulsed delivery to the early inspiration the negative contribution can be avoided. This finding may be the main explanation for the varying response in oxygenation detected in patients as a response to NO inhalation.</p><p>When the NO therapy aimed for the relief of induced pulmonary hypertension in pigs, no difference was observed between NO delivery as a short pulse or given to the whole inspiration. Maximum vasodilatation was observed with 105 nmol/min delivery rate. A larger delivery rate only contributed to an abrupt increase in pulmonary pressure at cessation of the delivery.</p><p>The NO uptake from alveoli to tissue depends on the alveolar NO partial pressure. In a simulation this partial pressure was shown to be independent of the administration mode. Also the relationship between the NO uptake and delivery setting was not explicit. With pulsed delivery, expired NO can be reduced which was confirmed by the experimental results. This is important when the NO therapy is given in rebreathing circuit.</p>

<p>We have developed a methacholine provocation method, which detects bronchial responsiveness in more than 80% of healthy subjects. The method enables us to detect differences in bronchial responsiveness within the normal range. </p><p>With this method FEV1 and Gaw had similar sensitivity in detecting small differences in bronchial responsiveness. Differences, between protocols when using doubling or fourfold concentration steps emphasize the importance to strictly adhere to a predefined protocol. </p><p>Deep inhalation associated with the FEV1 manoeuvre decreases bronchial tone induced by methacholine for up to 6 minutes, which emphasizes the importance of exact timing between successive FEV1 measurements in bronchial provocation tests. There is a substantial overlap in bronchial responsiveness between healthy and asthmatic subjects and a deep inhalation at the end of the methacholine test challenge could not discriminate between asthmatic and non-asthmatic subjects.</p><p>Inhalation of dust in a swine confinement building causes an intense airway inflammatory reaction with an extensive migration of inflammatory cells, predominantly neutrophils, into the upper and lower airways. Bronchial responsiveness to methacholine increased by about 3 doubling concentration steps and was normalized one week after exposure. However, exposure to dust in a swine confinement building did not yield increased bronchial responsiveness to eucapnic hyperventilation with dry air which is often observed in asthmatic subjects. Exhaled NO was approximately doubled five hours after exposure and in the present study we found no relationship between exhaled NO levels and bronchial responsiveness in healthy subjects. </p><p>Protection with half-mask inhibited the dust induced increase of exhaled NO whereas the increase in bronchial responsiveness was influenced only to a minor extent.</p><p>These findings, do not support the hypothesis that the increased bronchial responsiveness following organic dust exposure is directly caused by the inflammation. Instead, a possible direct effect on the smooth muscle and swelling of the airway mucosa and increased secretions due to the general inflammatory reaction probably leads to airway narrowing enhancing the post-exposure bronchial response to methacholine. </p>

<p>The development of diabetes mellitus depends on the balance between beta-cell proliferation and death. As mitogen-activated protein kinases (MAPK) may control this balance, the aim of this study was to investigate the events leading to MAPK activation in beta-cells and the consequences of these events. Overexpression of the SH2-domain containing adaptor protein Shb resulted in the assembly and activation of multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpression cells. This was paralleled by an attenuated activation of the MAP kinases ERK1/2. Thus, Shb-induced alterations in the IRS-1/PI3K/Akt/ERK pathway might explain the increased proliferation and apoptosis of beta-cells overexpressing Shb.</p><p>The importance of the MAP kinase p38 in nitric oxide- and cytokine-induced beta-cell death was also investigated. Knock-down of p38 expression resulted in a lowered cell death rate in response to a nitric oxide donor. In transient transfections MKK3 over-expression resulted in increased p38 phosphorylation in RIN-5AH cells. In addition, a short-term MKK3 expression resulted in increased cytokine-induced cell death. A nitric oxide synthase inhibitor abolished the MKK3-potentiating effect on cytokine-induced cell death and inhibitors of phosphatases enhanced MKK3-stimulated p38 phosphorylation. Finally, as the dominant negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide.</p><p>In further support for an MKK3/6-indepedent mechanism, the adaptor protein TAB1 significantly increased the cytokine- and nitric oxide-stimulated phosphorylation of p38. The TAB1-mediated activation of p38 was paralleled by a compensatory inhibition of ERK and JNK. In summary, p38 MAPK, activated mainly by TAB1, promotes, at least in part, beta-cell death in response to cytokines or nitric oxide.</p>

<p>Renal arterioles represent the most important effecter site in the control of renal perfusion and filtration. Adenosine (Ado), angiotensin II (Ang II) and nitric oxide (NO) interact in modulating arteriolar tone. The present work investigates the mechanism of this interaction. We tested the hypothesis that AT₁ receptor (AT₁AR) mediated NO release in isolated perfused afferent arterioles. Further, special attention was given to mechanisms of Ado-Ang II -interactions.</p><p>We found (I) that Ang II specifically induces NO release via AT₁AR in arterioles. The effect is important in view of high renin and Ang II concentrations in these vessels. (II) Ado modulates the Ang II response by acting on vasoconstrictor A₁AR and vasodilator A₂AR. Vice versa, Ang II critically enhances the constriction to Ado, which supports the assumption of its modulating action in the tubuloglomerular feedback (TGF). (III) The synergistic effect of Ang II and Ado on arteriolar contraction is concurrent with an increase in the cytosolic calcium. Further, (IV) Ado increases the calcium sensitivity of the contractile machinery in arteriolar smooth muscle cells most probably by enhancement of the phosphorylation of the myosin light chain regulatory unit. RhoA kinase, protein kinase C and p38 MAP are involved in the Ado effect, which is not receptor mediated and depends on the Ado uptake into vascular cells. Remarkably, the enhancing action of Ado is most likely limited to Ang II; since Ado does not influence endothelin-1 and norepinephrine induced contractions.</p><p>These novel results extend our knowledge about the synergistic action of Ang II and Ado in the control of renal filtration. Ado, the key factor in mediation of the TGF, develops a significant vasoconstrictor action only in the presence of Ang II. On the other hand, the Ang II induced vasoconstriction is modulated by Ado via receptor and non-receptor mediated intracellular signaling pathways.</p>

<p>Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). </p><p>In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function <i>in vitro</i>, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, <i>in vivo</i>. </p><p>Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, <i>in vitro</i>. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ.</p><p>In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. </p><p>Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model. </p>

The vascular endothelium is a single cell layer that lines the lumen of the entire vasculature. It is the site of synthesis of nitric oxide (NO), a vasodilatory compound synthesized by endothelial nitric oxide synthase (eNOS). NO causes intracellular calcium sequestration of the vascular smooth muscle cells, relaxing and dilating the arteries. Age profoundly affects endothelium-dependent vasodilation, leading to specific losses of NO. We sought to determine what causes the age-specific loss of endothelial NO. This was accomplished by investigating whether there are differences in markers of eNOS post-translational regulation elements in the aortic endothelium of young (2-4 months; corresponding to an adolescent human adult) and old (32-34 months; corresponding to a 65-75 year-old human). F 344 x Brown Norway hybrid rats. Results show that maximal eNOS activity significantly declines with age (n=4;p���0.05) though there was no change in eNOS protein levels in the aortic endothelium. Endothelial NOS exists in two distinct subcellular fractions. No alterations were detected in the soluble, inactive fraction while significantly less eNOS protein is detected in the active, plasma membrane fraction of the endothelium (n=4;p���0.02). Endothelial NOS activation is also controlled by its phosphorylation state. In this work we demonstrate that free ceramides and ceramide-activated phosphatase (PP2A) activity are significantly elevated with age in the endothelium and correlate with specific alterations in eNOS phosphorylation status consistent with its inactivation. These changes were concomittent with an age-associated decline in endothelial glutathione (GSH) and increased sphingomyelinase activity which liberates ceramides from membrane sphingolipids. In previously published reports we demonstrated that the dithiol compound R-��-lipoic acid (LA) increased maximal NO synthesis in cultured endothelial cells and that LA improved age-associated loss of eNOS stimulatory phosphorylation in rats. Therefore, we administered pharmacologic doses of LA (40 mg/kg, i.p. over 24 h) to old rats to determine whether it restored NO-dependent vasomotor function. Results show that LA significantly increased endothelial GSH (p���0.05 compared to saline controls), decreased sphingomyelinase activity and reversed the age-related increase in ceramide (p���0.01) in old animals. Finally, LA significantly improved endothelium-dependent vasodilation, suggesting that it might be a good therapeutic agent for age-related vascular endothelial dysfunction. / Graduation date: 2005

Development and studies of new electrochemical methods for the detection of various biologically and environmentally relevant substances are the focus of this dissertation. A dual amperometric sensor, capable of the simultaneous, real-time determination of NO and O,2, has been developed and optimized. Many tests were performed in order to reduce cross-talking between the two sensors, and an electro-deposited polymer, poly-5-amino-1-naphthol, was shown to reduce the cross-talking to insignificant levels. The use of bismuth-based electrodes in the detection of various metals has been explored. A bismuth bulk electrode has been developed, optimized, and used for the individual and simultaneous determination of Pb(II), Cd(II), and Zn(II). The fundamental electrochemistry of several bismuth-based electrodes in the system used for Cr(VI) analysis has also been explored, and many interactions among the electrode material, ligand, and analyte were observed, particularly the formation of a soluble bismuth-ligand complex. Electrochemical analysis of Cr(VI) was attempted at all of the bismuth-based electrodes, with success at the thin bismuth film electrode. A series of surface modifications were made to the glassy carbon substrate, in an attempt to remove any co-adsorbed contaminants and to understand the sensitivity of the chromium detection process. Inevitably, it was found that the contaminating source was contaminants in the nitrogen gas used for solution deaeration. Upon switching to argon, detection became highly reproducible and showed strong linearity with the Cr(VI) concentration.

Thesis (M.S.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.