<p>S-nitrosylation, the prototypic mechanism of redox-based signal transduction, involves the covalent attachment of a nitrogen monoxide group to a Cys-thiol side chain. S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions and protein stability. The primary focus of my dissertation was to solve a problem of great importance in the field of S-nitrosylation, which is, to identify denitrosylase(s) i.e., enzymes that remove NO groups from S-nitrosothiols. Recent progress in elucidating the cellular regulation of S-nitrosylation has led to the identification of two physiologically relevant denitrosylating activities that remove the NO group from S-nitrosylated substrates. Thioredoxin/thioredoxin reductase (Trx system) functions as an NADPH-dependent denitrosylase across a broad range of S-nitrosylated proteins (SNO-proteins). S-nitroso-glutathione reductase (GSNOR), which is highly conserved across phylogeny, metabolizes GSNO utilizing NADH as a reducing coenzyme, thereby shifting equilibria between GSNO and SNO-proteins. This dissertation describes the discovery of two novel denitrosylases: one from yeast and the other from mammals. Using technique of column chromatography we have purified these novel denitrosylases to homogeneity and have demonstrated a principal contribution of these enzymes towards S-nitrosothiol metabolism.</p> / Dissertation

The aim of the research presented in this thesis is to investigate and develop novel nitric oxide (NO) delivery systems, specifically designed for application in medical areas. The initial work has focused on utilising metal organic frameworks (MOFs) as a delivery system for this radical gas, NO. Due to their high porosity, high thermal stability and the presence of coordinated unsaturated metal sites (CUSs) when fully activated, the CPO-27 (Coordination Polymer of Oslo) family of MOFs has been selected as a suitable host framework. CPO-27 (Ni), CPO-27 (Mg) and CPO-27 (Zn) have been prepared using reflux and room temperature processes without recourse to the use of any toxic or harmful solvents. The resulting products are characterised by powder XRD (X-ray diffraction) and SEM (Scanning electron microscopy), and their NO adsorption, storage and release properties are reported. The results indicate that the crystallinity, particle size and NO adsorption, storage and release performance are comparable to those of equivalent samples synthesised via traditional solvothermal methods, paving the way for a more easily scalable and environmentally friendly synthetic procedure for these types of MOF. Depending on which metal is employed; the NO uptake, storage and release varies, the more toxic nickel based framework shows enhanced performance in terms of concentration and duration of NO released against either the magnesium or zinc counterparts. In order therefore to reduce the risk of toxicity whilst retaining good performance, Ni (II) ions were doped into the 3D framework of CPO-27 (Mg) and CPO-27 (Zn) using novel water-based reflux and room temperature crystallization methods. Several characterization techniques strongly support the effective incorporation of Ni (II) ions into the 3D framework. Nitric oxide (NO) adsorption/release data, as well as in vitro tests demonstrate that NO dosage and biological response can be tuned via the Ni doping process allowing enhanced performance without the high toxicity of pure Ni MOFs. Such materials would be extremely advantageous and more applicable for use in medical fields. NONOates and other NO-complexes have also been investigated as alternative NO delivery systems. This study has focused on developing NO-drug complexes using a variety of different compounds commonly used by clinicians, namely the antiseptic (chlorhexidine, CHx), the antibiotic (ciprofloxacin) and diuretic (furosemide). A unique high pressure NO loading methodology has been developed to coordinate nitric oxide to these drug molecules and their NO release performance has been evaluated. The resulting NO-drug complexes are characterised using a series of spectroscopic techniques and the collected data highlights that the radical gas coordinates with the secondary amine groups present in the drug molecules. The interaction between the amine group and the gas is reversible; in fact the release of NO from these complexes can be triggered using water (11% RH) and/or UV-light. In addition, chlorhexidine has been incorporated into the pores of the CPO-27 framework. The amount of antiseptic incorporated was determined using a variety of characterisation techniques. The controlled release of significant concentrations of CHx from the CPO-27 materials are achieved by exposing each CHx loaded sample to a water solution, in doing so topical conditions are simulated. The CHx loaded samples have also been activated and NO loaded following the novel high pressure procedure specifically developed during this research. The resulting NO loaded material released the radical gas in the presence of water and/or UV-light. By incorporating the CHx into the MOF and NO loading this complex the duration and release of NO was greatly enhanced over that of either of the components alone. On formulating the CHx loaded 3D frameworks into pellets, or even into a polyurethane polymer film, their ability to release the antiseptic under simulated topical conditions was maintained. The NO-CHx-CPO-27 composite film that has been prepared has proven to be able to simultaneously store and release both NO and CHx. Each component of the complex has more than one function and the quantity and duration of release of NO is again higher and longer than either of the two moieties alone. The release of these two antibacterial agents from a MOF is novel and is very exciting as it opens up the possibility of engineering products with multiple actions to fight infection. Owing to their high stability and shape persistence properties, the CC3 cage series (CC3, RCC3, FT-RCC3 and AT-RCC3) was chosen as the basis of an investigation into the potential use of porous organic cages as delivery systems for nitric oxide gas. NO has been stored in these porous materials through coordination to amine groups forming Nnitrosamine groups. Release of NO from these types of compounds can be triggered by various mechanisms including water and UV-light, the amine group being regenerated after the release of NO. The release performance significantly increased when the materials were exposed to UV-light and/or suspended in water. As a result of this investigation, these covalent organic molecular cages can now be added to the existing list of NO-based therapies available to medical professionals.

Abstract The aim of the study was to evaluate pulmonary endogenous and inhaled nitric oxide (NO) in neonates with severe respiratory failure. Infant autopsy documents were reviewed for fulminant early-onset bacterial pneumonia. 12 infants with the onset at &lt; 72 h of age and three control groups were identified. Immunohistochemistry revealed that 11 of the infants with early-onset pneumonia (92%) had no or faint inducible nitric oxide synthase (NOS2) staining in their alveolar macrophages (AM). All control infants, regardless of their postnatal age, had NOS2-positive AM. The marker of NO-toxicity, nitrotyrosine, was low in all specimens. To confirm this finding, airway specimens of 21 newborns requiring mechanical ventilation were examined. Seven of them had fulminant early-onset pneumonia with maternal ascending intra-uterine infection (IUI). The controls had no infection at birth despite IUI or neither infection nor IUI. In early-onset pneumonia, NOS2 and nitrotyrosine immunoreactivity were low at birth and increased during the recovery phase (p &lt; 0.05). Analyses of interleukin-1 and surfactant protein A showed the same pattern of age-dependent change. Of the autopsied infants, 12 had received inhaled NO (iNO) before death. Each case was paired with a matched control. Additional five infants without respiratory failure prior to death were also studied. The iNO-treated ones tended to have more intensive NOS2 staining in the bronchiolar epithelium and adjacent tissue than the controls. No differences in other NOS isoforms or nitrotyrosine were detected. A novel method for exhaled NO measurements of intubated infants was developed. Six preterm and six term newborns were prospectively recruited for expired and nasal NO measurements. During the first week of life, the preterm infants showed a different pattern of exhaled NO excretion compared to the term infants. For the pilot intervention study on very early iNO, the eligible patients had a birth weight &lt; 1500 g and progressive, therapy-resistant respiratory failure before five hours of age. Five infants received iNO, showed immediately improved oxygenation and survived without deleterious side effects. Deficient production of NO in small premature infants is associated with severe infection and respiratory failure. Very early iNO therapy may be exceptionally effective in a select group of infants, and did not appear to cause oxidation lung injury.

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS)±epinephrine, norepinephrine, and dopamine at 5×10-6M concentrations for 24h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5-10ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by β-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that β-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine-NO axis in disease states.

published_or_final_version / abstract / toc / Pharmacology / Master / Master of Philosophy

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

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