The design of new, improved NO-donor drugs is an important pharmacological objective due to the biological importance of nitric oxide. N-Hydroxyguanidines represent a useful class of NO donors where the mechanism of action is based on the biosynthetic pathway for NO. Thirty new N-arylalkyl-N’-hydroxyguanidines were synthesized and their vasodilatation activity examined by myography in rat aortic rings. The observed relaxations were reversed by ODQ, which is an inhibitor of the guanylate cyclase, implying that this was an NO dependent vasodilatation. The most active compounds were also tested in the isolated perfused kidney (IPK) giving the vasodilatation properties. Preliminary results indicated that N-phenyl-N’- hydroxyguanidine showed the best pharmacological profile with EC₅₀= 19.9 μM and ca. 100% reversibility with ODQ. A series of N-phenylalkyl-N’-hydroxyguanidines were synthesised. NO donor activity was found to be fairly constant up to three methylene groups, and then decreased. Substitutions in the benzene ring of N-phenylethyl-N’-hydroxyguanidine demonstrated that various electron-withdrawing and electron-donating groups in the para position did not significantly affect the NO donor activity of this series of analogues. The nitro and trifluoromethyl substituted compounds gave the best biological profiles. Additionally, a novel heterocyclic, N–furfuryl-N’–hydroxyguanidine possessed very promising vasodilatation properties. In general, almost all the N-arylalkyl-N’-hydroxyguanidines behaved as potent NO donors in the rat aorta assay. In order to establish the influence of the free NH₂ group in the hydroxyguanidine functionality on the vasodilatation properties, N,N-dimethyl and N-methyl-N’- hydroxyguanidines were successfully synthesised. Unfortunately, they have not been tested yet in the biological assay. However, their NMR spectra showed some unusual features and their detailed analysis and X-ray data are presented herein. In addition a series of hydroxamic acids was synthesised and the NO donor activity investigated using the same biological methodology. It was found that the 3-phenylpropionohydroxamic acid was the most potent compound with EC₅₀ = 6 μM and ODQ = 96%. However, behavior in the IPK indicated that hydroxamic acids did not undergo the same biological pathway as in the rat aorta. Two different types of enzyme-activated pro-drugs were designed using N-hydroxyguanidines as the NO donating molecule. Synthetic studies towards these targets were carried out using various synthetic approaches. The desired molecules have not yet been synthesised but the chemistry explored so far has indicated potentially more successful approaches that could be attempted.

Master of Science / Department of Kinesiology / B.J. Wong / Recent data suggests nNOS mediates the NO-component of reflex cutaneous vasodilation with passive heat stress. Our hypothesis was nNOS, but not eNOS, inhibition would attenuate reflex cutaneous vasodilation during dynamic exercise. Protocol 1: subjects performed a VO[subscript]2 peak test on a supine cycle ergometer. Protocol 2: with experimental arm at heart level subjects cycled in supine posture at 60% VO[subscript]2 peak to raise core temperature (Tc) 0.8-1.0°C (35-45 min). In protocol 2 subjects were equipped with 4 microdialysis fibers on the forearm and each randomly assigned as: 1) lactated Ringer’s (control); 2) 5mM NPLA (nNOS inhibition); 3) 10mM L-NIO (eNOS inhibition); and 4) 20mM L-NAME (non- selective NOS inhibition). At the end of protocol 2 all sites were locally heated to 43°C and infused with SNP to elicit maximal dilation. Mean arterial pressure (MAP), skin blood flow via laser- Doppler flowmetry (LDF), and Tc via ingestible telemetric pill were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to maximum. In protocol 2 there was no significant difference between control (62±5 %CVCmax) and NPLA (61±6 %CVCmax). L-NIO (38±4 %CVCmax) and L-NAME (41±7 %CVCmax) significantly attenuated CVC compared to control and NPLA (p<0.001 all conditions). There was no difference between L-NIO and L- NAME. We conclude eNOS, not nNOS, contributes to reflex cutaneous vasodilation during dynamic exercise.

O óxido nítrico (NO) é o principal agente vasodilatador endógeno que regula o tônus e a homeostase vascular. Dentre os compostos doadores de NO, estão os complexos nitrosilos de rutênio. No presente estudo, o doador de NO estudado, RuBPY, não apresenta citotoxicidade para células do músculo liso vascular (MLV) ao contrário do NPS. O RuBPY apresenta eficácia semelhante ao NPS em relaxar o MLV de aorta de ratos, porém o NPS é mais potente. Ambos compostos liberam NO do tipo radicalar (NO) no meio intracelular, mas o NPS libera também íon nitroxil (NO-). O sequestrador da espécie NO (hidroxocobalamina) reduziu mais a resposta relaxante estimulada com RuBPY do que com o NPS. Nenhum dos dois compostos precisa ser reduzido quimicamente para liberar NO, uma vez que houve relaxamento quando utilizamos alta concentração de KCl como agente contrátil. Porém, este relaxamento foi inibido, o que mostra a importância dos canais para K+ no relaxamento induzido pelos doadores de NO. O bloqueador não seletivo de canais para K+ (TEA), inibiu somente o relaxamento ao RuBPY. A via NO-GCs-GK é ativada por ambos doadores de NO, para induzir relaxamento. A inibição da degradação do GMPc potencializou o relaxamento estimulado com RuBPY e NPS. O armazenamento de Ca+2 no retículo sarcoplasmático (RS) via ativação da SERCA é importante somente para o relaxamento induzido com RuBPY. O composto RuBPY inibiu a resposta contrátil estimulada com fenilefrina devido ao armazenamento de Ca+2 no RS e também por inibir o influxo capacitivo de Ca+2. A presença do endotélio vascular não alterou o relaxamento induzido pelo RuBPY, porém potencializou o relaxamento induzido pelo NPS. A análise da liberação de NO por amperometria demonstrou que o RuBPY libera NO somente em presença do tecido aórtico de ratos. Portanto, não houve liberação espontânea de NO, por fotólise pela luz visível ou por redução química. É necessária a presença de heme-proteínas como a guanilil-ciclase solúvel (GCs) inibida pelo ODQ, para haver a conversão do nitrito presente no RuBPY, a NO. Pela quantificação da fluorescência emitida pela sonda DAF-2DA, RuBPY liberou cerca de 3,5 vezes mais NO do que o NPS. Pela medida do potencial de membrana, demonstramos que o RuBPY induz hiperpolarização de membrana de células isoladas do MLV da aorta de rato. RuBPY tem efeito hipotensor dose-dependente, em ratos hipertensos renais, o que não ocorre em animais normotensos. A redução da pressão arterial em ratos hipertensos é maior do que nos normotensos. Em estudos iniciais de farmacocinética, verificamos que o composto RuBPY é absorvido por via oral e é distribuído entre alguns tecidos após ser administrado aos ratos, por gavagem. / Nitric oxide (NO) is the main endogenous vasodilator agent that regulates vascular tone. Among the compounds which are able of releasing NO, are the nitrosyl ruthenium complexes. The NO donor studied, RuBPY, does not present cytotoxicity in smooth muscle cells (SMC), in contrast to SNP. RuBPY has similar efficacy to SNP in inducing rat aorta relaxation, although SNP is more potent. Both compounds release intracellular radicalar NO (NO), and SNP also release ion nitroxyl (NO-). The NO scavenger (hydroxocobalamine) had greater effect on the relaxation induced by RuBPY than by SNP. Both compounds do not need to be chemically reduced to release NO, as demonstrated in aorta relaxation after pre-contraction with high concentrations of KCl. However, this relaxation was impaired, showing the importance of K+ channels to induce relaxation by NO released from these compounds. By using non-selective blocker for K+ channels (TEA), only the relaxation induced by RuBPY was inhibited. The NO-sGC-GK pathway is activated by NO donors to induce relaxation. Inhibition of cGMP degradation, potentiated the effect of RuBPY and SNP. Storage of Ca+2 in the sarcoplasmic reticulum (SR) via activation of SERCA is important only for the relaxation induced by RuBPY. The contractile response induced by phenylephrine was inhibited by RuBPY due to the storage of Ca+2 in RS and also by inhibiting the capacitive influx of Ca+2. The presence of endothelium had no effect on the relaxation induced by RuBPY, but it potentiated the relaxation induced by SNP. RuBPY released NO only in the presence of the rat aorta. The complex RuBPY did not spontaneously release NO, by photolysis by visible light, or by chemical reduction. RuBPY requires the presence of heme-protein such as guanylyl-cyclase, inhibited by ODQ, to convert nitrite to NO. The amount of NO released from RuBPY was about 3.5 times greater than that released from SNP. RuBPY induced membrane hyperpolarization of SMC. RuBPY has hypotensive effect in renal hypertensive rats in a dose-dependent way, which does not occur in normotensive rats. The decreased of blood pressure in hypertensive rats was greater than in normotensive rats. Initial studies of pharmacokinetics demonstrated that RuBPY is orally absorbed and it is also distributed in some tissues after being administered by gavage to rats.

Xue, Hongmei. / "December 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yip Kwok Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 231-260). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Mast cells --- p.2 / Chapter 1.2. --- "Mast cell origin, growth and development" --- p.2 / Chapter 1.2.1. --- Stem cell factor --- p.4 / Chapter 1.2.2. --- Interleukins --- p.6 / Chapter 1.3. --- Mast ceII heterogeneity --- p.7 / Chapter 1.4. --- Mast ceII mediators --- p.9 / Chapter 1.4.1. --- Pre-Synthesized mediators --- p.9 / Chapter 1.4.1.1. --- Histamine --- p.10 / Chapter 1.4.1.2. --- Protease --- p.11 / Chapter 1.4.2. --- Newly synthesized mediators --- p.13 / Chapter 1.4.2.1. --- Prostanoid --- p.14 / Chapter 1.4.2.2. --- Cysteinyl Leukotriene --- p.15 / Chapter 1.4.3. --- Mast cell-derived cytokines and growth factors --- p.16 / Chapter 1.5. --- Mast cell activation --- p.17 / Chapter 1.5.1. --- FceRI-dependent mast cell activation --- p.18 / Chapter 1.5.1.1. --- FceRI and IgE aggregation --- p.19 / Chapter 1.5.1.2. --- Protein-tyrosine kinase activation --- p.21 / Chapter 1.5.1.3. --- Phospholipase activation and calcium ion mobilization --- p.22 / Chapter 1.5.1.4. --- GTPase and MAPK activation --- p.24 / Chapter 1.5.2. --- Non-immunogical mast cell activation --- p.26 / Chapter 1.6. --- Roles of mast cell in inflammatory disease --- p.27 / Chapter 1.7. --- Nitric oxide --- p.28 / Chapter 1.8. --- Nitric oxide synthase --- p.30 / Chapter 1.9. --- Nitric oxide signaling in cellular level --- p.31 / Chapter 1.9.1. --- Direct effects of NO --- p.32 / Chapter 1.9.2. --- Indirect effects of NO --- p.34 / Chapter 1.10. --- Mast cell and nitric oxide --- p.35 / Chapter 1.11. --- Aim of Study --- p.37 / Chapter 2. --- Materials and Methods --- p.43 / Chapter 2.1. --- Material --- p.44 / Chapter 2.1.1. --- Human buffy coat for mast cell culture --- p.44 / Chapter 2.1.2. --- Materials for cell isolation and cell counting --- p.44 / Chapter 2.1.3. --- Materials for mast cell culture --- p.45 / Chapter 2.1.4. --- Material for buffers --- p.45 / Chapter 2.1.5. --- Materials for cytospin and May-Griinwald-Giemsa staining --- p.46 / Chapter 2.1.6. --- Materials for immunocytochemical staining --- p.46 / Chapter 2.1.7. --- Mast cell secretagogues --- p.47 / Chapter 2.1.8. --- Nitric oxide donors --- p.47 / Chapter 2.1.9. --- Soluble Guanylyl Cyclase activators and cGMP analogues --- p.47 / Chapter 2.1.10. --- Drugs involved in NO-sGC-cGMP pathway --- p.48 / Chapter 2.1.11. --- Materials for histamine assay --- p.48 / Chapter 2.1.12. --- Materials for Enzyme Immunosorbent Assay (EIA) --- p.49 / Chapter 2.1.13. --- Pro-inflammatory cytokines --- p.49 / Chapter 2.1.14. --- Materials for RNA extraction and RT-PCR --- p.49 / Chapter 2.1.15. --- Materials for Immunofluorescence staining --- p.50 / Chapter 2.1.16. --- Anti-asthmatic compounds --- p.51 / Chapter 2.1.17. --- Buffer and stock solution --- p.51 / Chapter 2.1.17.1. --- Buffer ingredients --- p.51 / Chapter 2.1.17.2. --- Stock solution --- p.52 / Chapter 2.2. --- Methods --- p.52 / Chapter 2.2.1. --- CD34+ cell isolation from human buffy coat --- p.52 / Chapter 2.2.2. --- CD34+ cell culture --- p.53 / Chapter 2.2.3. --- Human mast cell line (HMC-1 cells) culture --- p.54 / Chapter 2.2.4. --- Mast cell heterogeneity identification --- p.54 / Chapter 2.2.4.1. --- Cell smear preparation --- p.54 / Chapter 2.2.4.2. --- May-Gruwald-Giemsa staining --- p.55 / Chapter 2.2.4.3. --- Immunocytochemical staining --- p.55 / Chapter 2.2.5. --- Histamine release and measurement --- p.56 / Chapter 2.2.5.1. --- Histamine release --- p.56 / Chapter 2.2.5.2. --- Spectroflurometric determination of histamine content --- p.57 / Chapter 2.2.5.3. --- Calculation of histamine level --- p.57 / Chapter 2.2.6. --- Prostaglandin D2 (PGD2) measurement --- p.58 / Chapter 2.2.6.1. --- PGD2 production --- p.58 / Chapter 2.2.6.2. --- EIA methods for PGD2 measurement --- p.58 / Chapter 2.2.6.3. --- Calculation of PGD2 concentration --- p.59 / Chapter 2.2.7. --- Cysteinyl Leukotrienes (Cys-LTs) measurement --- p.59 / Chapter 2.2.7.1. --- Cys-LTs production --- p.59 / Chapter 2.2.7.2. --- EIA methods for Cys-LTs measurement --- p.60 / Chapter 2.2.7.3. --- Calculation of Cys-LTs concentration --- p.60 / Chapter 2.2.8. --- Tumor necrosis factor-alpha (TNF-α) measurement --- p.61 / Chapter 2.2.8.1. --- TNF-α production --- p.61 / Chapter 2.2.8.2. --- EIA methods for TNF-α measurement --- p.61 / Chapter 2.2.8.3. --- Calculation of TNF-α concentration --- p.62 / Chapter 2.2.9. --- Interleukin-8 (IL-8) measurement --- p.62 / Chapter 2.2.9.1. --- IL-8 production --- p.62 / Chapter 2.2.9.2. --- ELISA for IL-8 measurement --- p.62 / Chapter 2.2.9.3. --- Calculation of IL-8 concentration --- p.63 / Chapter 2.2.10. --- Data presentation --- p.63 / Chapter 2.2.11. --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.64 / Chapter 2.2.11.1. --- RNA extraction --- p.64 / Chapter 2.2.11.2. --- Reverse Transcriptase reaction for cDNA synthesis --- p.65 / Chapter 2.2.11.3. --- Polymerase Chain Reaction --- p.66 / Chapter 2.2.11.4. --- Agarose Gel Electrophoresis --- p.67 / Chapter 2.2.11.5. --- Data representation in RT-PCR experiment --- p.67 / Chapter 2.2.12. --- Immunofluorescence staining --- p.67 / Chapter 2.2.12.1. --- Cell smear preparation --- p.68 / Chapter 2.2.12.2. --- Immunofluorescence staining --- p.68 / Chapter 2.3. --- Statistical analysis --- p.69 / Chapter 3. --- Effect of Nitric Oxide Donors on Mast Cell Activation --- p.70 / Chapter 3.1. --- Introduction --- p.71 / Chapter 3.1.1. --- Mechanisms of NO release from NO donors --- p.71 / Chapter 3.1.2. --- Experimental aims --- p.77 / Chapter 3.2. --- Materials and methods --- p.77 / Chapter 3.3. --- Results --- p.78 / Chapter 3.3.1. --- Development of mast cells from buffy coat --- p.78 / Chapter 3.3.2. --- Morphological features of cultured mast cells --- p.78 / Chapter 3.3.3. --- Phenotype of cultured mast cells --- p.79 / Chapter 3.3.4. --- Effects of NO donors on immunologically stimulated mediators release --- p.79 / Chapter 3.3.4.1. --- SIN-1 and NOR-3 --- p.80 / Chapter 3.3.4.2. --- SNP and SNAP --- p.80 / Chapter 3.3.4.3. --- Diazeniumdiolates (NONOates) --- p.80 / Chapter 3.3.5. --- Effects of NO scavenger on NO donors mediated inhibition of immunologically stimulated mediators release --- p.82 / Chapter 3.3.6. --- Discussion --- p.83 / Chapter 4. --- Interaction between NO donors and pharmacological agentsin modulating mast cell activation --- p.123 / Chapter 4.1. --- Introduction --- p.124 / Chapter 4.1.1. --- Modulators of NO-sGC-cGMP pathway --- p.125 / Chapter 4.1.2. --- Anti-asthmatic compounds --- p.128 / Chapter 4.1.3. --- Experimental aims --- p.130 / Chapter 4.2. --- Materials and methods --- p.131 / Chapter 4.3. --- Results --- p.132 / Chapter 4.3.1. --- Effect of sGC activators on immunologically stimulated histamine release and the inhibitory action of DEA/NO --- p.132 / Chapter 4.3.2. --- Effect of cGMP analog on immunologically stimulated histamine release --- p.133 / Chapter 4.3.3. --- "Effects of the sGC inhibitor, ODQ, on DEA/NO induced inhibition on immunologically stimulated mediators release" --- p.134 / Chapter 4.3.4. --- Effects of anti-oxidants on the actions of NO donors in modulating immunologically stimulated mediators release --- p.134 / Chapter 4.3.5. --- The effects of NO donors on salbutamol mediated inhibition of immunologically stimulated histamine release from human mast cells --- p.135 / Chapter 4.3.6. --- The effects of NO donors on theophylline mediated inhibition of immunologically stimulated histamine release from human mast cells --- p.136 / Chapter 4.3.7. --- The effects of NO donors and DSCG on immunologically stimulated histamine release from human mast cells --- p.137 / Chapter 4.4. --- Discussion --- p.137 / Chapter 4.5. --- Further studies --- p.150 / Chapter 5. --- Human mast cells as a source of nitric oxide --- p.178 / Chapter 5.1. --- Introduction --- p.179 / Chapter 5.1.1. --- Nitric oxide synthases expression in mast cell --- p.180 / Chapter 5.1.2. --- Modulation of NOS expression --- p.182 / Chapter 5.1.3. --- Experimental aims --- p.186 / Chapter 5.2. --- Materials and methods --- p.186 / Chapter 5.3. --- Results --- p.187 / Chapter 5.3.1. --- NOS expression in human mast cell-line HMC-1 --- p.187 / Chapter 5.3.1.1. --- Basal --- p.187 / Chapter 5.3.1.2. --- Effect of cytokines --- p.188 / Chapter 5.3.2. --- NOS expression in cultured CD34+ derived human mast cells --- p.189 / Chapter 5.3.2.1. --- Basal --- p.189 / Chapter 5.3.2.2. --- Effect of cytokines --- p.189 / Chapter 5.3.2.3. --- Effect ofIgE and anti-IgE --- p.190 / Chapter 5.4. --- Discussion --- p.191 / Chapter 5.5. --- Further studies --- p.200 / Chapter 6. --- Conclusion --- p.218 / Chapter 7. --- References --- p.230

"Gaseous nitric oxide, in concentrations between 0 and 20 ppm, is currently being used to treat patients with post-surgical complications and respiratory disorders. Currently available instruments are expensive and have problems that limit their usefulness for this application. This thesis discusses the development of an inexpensive, direct and continuous sensor for the measurement of inhaled nitric oxide. The prototype sensor incorporates a 0.125 cm, gas permeable, flow-thru liquid cell into a probe that can be incorporated into a ventilator circuit. Sensor operation is based on the complexation reaction of NO with cytochrome-c (Fe III), a biologically derived heme. The complex is monitored spectrophotometrically in the visible region of the spectrum at 563 nm by an optical spectrograph card. LabVIEW is used for all hardware control, signal acquisition, data processing, display and storage. The sensor has a sensitivity of 2x10-4 Abs/ppm, where Abs denotes absorbance units, a minimum detectable limit of 1.5 ppm, resolution of 0.5 ppm, is stable over the course of 8 hours, has less than 1 ppm error and a response time of less than 2 minutes. All aspects of sensor design and development will be discussed."

by Wong Ho Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 107-129). / Abstract --- p.i / List of Abbreviations --- p.ii / Contents --- p.iii / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Introduction of aging in central nervous system --- p.1 / Chapter 1.2 --- Introduction of hippocampus / Structure of the hippocampus --- p.4 / Function of hippocampus --- p.6 / Chapter 1.3 --- A literature review of aging in hippocampus / Cell loss in aging --- p.8 / Ultrastructural changes in aging --- p.9 / Changes in neurotransmitter system --- p.10 / Neuroglial change --- p.11 / Change in potentiation --- p.13 / Chapter 1.4 --- A literature survey of Nitric Oxide Synthase (NOS) / General introduction of Nitric Oxide Synthase --- p.15 / Introduction of nNOS --- p.15 / Introduction of iNOS --- p.16 / Introduction of eNOS --- p.17 / Similarities and differences among isoforms --- p.18 / Role of NO/NOS in neurotransmission --- p.19 / Role of NO in neurotoxicity --- p.23 / Chapter 1.5 --- Aim of study --- p.25 / Chapter Chapter 2. --- Change of nNOS in aging / Chapter 2.1 --- Purpose and approach --- p.27 / Chapter 2.2 --- Basic principle of the techniques / Basic principle of immunohistochemistry --- p.28 / Basic principle of RT-PCR --- p.28 / Chapter 2.3 --- Experimental procedure / nNOS immunohistochemistry --- p.32 / RT-PCR of nNOS --- p.34 / Chapter 2.4 --- Result / nNOS immunohistochemistry --- p.38 / RT-PCR of nNOS --- p.44 / Chapter Chapter 3. --- Expression of iNOS in aging / Chapter 3.1 --- Purpose and approach --- p.50 / Chapter 3.2 --- Experimental procedure / iNOS immunohistochemistry --- p.50 / RT-PCR analysis of iNOS --- p.51 / Chapter 3.3 --- Result / iNOS immunohistochemistry --- p.52 / RT-PCR analysis of iNOS --- p.56 / Chapter Chapter 4. --- Verification of the RT-PCR product of iNOS / Chapter 4.1 --- Purpose and approach --- p.58 / Chapter 4.2 --- Basic principle --- p.58 / Chapter 4.3 --- Experimental procedure / Elution of PCR product from PAGE gel --- p.60 / Restriction digestion of the eluted PCR product --- p.61 / Chapter 4.4 --- Result --- p.62 / Chapter Chapter 5. --- Identification of the iNOS-positive cells / Chapter 5.1 --- Purpose and approach --- p.64 / Chapter 5.2 --- Experimental procedure --- p.64 / Chapter 5.3 --- Result --- p.65 / Chapter Chapter 6. --- Quantitation of astrocyte in aging hippocampus / Chapter 6.1 --- Purpose and approach --- p.67 / Chapter 6.2 --- Experimental procedure --- p.68 / Chapter 6.3 --- Result --- p.69 / Chapter Chapter 7. --- Detection of apoptosis in aging / Chapter 7.1 --- Introduction of apoptosis --- p.74 / Chapter 7.2 --- Purpose and approach --- p.75 / Chapter 7.3 --- Basic principle --- p.76 / Chapter 7.4 --- Experimental procedure / TUNEL method --- p.77 / DNA gel electrophoresis --- p.78 / Chapter 7.5 --- Result / TUNEL method --- p.80 / DNA gel electrophoresis --- p.82 / Chapter Chapter 8. --- Discussion / Chapter 8.1 --- Pattern of neuronal NOS in aging / Localization of nNOS --- p.84 / Decrease in staining of nNOS in the hippocampus during aging --- p.87 / No change in nNOS mRNA level --- p.88 / nNOS in aging - past and present works --- p.89 / Implication of the result --- p.91 / Chapter 8.2 --- Increased iNOS expression in aging / Neurotoxicity of iNOS --- p.93 / Circumstances of iNOS expression --- p.95 / Discussion of the present study --- p.96 / Chapter 8.3 --- Detection of apoptosis in aging --- p.103 / Chapter Chapter 9. --- Conclusion --- p.106 / Biblography --- p.107 / Appendix --- p.130 / Acknowledgements --- p.134

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

Experiments described in this thesis address the pathogenesis of aortic valve sclerosis/stenosis using a bench to bedside approach. In particular, the thesis begins with development of a technique using ultrasonic backscatter analyses to quantitate the early stages of aortic stenosis. Subsequent chapters utilized this methodology to quantitate aortic valve structural changes in a model and intervention study of aortic stenosis in rabbits. The last chapters are human studies designed to identify factors associated with presence of aortic sclerosis/stenosis; with particular interest in potential association of endothelial dysfunction/inflammation/platelet aggregation with abnormal aortic valve structure quantitated by ultrasonic backscatter. In Chapter 1 (Introduction) the relevant literature is reviewed. Development of ultrasonic backscatter to quantitate aortic sclerosis (Chapter 2) Aortic valve sclerosis (ASc) is detected when there is visual assessment of focal increases in echogenicity of the aortic valve most commonly assessed by echocardiography. However, there is no previously described method to quantitate degree of aortic valve structural abnormality as ASc is not associated with marked hemodynamic obstruction quantifiable by Doppler echocardiography. The current study used ultrasonic backscatter to quantitate aortic valve structural abnormality in patients assessed as having ASc based on valve appearances, compared to young healthy volunteers with normal aortic valves. The results of the study indicate: 1) that the mean levels of aortic valve backscatter in ASc patients are approximately 60% greater than in young healthy volunteers (ie aortic valve backscatter scores ≥ 16dB are not consistent with normal aortic valve structure), 2) ultrasonic backscatter scores in ASc patients are directly correlated with subjective scoring of sclerosis and with a positive trend with transvalvular pressure gradients in patients with mild-moderate aortic stenosis, and most importantly, 3) ultrasonic backscatter is a reproducible technique, with mean differences between estimates based on repeat echocardiograms of 2.3 ± 1.7 (9.1%). These results indicate that ultrasonic backscatter could be used as a quantitative measure of aortic valve structural abnormality in epidemiology and for examination of interventions. In vivo studies Development of an animal model of aortic stenosis with vitamin D2 (Chapter 3) The aim of the study was to develop an appropriate animal model for AS. The study used vitamin D2 alone at 25,000IU/4 days weekly (vit-D2) for 8 weeks to induce AS in rabbits. Results showed that: 1) rabbits in the vit-D2 group had significantly increased in transvalvular velocity and pressure gradients compared to rabbits in the control group (normal chow + drinking water); this was consistent for aortic valve ultrasonic backscatter scores; 2) aortic valve immunohistochemistry/histology showed marked calcification, neutral lipids, macrophage, and leukocyte infiltrations for rabbits in the vit- D2 group (ie consistent with histology of human AS); 3) significant elevation of asymmetric dimethylarginine (ADMA) concentrations in the vit-D2 group occurred compared to controls over the 8 weeks treatment period; the change in ADMA concentrations correlated significantly with the change in transvalvular pressure gradients for rabbits in the vit-D2 group; 4) rabbits in the vit-D2 group had significantly impaired endothelium-dependent acetylcholine-induced aortic relaxation, and this effect was completely abolished by the nitric oxide synthase inhibitor (L-NAME); 5) the addition of 0.5% cholesterol-supplemented diet to the vitamin D2 regimen did not accentuate the development of AS. Thus, treatment with vitamin D2 at 25,000IU/4 days weekly for 8 weeks significantly induced AS with similar aortic valve pathology to that of human AS; therefore, the model is suitable for use in examining potential therapeutic interventions in AS. Effects of ramipril on development of AS in rabbits (Chapter 4) Using this animal model, this study aimed to examine the effects of the angiotensinconverting enzyme inhibitor (ACEi) ramipril on development of AS. Rabbits (n=28) treated for 8 weeks were divided into 2 groups: (a) vitamin D2 alone (n=10) (normal chow + 25,000IU vitamin D2 in drinking water); (b) vitamin D2/Ramipril (n=12) (normal chow+25,000IU vitamin D2/Ramipril (0.5mg/kg) in drinking water). Six further rabbits constituted a normal reference group (no treatment was given). The results for comparisons between vitamin D2/ramipril vs vitamin D2 alone were as follows: 1) ramipril-treated rabbits had significantly less severe hemodynamic obstructions (p<0.05, for both) as assessed by transvalvular velocity, and aortic valve area; with borderline reduction in aortic valve backscatter (p=0.08); 2) ramipril significantly reduced plasma ADMA concentrations; 3) there was improvement in acetylcholine-induced aortic relaxation (p=0.056), with significant improvement in sodium nitroprusside-induced relaxation (p<0.05); 4) there was a strong inverse correlation between acetylcholineinduced aortic relaxation and aortic valve backscatter score (0<0.001), thus providing further evidence of the potential role of nitric oxide in retarding the development of AS in this model. These data provide a strong rationale for the inception of a randomized trial of ACE inhibition as a strategy for limitation of AS progression in humans. Human studies Aortic stenosis is associated with elevated plasma levels of asymmetric dimethylarginine (ADMA) concentrations in humans (Chapter 5). Given the findings that aortic stenosis induced by vitamin D2 in rabbits also caused elevation of plasma ADMA concentrations, a physiological inhibitor of nitric oxide synthase, a mediator and marker of endothelial dysfunction and an indicator of incremental cardiovascular risk. The study sought to determine whether plasma ADMA concentrations are elevated independently of pre-existing coronary risk factors in subjects with at least moderate aortic stenosis (n=42) compared to age-matched patients with normal aortic valves (n=42): as determined both by visual assessment and with aortic valve backscatter scores < 16dB. Results for this study were as follows: 1) plasma ADMA concentrations were not statistically different between the AS and non-AS group (median 0.59 vs 0.54 µmol/L, p=0.13, Mann-Whitney test) on univariate analysis; 2) backward stepwise multiple linear regression showed the presence of AS was a significant predictor of elevated ADMA concentrations (p=0.04, 95% CI =0.001, 0.072). 3) in addition, elevated plasma ADMA concentrations were also associated with history of atrial fibrillation (p=0.009, 95% CI=0.015, 0.100), and negatively associated with creatinine clearance (p=0.01, 95% CI=-0.002, 0.000), and the use of statin therapy (p=0.01, 95% CI=-0.081, -0.011). Therefore, in conclusion, this study found that AS is independently associated with elevation of ADMA concentrations, beyond that implied by “conventional” risk factors for endothelial dysfunction. The clinical status of AS as an incremental marker of cardiovascular risk may reflect ADMA-mediated endothelial dysfunction. Assessment of factors associated with ASc in a random ageing population study (Chapter 6). There have been few clinical studies of factors associated with ASc. Previous population studies have established that ASc is an independent correlate of incremental risk of coronary events. Having established that patients with AS have increased plasma ADMA concentrations (Chapter 5), it was now aimed to determine whether subjects with increased aortic valve backscatter scores (ASc) also have other markers of endothelial dysfunction/NO effects, independent of preexisting coronary risk factors. The study was designed to identify such anomalies, if they existed, on an incremental basis to other putative correlates of ASc, including coronary risk factors, renal dysfunction and vitamin D levels. Random selected subjects (n=253) aged between 51 to 77 years were evaluated. All patients underwent transthoracic echocardiography examination; aortic valve ultrasonic backscatter score (AVBS), was used to quantitate echogenicity of the aortic valve. Conventional coronary risk factors were identified on history. Integrity of NO generation/response was assessed via (i) plasma ADMA concentrations; (ii) inhibition of platelet aggregation by the NO donor sodium nitroprusside (SNP); (iii) aortic augmentation index (AIx), a measure of arterial stiffness/wave reflection. All putative correlations with AVBS were examined by univariate and stepwise multiple linear regression analyses. On the basis of echocardiographic appearances, ASc was present in 63 subjects (25.4%); mean AVBS scores was 14.9±4.6dB (SD) vs 11.2±3.9dB (SD) in the presence vs absence of ASc (p<0.001). Univariate analyses revealed that platelet responsiveness to NO was inversely correlated with AVBS (β=-0.16, p=0.02); but [ADMA] and AIx were not. On multiple linear regression, significant correlates of increased AVBS were: (i) advanced age (β=0.21, p=0.003), (ii) low body mass index (β=-0.23, p=0.001); and (iii) impaired platelet responsiveness to NO (β=-0.16, p=0.02). In Chapter 7, the implications of the overall findings in this thesis are discussed in relation to future perspective. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309350 / Thesis(Ph.D.) -- School of Medicine, 2008

Thesis (Ph.D.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 122-146.