Rozprawy doktorskie na temat „Nitric oxide Pathophysiology”

Introduction: Nitric oxide (NO) is an integral molecule implicated in the control of vascular function. It has been suggested that vascular dysfunction may lead to the development of acute mountain sickness (AMS), high-altitude cerebral oedema (HACE) and high-altitude pulmonary oedema (HAPE), though data to date remains scarce. Therefore, there is a clear need for further work to address the role of NO in the pathogenesis of high-altitude illness. Aims: There were two primary aims of the current work: (1) To examine whether hypoxia mediated changes in systemic NO metabolism are related to the development of AMS and sub-clinical pulmonary oedema and (2) to examine whether hypoxia mediated changes in the trans-cerebral exchange kinetics of NO metabolites are related to the development of AMS and headache. Hypothesis: We hypothesise that hypoxia will be associated with an increase in reactive oxygen species (ROS) formation, resulting in a decrease in vascular NO bioavailability (O2•- + NO → ONOO•-, k = 109 M.s-1). The reduction in NO will lead to vascular dysfunction and impaired oxygen (O2) delivery. Subsequent hypoxaemia will result in pulmonary vascular vasoconstriction and the development of sub-clinical pulmonary oedema within and mild brain swelling. Symptoms and reductions in NO bioavailability will be more pronounced in those who develop AMS since they are typically more hypoxaemic. Alternatively, a hypoxia mediated increase in NO, during vasodilatation, specifically across the cerebral circulation, may activate the trigminovascular system resulting in headache and by consequence, AMS. Methods: Study 1 – AMS symptoms, systemic venous NO concentration and nasal potential difference (NPD), used as a surrogate biomarker of extravascular lung oedema, were quantified in normoxia, after a 6hr passive exposure to 12% oxygen (O2) and immediately following a hypoxic maximal exercise challenge (≈6.5 hrs). Final measurements were 2 obtained two hours into (hypoxic) recovery. Study 2 – AMS, radial arterial and internal jugular venous NO metabolite concentrations and global cerebral blood flow (CBF), using the Kety-Schmidt technique, were assessed in normoxia and after a 9hr passive exposure to 12.9% O2. AMS was diagnosed if subjects presented with a combined Lake Louise score of ≥5 points and an Environmental Symptoms Questionnaire – Cerebral score of ≥0.7 points. Results: Hypoxia was associated with a reduction in total plasma NO, primarily due to a reduction in nitrate (NO3•) and a compensatory increase in red blood cell (RBC)-bound NO(P < 0.05 vs. normoxia) in both studies. Study 1 – Exercise reduced plasma nitrite (NO2•) (P< 0.05 vs. normoxia) whereas RBC-bound NO did not change. NO was not different in those who developed AMS (AMS+) compared to those who remained comparatively more healthy (AMS-) (P < 0.05). NPD was not affected by hypoxia or exercise and was not different between AMS+ and AMS- (P > 0.05). Study 2 – Hypoxia decreased arterial concentration of total plasma NO due primarily to a reduction in NO2•- and nitrate (NO3•-). Hypoxia did not alter the cerebral metabolism of RSNO, whereas the formation of RBC-bound NO increased. Discussion: These findings suggest that alterations in systemic or trans-cerebral NO metabolism are not implicated in the pathophysiology of AMS or sub-clinical pulmonary oedema. However, hypoxia was associated with an overall reduction in the total NO pool (NOx), whereas, selected alterations in more vasoactive NO metabolites were observed. Reductions in the partial pressure of O2 (pO2) were thought to be a key regulator in these changes. Overall net increases in RBC NO and corresponding reductions in plasma NO2• in the face of no alterations in NOx indicates that rather than being simply consumed, NO is reapportioned to other NO metabolites and this may be implicated in the pathophysiology of AMS.

Le travail présenté ici porte sur l’étude de la production et du transport du monoxyde d’azote (NO) dans le poumon humain. Le NO est une molécule dont l’implication dans des processus physiologiques n’a été mis en évidence qu’en 1987. Depuis, il a été démontré que le NO joue de nombreux rôles dans le corps humain. Le NO est un gaz labile (instable) dans les conditions physiologiques, il diffuse très facilement au travers des parois et il a une grande affinité pour l’hémoglobine. La production du NO est liée à 3 isoformes différentes de la protéine appelées synthases du NO ou NO synthases.<p>\<br>Doctorat en sciences, Spécialisation physique<br>info:eu-repo/semantics/nonPublished

Thesis (MScMedSc)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: Ataxia telangiectasia (AT) is a well-characterized neurodegenerative disease resulting from a genetic defect in the Atm gene causing an absence or very low expression of the ATM protein. As AT patients are prone to the development of insulin resistance and atherosclerosis, the aim or the current study was to investigate the importance of the ATM protein in the endothelium and its role in the signalling pathways of nitric oxide (NO) production. To accomplish this, the first objective was to establish an in-house endothelial cell isolation technique harvested from normal and insulin resistant animals. Unfortunately, these cultures, although staining positive with an endothelial cell specific stain, were not pure enough and did not express endothelial NO synthase (eNOS), the central enzyme in NO production. The remainder of the study utilized commercial aortic endothelial cells (AECs) and found that there was a significant increase in NO production when the ATM protein was inhibited by the specific inhibitor, Ku-60019. The beneficial impact of increased NO production includes maintaining vascular homeostasis, promoting angiogenesis, initiating DNA repair by activating p53 and inhibiting smooth muscle cell proliferation. On the other hand, reactive oxygen species (ROS) and reactive nitrogen species (RNS) also generated by high levels of NO, can exert both protective and harmful effects. Examples of these include cell death due to high concentrations of ROS. However, Ku-60019 did not result in increased cell death of AECs. We demonstrated for the first time, a relationship between endothelial ATM protein kinase and the generation of NO. The signalling pathways involved in NO production and glucose utilization form a network of interrelationships. Central to both pathways is the activity of two protein kinases, PKB/Akt and AMPK. Both these kinases are known to phosphorylate the eNOS enzyme to produce NO on the one hand and AS160 to induce GLUT 4 translocation and glucose uptake on the other hand. Activation of the ATM protein is postulated to be a prerequisite for PKB/Akt activation and it may also result in activation of AMPK. However, using insulin to stimulate ATM, we could not show that inhibition of ATM in endothelial cells affected expression or insulin-stimulated activation of PKB/Akt while the PI3-K inhibitor wortmannin, inhibited the latter. In addition, inhibition of ATM negatively regulated the phospho/total ratio of AMPK. We therefore postulate that the NO production elicited by inhibition of ATM, may not be as result of eNOS activity. A second important observation was that inhibition of ATM significantly enhanced phosphorylation of the p85 regulatory subunit of PI3-K. This would imply that ATM normally has an inhibitory effect on p85 phosphorylation and therefore PI3-K activation. We base this assumption on previous publications showing that Ku-60019 does not inhibit PI3K. This again indicates that ATM has a hitherto unexplored regulatory role in endothelial function.<br>AFRIKAANSE OPSOMMING: Ataxia telangiectasia (AT) is a goed-gekarakteriseerde neurodegeneratiewe siekte a.g.v. ‘n genetiese afwyking in the Atm geen wat lei tot ‘n afwesige of lae uitdrukking van die ATM proteïen. Aangesien AT pasiënte geneig is om insulienweerstandigheid en aterosklerose te ontwikkel, was die doel van hierdie studie om die belang van die ATM proteïen in die endoteel, en sy rol in die seintransduksiepaaie betrokke by stikstofoksied (NO) produksie, te ondersoek. Om dit te bereik, was die eerste mikpunt om ‘n eie endoteelsel isolasie-tegniek (ge-oes van normale en insulienweerstandige diere) te vestig. Ongelukkig was hierdie selkulture nie suiwer genoeg nie.Ten spyte daarvan dat hulle positief getoets het met ‘n endoteelsel-spesifieke kleurstof kon geen uitdrukking van eNOS, die sentrale ensiem verantwoordelik vir NO produksie, waargeneem word nie. Die res van die studie het van kommersiële aorta endoteelselle (AES) gebruik gemaak, en daar is gevind dat die inhibisie van die ATM proteïen met die spesifieke inhibitor, Ku-60019, tot ‘n beduidende toename in NO produksie gelei het. Die voordelige impak van verhoogde NO produksie sluit die handhawing van vaskulêre homeostase, bevordering van angiogenese, inisiëring van DNA herstel deur p53 aktivering en inhibisie van gladdespiersel proliferasie in. Reaktiewe suurstofspesies (ROS) en reaktiewe stikstofspesies (RNS) wat ook a.g.v.verhoogde NO gegenereer word, kan egter beide beskermende sowel as skadelike effekte uitoefen. Voorbeelde sluit seldood a.g.v. hoë ROS konsentrasies in. Ku-60019 het egter nie tot ‘n toename in seldood van die AES gelei nie. Hierdie studie het vir die eerste keer aangetoon dat daar ‘n verwantskap tussen die endoteel ATM proteïen kinase en die produksie van NO bestaan. Die seintransduksie paaie betrokke by NO produksie en glukose verbruik vorm ‘n interafhanklike netwerk. Die aktiwiteit van twee proteïen kinases, PKB/Akt en AMPK, is sentrale rolspelers in beide paaie. Albei hierdie kinases is daarvoor bekend dat hulle die eNOS ensiem fosforileer om NO te produseer, maar terselfdertyd ook lei tot AS160 fosforilering, wat tot GLUT 4 translokering en glukose opname lei. Dis is voorgestel dat aktivering van die ATM proteïen ‘n voorvereiste vir PKB/Akt aktivering mag wees en verder kan dit ook tot aktivering van AMPK lei. Ons kon nie aantoon dat inhibisie van ATM in endoteelselle die uitdrukking of insulien-geïnduseerde aktivering van PKB/Akt onderdruk nie, terwyl die PI3-K inhibitor, wortmannin, wel laasgenoemde geïnhibeer het. Verder het die inhibisie van ATM die fosfo/totale AMPK verhouding negatief gereguleer. Ons postuleer dus dat die NO produksie waargeneem tydens ATM inhibisie, moontlik nie die gevolg van eNOS aktiwiteit was nie. ‘n Tweede belangrike waarneming was dat die inhibisie van ATM die fosforilering van die p85 regulatoriese subeenheid van PI3-K beduidend laat toeneem het. Dit impliseer dat ATM normaalweg ‘n inhibitoriese effek op p85 fosforilering, en dus PI3-K aktivering, het. Hierdie aanname word gemaak n.a.v. vorige publikasies wat getoon het dat Ku-60019 nie PI3-K inhibeer nie. Dit dui weer eens daarop dat ATM ‘n tot nog toe onbekende regulatoriese rol in endoteelfunksie het.

Amendments inserted at back. "May 2002" Includes bibliographical references (leaves 237-290) Experiments described in this thesis address the potential role of inducible nitric oxide synthase (iNOS) in hibernating myocardium. Specifically it was sought to establish a cellular model of hibernating myocardium and investigate the expression, regulation and effects of iNOS in this model. Experiments were performed using primary cultures of neonatal rat ventricular myocytes.

In this thesis, the behavioral, electrographic and neurobiological effects of a period of kainic acid-induced status epilepticus (SE) on the C57BL/6J inbred mouse strain are characterised. The severity of epileptic behaviour was scored, used immunohistochemistry to investigate the anatomical distribution of c-Fos expression in the hippocampal formation following SE and recorded EEG during and after SE using an implantable, wireless telemetry device. Further to assessing the severity of SE, changes subsequent to seizures related to the emergence of chronic epilepsy were investigated, including reactive gliosis and synaptogenesis and epileptiform discharges in the EEG trace. I investigated the potential of a range of pharmacological agents for modulating the severity of induced seizures and disease progression. These included drugs targetting the NR2B subunit of the NMDA receptor (RO 25-6981), neuronal nitric oxide synthase (L-NPA), The post synaptic density protein 95 (Tat-NR2B9a) and inducible nitric oxide synthase (1400W). L-NPA, when administered prior to the induction of SE was found to profoundly suppress the emergence of epileptiform activity, including behavioural, electrographic and neurobiological indicators. Further, L-NPA’s modulation of the precipitating event lead to a decrease in neurobiological changes associated with epileptogenesis, such as reactive gliosis in the CA3 region of the hippocampus and 5 elevated synaptogenesis in th molecular layer of the hippocampus. This correlated with a marked decrease in epileptiform discharges in the EEG trace. A novel method of kainic acid administration was trialed, involving multiple small doses of the drug, titrated by the severity of behaviour. This method led to a decrease in mortality and an increase in the severity and inter-individual uniformity of SE, assessed by the analysis of behaviour, EEG and c-Fos expression in the hippocampus. Furthermore, this method induced neurobiological changes associated with epileptogenesis 3 days following SE and was associated with an increased frequency of epileptiform discharges for 7 days post SE.

The purpose of this study was to investigate which type(s) of somatic cells release nitric oxide (NO) in response to Escherichia coli lipopolysaccharide (LPS) and cytokines in vitro and how NO affects superoxide anion (O2-) production by bovine neutrophils and blood monocytes. Mammary epithelial cell line (FbE) released NO after stimulation with recombinant bovine interleukin-1beta (rBoIL-1beta). Moreover, monocytes produced NO in response to recombinant bovine interferon gamma (rBoIFN-gamma) alone or in combination with LPS in a dose- and time-dependent manner. Nitric oxide production was diminished by addition of inducible nitric oxide synthase (iNOS) inhibitors L-N 6-(1-Iminiethyl)lysine or aminoguanidine. However, NO release could not be induced in freshly isolated bovine neutrophils under the experimental conditions used, even after 96 h of incubation. Interestingly, when reverse transcriptase polymerase chain reaction (RT-PCR) with specific primers for iNOS was performed to study mRNA expression, iNOS expression was observed in both monocytes and neutrophils in response to LPS and rBoIFN-gamma.<br>Unlike neutrophils, monocytes were poor producers of superoxide anion under the experimental conditions. A neutrophil-monocyte co-culture system was set up to study the effect of monocyte derived-NO and iNOS inhibitors on superoxide anion production by neutrophils. Neither NO derived from activated monocytes nor iNOS inhibitors seemed to have an effect on bovine neutrophil ability to release O2-. These results suggest that mammary epithelial cells and mononuclear phagocytes are among the cell types responsible for the important quantities of NO released by somatic cells recovered from LPS-infused mammary quarters during endotoxin-induced bovine mastitis. In addition, NO or iNOS inhibitors have no effect on the ability of activated bovine neutrophils to produce superoxide anions.

La pneumopathie interstitielle (PI) est devenue la principale cause de décès de la sclérodermie systémique (ScS). Au cours de PI, l’activation immunitaire déclenche une forte production du monoxyde d’azote (NO) et l’augmentation de la concentration de NO dans l’air expiré des patients atteints de ScS avec PI suggère que cette méthode pourrait détecter précocement l’alvéolite, afin de traiter à temps la PI pour éviter son évolution vers la fibrose pulmonaire. En utilisant le modèle à deux compartiments séparant le NO alvéolaire (CANO) du NO bronchique, nous avons montré que la CANO est : (1) augmentée chez les patients atteints de ScS comparativement aux volontaires sains; (2) associée à l’alvéolite et (3) corrélée à la sévérité de la PI. De plus, une valeur de CANO = 10,8 ppb permettait d’affirmer la présence de la PI et une valeur = 3,8 ppb, d’écarter l’existence d’une PI avec une valeur prédictive= 95%. Le modèle bi-compartimenté néglige la distribution arborisée des voies aériennes et la diffusion axiale du NO, que prend en compte le nouveau « modèle de la trompette ». Les résultats de CANO des 2 méthodes sont comparables (rho=0,98, p&lt;0.001). Enfin, La capacité des sérums à induire la prolifération des fibroblastes pulmonaires et sa conversion en myofibroblaste est augmentée chez les patients atteints de ScS avec une CANO &gt; 5ppb par rapport à celle des patients atteints de ScS qui ont une CANO = 5 ppb et à celle des volontaires sains. Nos résultats suggèrent une relation possible entre l’inflammation alvéolaire et la fibrose pulmonaire au cours de la ScS<br>Interstitial lung disease (ILD) has become the main cause of death in systemic sclerosis (SSc). In ILD, immune activation leads to strong nitric oxide (NO) output by inducible NO synthase. Increased the whole fractional rate of NO in exhaled air has been reported in SSc patients with ILD and suggested that exhaled NO can be an accurate none-invasive marker of early alveolar inflammation in order to initiate in time treatment. The two compartment-model method partitioned exhaled NO into alveolar concentration (CANO) and conducting airway flux, We hypothesized that overproduction of NO in the lung eventually leads to ILD in SSc. We have found that CANO is significantly increased in SSc patients as compared with healthy controls. We have also demonstrated that high levels of CANO were related to alveolitis and the severity of ILD in SSc. Moreover, we have found that ILD could be ruled in (positive predictive value &gt; 95%) when CANO = 10.8 ppb, and ruled out when CANO values = 3.8 ppb (negative predictive value &gt; 95%). The two-compartment model neglected the trumpet shape of airway tree and the axial diffusion of NO that the advanced “trumpet model” takes account. We have found that CANO levels assessed by the two models were comparable (rho=0,98, p&lt;0.001). Finally, we have found that the serum ability to induce lung fibroblast proliferation and myofibroblast transition was increased in SSc patients with high levels of CANO (&gt;5ppb) as compared to SSc patients with low levels of CANO (=5ppb) and healthy controls. Our findings suggest a possible link between alveolar inflammation, and lung fibrosis in SSc. 1624 caractères avec espace

<p>A diagnosis of coeliac disease (CD) in adults relies on the presence of a structurally abnormal intestinal mucosa, followed by a clear clinical remission on a gluten-free diet. There is a clear need for a rapid, simple, safe and sensitive method to determine the type and intensity of inflammation in the gut mucosa in clinical practice. The overall aims of our studies were to develop and evaluate a new technique, “the mucosal patch technique”, to characterize rectal local inflammatory process after rectal food challenge in patients with CD<b>. In study 1</b> we evaluated the potential of the new technique. The technique was well tolerated and easily applied. Pronounced neutrophil and eosinophil involvement in ulcerative colitis (UC) was demonstrated. With the high sensitivity of the technique, low-degree mucosal neutrophil activation could also be quantified in patients with collagen colitis,UC in clinical remission and in patients with irritable bowel syndrome. <b>In study 2 and 3</b> the aim was to elucidate the dynamics of the rectal inflammatory response and nitric oxide (NO) production after rectal gluten challenge. We found a pronounced neutrophil activation in coeliac patients after rectal gluten challenge. This activation was apparent 4 hours after challenge and remains for at least 48 hours. A more modest eosinophil activation started 1-2 hours later and remained at least for 48 hours. The biphasic pattern of neutrophil and eosinonphil activation after challenge suggests a biphasic inflammatory reaction. The activation of neutrophils and eosinophils precedes a pronounced enhancement of mucosal NO production. Some of our coeliac patients displayed signs of an inflammatory reaction after rectal corn gluten challenge. <b>In study 4</b> the aim was to investigate the local inflammatory reaction to gluten and cow’s milk protein in CD patients in remission. The findings indicate that not only gluten sensitivity but also cow’s milk (CM) protein sensitivity is common in CD. The data support the hypothesis that CM sensitivity may contribute to persistent symptoms in coeliac patients on gluten-free diet.</p>

<p>Red blood cell-dependent hypoxic vasodilation is largely mediated via the delivery of NO through <em>S</em>-nitrosohemoglobin (Hb-SNO). Hb-SNO is regulated through allosteric and redox mechanisms that are not well understood. Part One of this dissertation explores the biochemical features of Hb micropopulations suggested to be involved in Hb-SNO synthesis. An NO-liganded mixed valency micropopulation was synthesized <em>in vitro</em> and identified spectroscopically as a ferric nitrosyl species (Fe(III)NO). Remarkably, this species was found to undergo a reaction that couples heme reduction and <em>S</em>-nitrosylation of β93C. The biochemical properties of this species were found to resemble those of Hb valency hybrids (VHy's) identified by others in previous work. The similarities between the two species are discussed, and a model for Hb-SNO formation, including the putative identification of the intermediates involved, is proposed. Part Two explores the insights provided by this chemistry as it is relevant to human pathophysiology in the context of Hb-SNO depletion of RBCs in stored blood. Hb-SNO and membrane <em>S</em>-nitrosothiols were found to be depleted after only two days of storage. The vasoactive function of stored RBCs has been shown in previous work to be impaired relative to controls. These findings, in conjunction with the this study, could implicate NO dysregulation as a primary component in the etiology of hypoxic diseases and the negative sequelae associated with blood transfusions. The results from Part Two suggest that NO blood gas measurements could serve an important role in improving clinical outcome of patients with hypoxic diseases. The goal of the work presented in Part Three is to begin to address this emerging need by developing an economical and pragmatic sensor platform for routine clinical use in both an outpatient and inpatient setting.</p><br>Dissertation

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

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin.<br>Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells.<br>The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation.<br>The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis.<br>The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets.<br>Leung Lai-han.<br>"July 2006."<br>Adviser: Ronald Ray Fiscus.<br>Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2006.<br>Includes bibliographical references (p. 175-191).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.

First study determined the effects of genetic deletion of nNOS on the levels of spontaneous apoptosis in brain of young-adult (2-3 months) and aged (12-18 months) mice, using nNOS-knockout mice with age-matched B6129SF2/J mice as wild-type control. The results indicate that aging resulted in 11-fold increase in levels of apoptotic-DNA-fragmentation in B6129SF2/J mouse brain. nNOS-knockout mice demonstrated dramatic (72-fold) increases in levels of apoptotic-DNA-fragmentation in young-adult, but not aged, brains. Aging resulted in decreased number of nNOS-positive cells, increased number of iNOS-positive cells and no change of eNOS-positive cells in control mice. The data suggest that nNOS may serve an anti-apoptotic/neuroprotective role in young-adult mouse brain. However, because of diminished nNOS and increased iNOS with aging, this neuroprotective effect may become less effective in aged mice.<br>Fourth study showed that new microchip-electrophoresis-technology can be successfully used to identify and quantify levels of apoptosic-DNA-fragments in brain slice cultures, similar to our previous studies with CE-LIF. Because of the much greater throughput of microchip-electrophoresis-system, compared to CE-LIF, this new technology should help accelerate the progress of apoptosis research.<br>In second study, apoptotic effects of genetic deletion of either eNOS or iNOS were studied using young-adult (1-4 months) and aged (12-24 months) eNOS- or iNOS-knockout mice with age-matched C57BL/6J wild-type control mice. The data show that both young-adult and aged iNOS-knockout mice had dramatically (8- to 36-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially noticeable in hippocampus and medulla oblongata. Both young-adult and aged eNOS-knockout mice also had dramatically (18- to 35-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially in cerebral cortex, hippocampus and medulla oblongata. The data suggest that both iNOS and eNOS provide neuroprotective effects, helping to limit the extent of spontaneous apoptosis in brain of young-adult and aged mice.<br>Nitric oxide (NO) has either pro-apoptotic or anti-apoptotic effects on neuronal cells, depending on concentration of NO produced by different source of NO synthases (NOSs) including neuronal-NO-synthase (nNOS/NOS-1), inducible-NO-synthase (iNOS/NOS-2) or endothelial-NO-synthase (eNOS/NOS-3) and possibly age of the individual. The present study determines if genetic deletion of nNOS, iNOS or eNOS alters levels of aging-induced apoptosis in vivo and hydrogen peroxide (H2O2)-induced-apoptosis in organotypic brain slice cultures using NOS-knockout mice. The quantitative ultrasensitive techniques using capillary-electrophoresis with laser-induced-fluorescent detector (CE-LIF) and Cell-Death---Detection-ELISA were used as novel ways to accurately measure the levels of apoptotic-DNA-fragmentation. Expressions of different forms of NOSs were determined by immunohistochemical-staining.<br>Third study determined H2O2-induced apoptosis in hippocampal and cerebellar slices from young-adult (8-10 weeks) and aged (12-24 months) C57BL/6J control mice, as well as iNOS- and eNOS-knockout mice (determined by Cell-Death-Detection-ELISA measuring levels of apoptotic-DNA-fragmentation). The data show spontaneous onset of apoptosis occurred in both hippocampal and cerebellar slices during culturing, beginning at 24 hours and progressively increasing for 48--72 hours. Staurosporine (positive-control) and H2 O2 both caused time-dependent increases in apoptosis in both hippocampal and cerebellar slices, compared to time-matched controls. Lastly, genetic deletion of iNOS greatly reduced levels of spontaneous apoptosis in young hippocampus and aged cerebellum, suggesting iNOS had contributed to induction of spontaneous apoptosis.<br>Chow Wing Han Vivian.<br>"Dec 2005."<br>Advisers: Siew Boon Chew Cheng; Ray Ronald Fiscus.<br>Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6218.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (p. 144-153).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.