Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Nitric oxide (NO) is an important chemical messenger in the cardiovascular system. Despite considerable progress in this field, there remains an on-going need for affordable and user-friendly NO measurement techniques. Therefore, in this study we aimed to develop and characterise NO-detection techniques not previously used in our laboratory, and, in addition, characterise an ex vivo method to measure the functional effects of the endothelium and NO production in the vasculature. Methods: Three different NO-detection techniques were compared: (i) Amperometric NO sensors. Here, NO-increasing effects of known NO synthase (NOS) activators were investigated (insulin, acetylcholine and biosynthetic human insulin). Three different NO sensors were evaluated on cultured endothelial cells and aortic tissue. Putative NOincreasing effects of shear stress were also investigated; (ii) Nitrite (NO2 -) + nitrate (NO3 -) sensors. Here, I aimed to measure NO release from cultured endothelial cells; (iii) Colorimetric NO2 - measurement assay with the Griess reagent. Here, NO2 - production by endothelial cells was measured with a plate reader. In the second part of the study an organ bath - isometric tension technique was established to measure endothelium-dependent function of aortic rings. Functional differences in aortic rings isolated from diet-induced obese rats compared to lean rats were investigated. Ring contraction was induced with phenylephrine and relaxation with acetylcholine. These investigations were further supported by western blot analyses of selected critical proteins. Lastly, the effects of perivascular adipose tissue (PVAT) on contraction and relaxation were investigated in endothelium-containing or denuded aortic ring segments. Results: Although some success was achieved with the amperometric sensors regarding calibration, any experimental results obtained were difficult to repeat due to instability of the sensors. With the NO2 -/NO3 - sensor we were not able to carry out any planned experiments due to failure to properly calibrate and standardise the sensors. Success was achieved with the Griess method. All the drugs used as positive controls (DEA/NO, fenofibrate, oleanolic acid and IL-1ß) proved to be potent inducers of NO2 - release from endothelial cells. Interestingly, the isometric tension studies showed a higher % relaxation in high fat (HF) diet aortic rings compared to those from lean animals. Western blot data showed downregulation of eNOS activation and iNOS expression in obese groups, which was suggestive of endothelial dysfunction. Interestingly, proteins associated with oxidative stress (p22phox and nitrotyrosine) were downregulated in obese groups. The presence of PVAT exerted anti-contractile effects on the rings from HF rats, however in denuded aortic rings, PVAT showed a significant pro-contractile response in both lean and HF groups. PVAT also exerted anti-relaxation effects in aortic rings from both lean and HF rats. Conclusion: We managed to successfully establish two new techniques for our laboratory (Griess method and the organ bath – isometric tension method) which can complement the more established techniques in our laboratory in order to aid us in future vascular research. Finally, the isometric tension technique used in the obese rat studies generated interesting data, which further assisted in characterising the dietinduced obesity rat model in our laboratory. / AFRIKAANSE OPSOMMING: Inleiding: Stikstofoksied (NO) is ‘n belangrike chemiese boodskapper in die kardiovaskulêre sisteem. Ondanks vordering in die veld, bestaan daar ‘n aangaande behoefte aan bekostigbare en gebruikersvriendelike NO-metingstegnieke. Gevolglik het ons in hierdie studie daarna gemik om NO-metingstegnieke wat nie vantevore in ons laboratorium beskikbaar was nie, te ontwikkel en karakteriseer. Verder het ons ten doel gehad om ‘n ex vivo model te karakteriseer om die funksionele effekte van vaskulêre endoteel en NO produksie te meet. Metodes: Drie verskillende NO-metingstegnieke was ondersoek: (i) Amperometriese NO sensors. Hier het ons die verhogende effekte op NO van bekende aktiveerders van NO sintetase (NOS) ondersoek (Insulien, asetielcholien en biosintetiese menslike insulien). Drie verskillende NO-sensors was ge-evalueer in gekultuurde endoteelselle en aortaweefsel. Die vermeende NO verhogende effekte van die wrywingskragte opgewek deur laminere vloei (“shear stress”) is ook ondersoek. (ii) Nitriet (NO2 -) + nitraat (NO3 -) sensors. Hier het ons beplan om NO-vrystelling deur gekultuurde endoteelselle te meet. (iii) Kolorimetriese meting van NO2 - met die Griess reagens. Hier het ons m.b.v. ‘n mikroplaat leser die NO2 - - vrystelling deur endoteelselle gemeet. In die tweede deel van die studie het ons ‘n orgaan bad–isometriese spanningstegniek opgestel om endoteelafhanklike funksie van aortaringe te meet. Funksionele verskille in aortaringe van vetsugtige rotte is vergelyk met kontrole rotte. Ringkontraksie is met fenielefrien geïnduseer en verslapping met asetielcholien. Hierdie ondersoeke is verder ondersteun deur Western blot analises van sleutelproteïene in die aortaweefsel. Laastens het ons die effekte van perivaskulêre vetweefsel (PVAT) op kontraksie en verslapping in aortaringe met of sonder intakte endoteel ondersoek. Resultate: Alhoewel ‘n mate van sukses behaal was met die kalibrasie van die amperometriese sensors, was eksperimentele resultate moeilik om te herhaal a.g.v. sensor-onstabiliteit. Geen eksperimente kon met die NO2 -/NO3 - sensors uitgevoer word nie weens ‘n onvermoë om ordentlike kalibrasie en standardisering uit te voer. Ons het egter wel sukses behaal met die Griess-metode. Al die middels wat as positiewe kontroles gebruik was (DEA/NO, fenofibraat, oleanoliese suur and IL-1ß) het geblyk kragtige induseerders van NO2 - produksie vanaf endoteelselle te wees. Die isometriese spanningsstudies het ‘n hoer % verslapping getoon in die hoë vet (HF) dieet aortaringe in vergelyking met die kontroles. Western blot data het ‘n afregulering van eNOS en iNOS getoon in die HF diere, wat aanduidend is van endoteel disfunksie, terwyl proteïene geassosieer met oksidatiewe stress (p22phox en nitrotirosien) afgereguleer was in die HF groep. Die aanwesigheid van PVAT het ‘n anti-kontraktiele effek gehad op die ringe van die HF groep. Toe die endoteel egter verwyder was, het PVAT in beide kontrole en HF ringe ‘n beduidende pro-kontraktiele effek gehad. Verder het PVAT ook anti-verslappingseffekte op aortaringe beide kontrole en HF rotte uitgeoefen. Gevolgtrekking: Ons het daarin geslaag om twee nuwe tegnieke vir ons laboratorium suksesvol te vestig (Griess metode en die orgaanbad-isometriese spanningstegniek) wat in die toekoms die meer gevestigde tegnieke in ons laboratorium kan komplementeer. Laastens het die isometriese spanningstegniek wat in die dieetstudies gebruik is, data opgelewer wat ons verder sal help om die vetsug model in ons laboratorium te karakteriseer.

Thesis (MScMedSc)--Stellenbosch University, 2015. / 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. / 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.

Thesis (PhD)--Stellenbosch University, 2012. / Salinity stress is one of the major environmental factors that lead to poor crop yield. This is due to overproduction of reactive oxygen species (ROS) which consequently lead to oxidative stress. Although these ROS may be required for normal physiological functions, their accumulation acts as a double edge sword, as they also cause oxidative damage to nucleic acids, lipids and proteins of plant cell membranes. Plants have evolved with an efficient antioxidant defensive system in order to protect and detoxify harmful effects of ROS. Ascorbate peroxidase (APX) is regarded as one of the major scavengers of H2O2. Although some studies have described the role of nitric oxide (NO) in diverse physiological processes in plants, there is still much to know as regards to modulation of APX activity by nitric oxide in salinity-induced stressed plants. For the purposes of this study, the effect of salt and exogenously applied NO on APX, dehydroascorbate reductase and antioxidant metabolite content was determined. This study investigated the use of NO donor 2,2'-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) and diethylenetriamine (DETA) on soybean. The data obtained from this study shows that application of DETA/NO resulted in an increase of NO nodular content and also regulated APX activity. The NO-induced changes in APX enzymatic activity were coupled to altered nodule H2O2 content. Further analysis of APX enzymatic activity identified three APX isoforms for which augmented enzymatic activity occurred in response to NO. By supplementing salinity-induced stress soybeans with NO, this study shows that tolerance to salt stress is improved. The underlying mechanism of the NO-mediated tolerance to salt is shown to be its role in modulating the plant antioxidant defense system thus maintaining redox status under salinity-induced stress. Here, although there was increased APX activity in salt stressed plant, supplementing the salinity-induce stressed plants with NO resulted to even higher APX activity which was sufficient to detoxify ROS. Furthermore, this study shows that the NO-mediated effect is not limited in antioxidant enzymes but also involves regulating antioxidant metabolite ratio through modulating the antioxidant enzymes that are involved in the ascorbate -glutathione cycle.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2007. / Nitric oxide (NO) is a major signaling molecule in the heart with various biological effects. The putative role of NO as a cardioprotective agent against ischaemiareperfusion injury and in ischaemic preconditioning (IP) has made it one of the fastest growing fields in basic cardiovascular research. However, NO may also be associated with harmful effects, especially when released in excessive amounts. Little is known about the relative contributions to NO-production by the cardiac microvascular endothelial cells (CMECs) and the adjacent cardiomyocytes. Furthermore, the respective roles of endothelial NOS (eNOS) and inducible NOS (iNOS) are not well characterized in these cell types, particularly in hypoxia. In order to gain a better understanding of the role of NO in the hypoxic/ischaemic heart, the aims of this study were to: (1) develop an isolated cardiomyocyte model in which hypoxia and early IP can be induced and the role of NO assessed; (2) measure NOproduction in cardiomyocytes and CMECs under baseline and hypoxic conditions; and (3) evaluate the expression, regulation and activation of eNOS and iNOS in cardiomyocytes and CMECs (baseline and hypoxia) and establish the relationship with NO-production under these conditions. Cardiomyocytes isolated from adult rat hearts and commercially purchased rat CMECs were used as cell models.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Physics / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Described herein are three projects centered on the synthesis of conducting polymer derivatives for various applications. The first is the novel synthesis of 9,9-dioctylfluorene-co-benzothiadiazole [F8BT] oligomers through solid phase synthesis for the study of the thermodynamics and kinetics of electron transfer in the polymer. The second endeavor involves the synthesis of a series of 4”,3’’’-dialkyl-2,2’:5’,2”:5”,2’’’:5’’’,2’’’’:5’’’’,2’’’’’-sexithiophenes for the studies on crystal packing and surface deposition of organic p-type semiconducting materials. Lastly is described the development of a conducting metallopolymer based on the ligand 2,6-Bis(4-(2,2’-bithiophen-5-yl)-1H-pyrazol-1-yl)pyridine for use in a drug delivery system. / text

Oleic acid (18:1), a monounsaturated fatty acid (FA), is synthesized upon desaturation of stearic acid (18:0) and this reaction is catalyzed by the plastidal enzyme stearoyl-acyl carrier protein-desaturase (SACPD). A mutation in the SSI2/FAB2 encoded SACPD lowers 18:1 levels, which correlates with induction of various resistance (R) genes and increased resistance to pathogens. Genetic and molecular studies have identified several suppressors of ssi2 which restore altered defense signaling either by normalizing 18:1 levels or by affecting function(s) of a downstream component. Characterization of one such ssi2 suppressor mutant showed that it is required downstream of low 18:1-mediated constitutive signaling and partially restores altered defense signaling in the ssi2 mutant. Molecular and genetic studies showed that the second site mutation was in the Nitric Oxide Associated (NOA) 1 gene, which is thought to participate in NO biosynthesis. Consistent with this result, ssi2 plants accumulated high levels of NO and showed an altered transcriptional profile of NO-responsive genes. Interestingly, the partial defense phenotypes observed in ssi2 noa1 plants were completely restored by an additional mutation in either of the two nitrate reductases NIA1 or NIA2. This suggested that NOA1 and NIA proteins participated in NO biosynthesis in an additive manner. Biochemical studies showed that 18:1 physically bound NOA1, in turn leading to its degradation in a protease-dependent manner. In concurrence, overexpression of NOA1 did not promote NO-derived defense signaling in wild-type plants unless 18:1 levels were lowered. Subcellular localization showed that NOA1 and the 18:1-synthesizing SSI2 were present in close proximity within the nucleoids of chloroplasts. Indeed, pathogen- or low 18:1- induced accumulation of NO was primarily detected in the chloroplasts and their nucleoids. Together, these data suggested that 18:1 levels regulate NO synthesis and thereby NO-mediated retrograde signaling between the nucleoids and the nucleus. Since cellular pools of glycerol-3-phosphate (G3P) regulate 18:1 levels, I next analyzed the relationship between G3P and 18:1. Interestingly, unlike 18:1, an increased G3P pool was associated with enhanced systemic immunity in Arabidopsis. This was consistent with G3P-mediated transcriptional reprogramming in the distal tissues. To determine mechanism(s) underlying G3P-conferred systemic immunity, I analyzed the interaction between G3P and a lipid transfer protein (LTP), DIR1. In addition, I monitored localization of DIR1 in both Arabidopsis as well as tobacco. Contrary to its predicted apoplastic localization, DIR1 localized to endoplasmic reticulum and plasmodesmata. The symplastic localization of DIR1 was confirmed using several different assays, including co-localization with plasmodesmatal-localizing protein, plasmolysis and protoplast-based assays. Translocation assays showed that G3P increased DIR1 levels and translocated DIR1 to distal tissues. Together, these results showed that G3P and DIR1 are present in the symplast and their coordinated transport into distal tissues is likely essential for systemic immunity. In conclusion, this work showed that low 18:1-mediated signaling is mediated via NO, synthesis of which is likely initiated in the plastidal nucleoids. In addition, my work shows that G3P functions as an independent signal during systemic signaling by mediating translocation of the lipid transfer protein, DIR1.