<p>Regulation of the flux-generating enzyme complex pyruvate dehydrogenase (PDHc) was examined in the context of its physiological function in human skeletal muscle. In the first two studies, the role of PDHc in intramuscular fuel selection and the mechanisms regulating PDHc transformation from its inactive form (PDHb) to its active form (PDHa) and PDHa activity were examined. In a third study, the role of PDHc in muscle lactate production during exercise and recovery was assessed and the factors controlling transformation to PDHa were also examined.</p> <p>In the first study, 5 subjects were examined during rest and cycling exercise at 75% of VO₂max after 3 days of consuming a low-carbohydrate (LCD) or high-carbohydrate (RCD) diet In the second study, 8 subjects were examined at rest and during cycling exercise at 40% and 80% VO₂max while they were infused with sodium acetate (ACE) or sodium bicarbonate (BIC). At rest, consumption of a LCD and ACE infusion increased the intramuscular acetylCoA-to-CoASH ratio and citrate, as a result of increased oxidation of available fat fuels and acetate, respectively. Elevation of the acetylCoA-to-CoASH ratio and citrate inhibited PDHa and phosphofructokinase activity, respectively. Consequently, the rates of pyruvate oxidation by PDHa and pyruvate production by glycolysis were reduced preventing the oxidation of intramuscular glucose. The resting results of these two studies were consistent with the operation of a reciprocal cycle of glucose and fat oxidation for intramuscular energy production. The events leading to glucose restriction were initiated by changes in acetylCoA accumulation which caused an increase in the acetylCoA-to-CoASH ratio and citrate concentration which induced the transformation of PDHa to PDHb and inhibition of glycolytic flux, respectively.</p> <p>In contrast, during exercise at 40%, 75% and 80% VO₂max, transformation from PDHb to PDHa was determined by Ca²⁺ and was not restricted in any of the conditions except the LCD condition. Lower PDHa activity and incomplete transformation to PDHa occurred in this condition, placing a restriction on intramuscular glucose utilization. However, the acetylCoA-to-CoASH ratio actually decreased in this condition, suggesting that the lower rate of transformation was independent of alterations in the acetylCoA-to-CoASH ratio. In contrast, when transformation to PDHa was not limited in any other condition there was an increase in the acetylCoA-to-CoASH ratio which should have limited transformation. Thus, during exercise, the restriction on muscle glucose oxidation occurred only after a LCD indicating that this reciprocal cycle of fuel selection and utilization was also operating during exercise and that PDHc transformation was an integral part of its operation. However, during exercise, the regulatory factors controlling PDHa differed from those that determined the activity of this enzyme at rest. Lower PDHa during exercise in the LCD condition was consistent with an increase in the PDH-kinase/PDH-phosphatase activity ratio, in the 3 day period before exercise, increasing the occupancy of monophosphate esters on both the transformation site and the inhibitory sites of the Elα components. Incomplete removal of these additional phosphate esters by PDH-phosphatase during exercise may have then resulted in lower PDHa and glucose conservation later on during exercise.</p> <p>In a third study, the role of PDHa in muscle lactate production during exercise and recovery was examined and the factors controlling PDHc transformation were determined in 7 subjects. During repeated 30 second bouts of maximal isokinetic cycling exercise, complete transformation to PDHa occurred concurrently with muscle lactate accumulation and increased mitochondrial oxidation, indicating that lactate production was not dependent on the development of tissue hypoxia. Instead, during exercise muscle lactate production resulted from a rate of glycolytic pyruvate production that was greater than PDHa activity. Conversely, during recovery, net lactate oxidation occurred as the lactate dehydrogenase equilibrium shifted more toward pyruvate production and PDHa remained partially active due to attenuation of the acetylCoA-to-CoASH ratio, reductions in the ratios of NADH-to-NAD and ATP-to-ADP and elevated concentrations of hydrogen ion and pyruvate.</p> <p>The present studies extend the analysis of PDHc regulation from in vitro and in situ studies to human skeletal muscle in vivo. The findings of the present studies suggest that the during muscle contraction the most important factor regulating PDHc transformation in human muscle is probably Ca⁺, while the other regulatory factors perform secondary roles. In contrast, during rest and recovery from maximal exercise, PDHc transformation to PDHa and PDHa activity are determined by the intramuscular ratios of acetyICoA-to-CoASH, NADH-to-NAD, ATP-to-ADP and the concentrations of hydrogen ion and pyruvate.</p> / Doctor of Philosophy (PhD)

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology)--Stellenbosch University, 2008. / For many centuries, scientists have engaged in a theoretical debate concerning the etiology of mood disorders, with very few ancient scholars speculating about the importance of genetic factors and affective temperaments as factors in the etiology of depression. Mood, emotion and cognition have been shown to be modulated by the serotonergic midbrain raphe system; implicated in the pathogenesis of psychiatric disorders like those of the affective spectrum. Evidence from neuroscience, genetics, and clinical investigation demonstrate that depression is a disorder of the brain. Brain imaging research is revealing that in depression, neural circuits responsible for moods, thinking, sleep, appetite, and behavior fail to function properly, and that the regulation of critical neurotransmitters is impaired. Genetics research, including studies of twins, indicates that genes play a role in depression. Vulnerability to depression appears to result from the influence of multiple genes acting together with environmental factors. Other research has shown that stressful life events, particularly in the form of loss such as the death of a close family member, may trigger major depression in susceptible individuals. Depression and anxiety have often been successfully treated by means of selective serotonin reuptake inhibitors. However, selective serotonin reuptake inhibitors do not solve all the problems inherent to the treatment of depression, for approximately 30 % of depressed patients do not respond to treatment and 20 % experience relapses whilst on treatment. Of consideration is the fact that the majority of drugs today are based on proteins, with 50 % of therapeutics on the market targeting cell membrane proteins. Up to this day the precise pathophysiology of mood disorders remains obscure, as does the neurobiology of normal mood regulation. Accordingly, there is a need for methods to identify the structural and/or signaling components which lead to changes in the brain, particularly the hippocampus, of subjects having mood disorders such as bipolar depressive disorder, chronic major depressive disorder and the like. Similarly, there is a need for the early detection, screening and diagnosis of individuals at risk for a mood disorder. As the serotonin tranpsorter is the primary target for therapeutic intervention in the treatment of numerous psychiatric disorders and considering the fact that at the structural level this protein’s function as transporter in membranes remains incompletely understood, investigating its function in psychiatric disorders are of importance . The objective of this study was to determine the role of the serotonin transporter in wild type and serotonin knockout rats, with regards to the hippocampus. Rat hippocampi were fractionated into cytosolic and membrane components, which were run and further separated in two dimensions. Firstly separation occurred by isoelectrical focusing (pI), follwed by gel iii electrophoresis (molecular weight). Gels were compared to see whether protein spots have changed between animals that have been differentially bred. Differentially expressed protein spots, as determined by PD Quest software, were excised, digested and analyzed by means of mass spectrometry. Our results indicated that metabolic, structural and cell signaling proteins were differentially expressed in both the ventral and dorsal hippocampus of the serotonin knockout rat. Futhermore, cellular stress proteins were found to be only differentially expressed in the ventral hippocampus. The majority of proteins identified in both hippocampal areas as well as both fractions, were assigned to energy metabolism. The cytosolic protein profile mirrored the pattern of the membrane protein profile. In conclusion, this proteomic study identified various protein groups that interacted with one another, thus establishing compensation for disrupted serotonin homeostasis.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The worldwide escalation in the incidence of obesity and its strong association with insulin resistance, type 2 diabetes and the cardiovascular complications that accompany these disease states have elicited interest in the underlying mechanisms of these pathologies. Preliminary data generated in our laboratory showed that obesity is associated with abnormalities in the insulin signalling pathway. Specifically, we found a down-regulation of protein kinase B (PKB/Akt), which is known to mediate the metabolic effects of insulin. One of the downstream targets of PKB/Akt is glycogen synthase kinase-3 (GSK-3), which is inhibited by this phosphorylation. Detrimental effects of unopposed activity of GSK-3 have recently been described. This may play a pivotal role in some of the adverse consequences of insulin resistance in the heart. Hypothesis: Chronic inhibition of GSK-3 will induce myocardial hypertrophy or exacerbate the development of existing hypertrophy in a pre-diabetic model of diet induced obesity and insulin resistance. Objectives: (1) Assess the extent of the development of myocardial hypertrophy in a rat model of diet induced obesity (DIO) and insulin resistance. (2) Assess the effect of inhibition of GSK-3 protein on the development of myocardial hypertrophy. Methods: Two groups of age-matched male Wistar rats were used. Control animals received standard rat chow, while obese animals received a high caloric diet for 20 weeks. After 12 weeks, half of the animals in both groups received GSK-3 inhibitor treatment (CHIR118637, 30mg/kg/day, Novartis). At the end of 20 weeks, three series of experiments were conducted. (i) The animals were subjected to echocardiography to determine in vivo myocardial function, and biometric, metabolic and biochemical parameters were evaluated. (ii) The ability of the cardiomyocytes to accumulate deoxy-glucose after stimulation with insulin was determined, and (iii) the localization of key proteins was monitored using fluorescence microscopy and cell size was determined using light microscopy and flow activated cell sorter analysis. Results and discussion: The high caloric diet increased body weight (p<0.005) and intraperitoneal fat mass (p<0.01) when compared to controls. Complications associated with obesity, such as impaired glucose tolerance (p<0.05), hyperinsulinemia (p<0.0005) and an increased HOMA-IR index (p<0.01) were observed. Additionally, cardiomyocytes from the DIO animals had a significantly impaired response to insulin, specifically when 10nM (p<0.05) and 100nM (p<0.05) of insulin were used as stimulus. We also found a dysregulation in PKB/Akt, indicated by a down-regulation of phosphorylated PKB/Akt (p<0.01). The diet promoted the development of myocardial hypertrophy, since the ventricular weight (p<0.05) and ventricular weight to tibia length ratio were increased (p<0.01). Echocardiography experiments showed an increase in end diastolic diameter in the DIO animals (p<0.05). Additionally, there was an increase in the cardiomyocyte cell width in the DIO rats (p<0.0001) and a tendency for peri-nuclear localization of NFATc3. GSK-3 inhibition promoted the development of insulin resistance in control animals, as indicated by an increase in the body weight (p<0.05), serum insulin levels (p<0.01) and HOMA-IR index (p<0.01). In the DIO animals, the GSK-3 inhibitor treatment improved insulin resistance, as a decrease in serum insulin concentration (p<0.05) was observed. The cardiomyocytes from the treated DIO animals also showed an increase in glucose uptake (p<0.05) when stimulated with 100nM of insulin. The GSK-3 inhibitor promoted the development of myocardial hypertrophy in the control animals, indicated by an increase in ventricular weight (p<0.05) and cardiomyocyte cell width (p<0.0001), but did not exacerbate hypertrophy in the DIO animals. Conclusion: Both the high caloric diet and the GSK-3 inhibitor promoted the development of insulin resistance and myocardial hypertrophy in the rats. In the DIO animals the GSK-3 inhibitor treatment ameliorated insulin resistance and did not promote the further development of myocardial hypertrophy. / AFRIKAANSE OPSOMMING: Inleiding: Die huidige styging in vetsugtigheid en die sterk assosiasie daarvan met insulien weerstandigheid, tipe 2 diabetes en kardiovaskulêre komplikasies soos hipertrofie, het ‘n belangstelling in die onderliggende meganismes van hierdie siektetoestande ontlok. Voorlopige data uit ons laboratorium het getoon dat vetsug geassosieerd is met abnormaliteite in die insulien seintransduksie-pad soos byvoorbeeld ‘n afregulering van miokardiale proteïen kinase B (PKB/Akt), wat bekend is om die metaboliese effekte van insulien te medieer. Een van die proteïene wat deur PKB/Akt gefosforileer en daardeur geïnhibeer word, is glikogeen sintase kinase-3 (GSK-3). Negatiewe effekte van onge-opponeerde aktiwiteit van GSK-3 is beskryf en dit mag ‘n sleutelrol speel in sommige van die nadelige gevolge van insulien weerstandigheid in die hart. Hipotese: Chroniese onderdrukking van GSK-3 sal miokardiale hipertrofie ontlok of die bestaande hipertrofie in ‘n pre-diabetiese model van dieet-geïnduseerde vetsug en insulien weerstandigheid vererger. Doelstellings: (1) Om die omvang van die ontwikkeling van miokardiale hipertrofie in ‘n rotmodel van dieet-geïnduseerde vetsug te ondersoek en (2) om die effek van inhibisie van GSK-3 op die ontwikkeling van hipertrofie te ondersoek. Metodes: Ouderdomsgepaarde manlike Wistarrotte is in hierdie studie gebruik. Die diere is vir ‘n periode van 20 weke aan verskillende diëte onderwerp, naamlik standaard kommersiële rotkos vir die kontrole diere en ‘n hoë kalorie dieet vir die eksperimenteel vet diere (DIO). Helfte van elke groep diere is vir 8 weke met ‘n GSK-3 inhibitor behandel (CHIR118637, 30mg/kg/day, Novartis). Na die 20 weke is 3 eksperimentele reekse uitgevoer: (i) Die diere is eggokardiografies ondersoek om in vivo miokardiale funksie te bepaal en biometriese, metaboliese en biochemiese parameters is evalueer. (ii) Die vermoë van kardiomiosiete om de-oksiglukose na insulien stimulasie te akkumuleer, is bepaal, en (iii) die lokalisering van sleutelproteïene is met behulp van fluoressensie mikroskopie en die selgrootte met behulp van ligmikroskopie bepaal. Resultate en bespreking: Die hoë kalorie dieet het gepaard gegaan met ‘n beduidende toename in liggaamsgewig (p<0.005) en intraperitoneale vetmassa (p<0.01) in vergelyking met diere op die kontrole dieet. Newe-effekte geassosieerd met vetsug nl. onderdrukte glucose toleransie (p<0.05), hiperinsulinemie (p<0.0005) en ‘n verhoogde HOMA-IR index (p<0.01) is ook waargeneem. Daar was ook ‘n beduidend ingekorte respons van glukose opname deur kardiomiosiete van die vet diere na stimulasie met 10nM (p<0.05) en 100nM (p<0.05) insulien. Disregulering van PKB/Akt is gevind in die vorm van ‘n afregulering van die fosforilering van die proteïen (p<0.01). Die dieet het ook gelei tot die ontwikkeling van miokardiale hipertrofie aangesien die ventrikulêre gewig (p<0.05) asook die verhouding van die ventrikulêre gewig teenoor tibia lengte beduidend toegeneem het (p<0.01). Eggokardiografie het ‘n toename in ventrikulêre end-diastoliese dimensie in die DIO diere aangetoon (p<0.05). Tesame hiermee het die breedte van kardiomiosiete van die DIO diere toegeneem (p<0.0001) en daar was ook ‘n peri-nukluêre lokalisering van NFATc3. Behandeling van kontrole diere met ‘n GSK-3 inhibitor het insulienweerstandigheid ontlok soos afgelei uit ‘n verhoging in liggaamsgewig (p<0.05), serum insulien-vlakke (p<0.01) en die HOMA-IR waarde (p<0.01). In teenstelling het behandeling van die DIO diere met die GSK-3 inhibitor tot ‘n verbetering van insulienweerstandigheid gelei aangesien ‘n verlaging in serum insulien konsentrasies gevind is (p<0.05). Kardiomiosiete vanaf die behandelde DIO diere het ook ‘n verhoogde insulien-gestimuleerde glukose opname met 100nM insulien getoon (p<0.05). Behandeling met die GSK-3 inhibitor het die ontwikkeling van miokardiale hipertrofie in die kontrole diere teweeggebring, soos aangetoon deur ‘n toename in die ventrikulêre gewig (p<0.05) en ‘n groter selwydte in kardiomiosiete terwyl dit geen invloed op die bestaande hipertrofie van die vet diere gehad het nie. Gevolgtrekking: Die huidige studie het getoon dat die betrokke dieet asook behandeling met ‘n GSK-3 inhibitor insulienweerstandigheid sowel as die ontwikkelling van miokardiale hipertrofie in rotte ontlok. In die DIO diere het die behandeling met die GSK-3 inhibitor bloedglukose en insulien-vlakke verlaag en het nie hipertrofie vererger nie.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Introduction: Obesity and its associated complications, such as the metabolic syndrome, hypertension and cardiovascular disease, are escalating worldwide. In recognition of this, untested remedies advertised as anti-diabetic agents are flooding the market. Many of these products have limited efficacy, limited tolerability and significant side-effects. One remedy, claiming to have anti-diabetic properties, is DiaviteTM. DiaviteTM, a herbal product, consisting solely of the dried and ground pods of the Prosopis glandulosa tree, which is currently marketed as a food supplement with blood glucose and blood pressure stabilizing properties, as well as having the ability to enhance glucose utilization. It is already freely available from agents as well as sold over the counter at pharmacies. The producers of DiaviteTM are now seeking registration for their product from the Medicines Control Council (MCC) and, therefore, require solid scientific evidence of its effects. Aims: The aims of our study were, on request of the producing company, to determine the efficacy of DiaviteTM (P. glandulosa) as an anti-diabetic agent and possible mechanisms of action of this plant product. Methology: We utilized rat models of streptozotocin (STZ)-induced type 1 diabetes and diet-induced obese (DIO) insulin resistance. Male Wistar rats were rendered (a) type 1 diabetic after a once-off intra-peritoneal injection of STZ at a dose of 40 mg/kg and (b) insulin resistant after being on a high caloric diet (DIO) for 16 weeks. Half the animals of the type 1 diabetes model as well as the insulin resistant model were placed on DiaviteTM treatment (25 mg/kg/day) for a period of 4 – 8 weeks, depending on the model. The STZ-induced type 1 diabetic rats were sacrificed and the pancreata harvested for histological analysis. Animals on the DIO diet were sacrificed and (i) intra-peritoneal fat weight determined (ii) isolated hearts subjected to ischaemia/reperfusion to determine infarct size and protein expression profiles and (iii) cardiomyocytes prepared to determine insulin sensitivity. At the time of sacrifice blood was collected for blood glucose and serum insulin level determination, for both models. In addition, a standard toxicology study was performed in Vervet monkeys over a 3 month period. Results: In our type 1 diabetic model (blood glucose > 10 mmol/L) with a β-cell reserve, DiaviteTM treatment lead to increased serum insulin levels (p < 0.001) in both control and STZ groups as well as increased small β-cell (0 - 2500 μm2) formation (p < 0.001) in the pancreas of the STZ animals. Hearts from DiaviteTM treated control and DIO insulin resistant animals presented with smaller infarct sizes (p < 0.05) after ischaemia/reperfusion compared to their controls. DiaviteTM treatment lead to the increase of basal (p < 0.01) and insulin-stimulated (p < 0.05) glucose uptake in cardiomyocytes prepared from DIO insulin resistant animals. DiaviteTM treatment also led to significantly suppressed PTEN expression and activity (p < 0.01) in the DIO insulin resistant animals. In addition, DiaviteTM treatment had (i) no obvious detrimental effects in our rat models and (ii) no toxicity over a 3 month period in vervet monkeys. Conclusion: Our present study has shown that DiaviteTM treatment lowers fasting blood glucose levels, stimulates insulin secretion and leads to the formation of β-cells. In addition, oral consumption of DiaviteTM elicits cardioprotection against an ischaemic incident. DiaviteTM treatment improves insulin sensitivity of cardiomyocytes. Furthermore, it has been established that DiaviteTM treatment has no obvious detrimental effects in either of our rat models and no short-term toxic effects over a 3 month period in Vervet monkeys (data not shown). We thus conclude that in our models, DiaviteTM proved safe and it seems as if DiaviteTM, after short-term use, is beneficial as a dietary supplement. / AFRIKAANSE OPSOMMING: Inleiding: Vetsug, en die gepaardgaande komplikasies, soos die metaboliese sindroom, hipertensie en kardiovaskulêre siektes, neem wêreldwyd toe. Daar is tans verskeie middels op die mark wat as anti-diabetiese middels geadverteer word. Baie van hierdie geadverteerde produkte het beperkte effektiwiteit en het verskeie newe-effekte. Een so ‘n middel, is DiaviteTM. DiaviteTM is 'n plantproduk, wat slegs uit die gedroogte en fyngemaakte peule van die P. glandulosa boom bestaan. Hierdie produk word tans bemark as 'n voedselaanvulling met beide bloedglukose en bloeddruk stabiliserende eienskappe, asook die vermoë om glukose gebruik te verbeter. DiaviteTM is reeds vrylik beskikbaar van agente sowel as verkrygbaar by verskeie apteke. Die produsente van DiaviteTM wil aansoek doen om registrasie vir hul produk by die Medisynebeheerraad (MCC) en hulle vereis daarom wetenskaplike bewyse van die gevolge van die gebruik van hierdie produk. Doel: Die doel van ons studie was om op versoek van die produksie maatskappy, die doeltreffendheid van DiaviteTM (P. glandulosa) as 'n anti-diabetiese behandeling te evalueer, sowel as die moontlike meganismes van werking van hierdie plantproduk. Metodes: Ons het gebruik gemaak van rot modelle van (i) streptozotocin (STZ)-geïnduseerde tipe 1 diabetes en (ii) dieet-geïnduseerde vetsugtig (DIO) insulienweerstandigheid. Manlike Wistar rotte was as (a) tipe 1 diabeties geklassifiseer na 'n eenmalige, intra-peritoneale inspuiting van STZ teen 'n dosis van 40 mg/kg en as (b) insulienweerstandig geklassifiseer, nadat hulle op 'n hoë kalorie dieet (DIO) vir 16 weke was. Die helfte van beide die tipe 1 diabetes en die insulienweerstandige groep diere was met DiaviteTM behandel (25 mg/kg/dag) vir 'n tydperk van 4 - 8 weke, afhangende van die model. Die STZ-geïnduseerde tipe 1 diabetes rotte is geslag en die pankreata geoes vir histologiese analise. Diere op die DIO dieet is geslag en (i) die intra-peritoneale vet gewig bepaal, (ii) die geïsoleerde harte blootgestel aan isgemie/herperfusie om die infarkt groottes vas te stel, sowel as die proteïenuitdrukkingsprofiele te bepaal en (iii) kardiomiosiete was berei om die insulien sensitiwiteit te bepaal. Ten tyde van die slagting is bloedmonsters geneem vir bloedglukose en serum insulien vlak bepaling, vir beide modelle. Additioneel, is 'n standaard toksologie studie met Vervet apies oor 'n 3 maande tydperk uitgevoer. Resultate: In die model van tipe 1 diabetes (bloed glukose > 10 mmol/L), met 'n β-sel reserwe, is gevind dat DiaviteTM behandeling tot verhoogde serum insulien vlakke (p < 0.001) in beide kontrole en STZ groepe lei. DiaviteTM behandeling lei ook tot ‘n hoër vlak van klein β-sel (0 - 2500 μm2) vorming (p < 0.001) in die pankreas van die STZ diere. Die harte van die DiaviteTM behandele kontrole en DIO groep het kleiner infarkt groottes (p < 0.05) getoon na isgemie/herperfusie in vergelyking met hul kontrole groepe. DiaviteTM behandeling het ook gelei tot verhoogde basal (p < 0. 01) en insulin-gestimuleerde (p < 0. 05) glukose opname in kardiomiosiete wat berei was van DIO insulinweerstandige diere. DiaviteTM behandeling het PTEN uitdrukking en aktiwiteit aansienlik onderdruk (p < 0.01) in die DIO insulienweerstandige groep diere. Daar is dus gevind dat DiaviteTM behandeling (i) geen duidelike nadelige invloed in ons rot-modelle en (ii) geen toksisiteit oor 'n 3 maande tydperk in Vervet apies getoon nie. Gevolgtrekking: Ons huidige studie toon dus dat DiaviteTM behandeling vastende bloedglukosevlakke verlaag, insulien sekresie stimuleer en die proses van β-sell vorming bevorder. Additioneel, is gewys dat wanneer DiaviteTM mondelings gebruik word, dit die hart beskerm teen isgemiese insidente. Ons het ook getoon dat DiaviteTM behandeling insuliensensitiwiteit van kardiomiosiete verhoog. Verder is daar vasgestel dat DiaviteTM behandeling geen ooglopende nadelige gevolge in beide ons rot-modelle getoon het nie en daar geen korttermyn-toksiese effekte oor 'n 3 maande tydperk in Vervet apies (data nie getoon) is nie. Ons kan dus aflei dat Diavite TM in ons modelle veilig is en na kort termyn gebruik, voordelig is as 'n dieetaanvulling.

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 (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.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Myocardial oxidative fuel supply is increased in obese conditions. How this metabolic environment and altered cardiometabolic phenotype associated with prediabetic obesity impacts on cardiac function and tolerance to ischaemia/reperfusion injury remains uncertain. While obese individuals are likely to be treated with PPARα agonists, controversy exists as to how activation of the PPARα receptor influences cardiovascular function and post-ischaemic recovery. Aims: To determine in a model of hyperphagia-induced obesity 1) whether protracted obesity is associated with left ventricular (LV) mechanical dysfunction; 2) the responsiveness of these hearts to insulin stimulation; 3) whether insulin can afford cardioprotection against ischaemia/reperfusion damage; and 4) how obesity and chronic PPARα agonist (K-111) treatment influences myocardial function, substrate metabolism, mitochondrial function and post-ischaemic outcomes. Methods: Male Wistar rats were fed standard rat chow or a high caloric diet. 1) In vivo LV mechanical function was assessed echocardiographically in 32 week fed animals. Ex vivo LV function was measured in the presence of glucose, insulin and/or fatty acid (FA); 2) Ex vivo myocardial insulin sensitivity was assessed by measuring insulin stimulated glycolytic flux in 16 week fed rats. Insulin was also administered prior to and during regional ischaemia to determine its effect on post-ischaemic function and infarct size; 3) K-111 was added to the drinking water during the last 10 weeks of feeding (feeding period of 18 weeks); a) Ventricular mitochondrial function was determined polarographically in the presence of either glutamate or palmitoyl-L-carnitine as substrates; b) Myocardial carbohydrate and lipid metabolism, and in a separate series of perfusions, myocardial infarct size were determined in the presence of physiological or high insulin (30 or 50μIU/ml) and FA (0.7 or 1.5mM) concentrations. Results: 1) Obese animals maintained normal in vivo LV mechanical function. Glucose perfused hearts from obese animals had depressed aortic outputs compared to the control group (32.58±1.2 vs. 46.17±0.91 ml/min; p<0.001) which was abolished by the presence of FA; 2) Hearts from obese animals had reduced insulin stimulated glycolytic flux rates (1.54±0.42 vs. 2.16±0.57 μmol/g ww/min, p<0.01). Although insulin reduced infarct size in the obese group (20.94±1.60 vs. 41.67±2.09 %, p<0.001), its cardioprotective effect was attenuated in the presence of FA; 3) By simulating the in vivo metabolic environment of control and obese animals in ex vivo perfusions, elevated insulin and FA levels associated with obesity increased infarct sizes in the obese group compared to the control group (47.44±3.13 vs. 37.17±2.63 %, p<0.05); 4) While chronic K-111 treatment reversed systemic metabolic abnormalities associated with obesity, neither obesity nor the drug influenced myocardial and mitochondrial function or postischaemic outcomes. K-111 was able to reduce palmitate oxidation in the obese group. Conclusion: Elevated levels of circulating FFA may be important in maintaining normal LV mechanical function in the obese condition. While obesity had no impact on myocardial mitochondrial function and post-ischaemic outcomes during comparable perfusion conditions, the specific metabolic environment associated with obesity may augment post-ischaemic injury. K-111 is effective in reducing obesity related metabolic abnormalities, but has no effects on myocardial function, mitochondrial function or ischaemic tolerance. / AFRIKAANSE OPSOMMING: Agtergrond: Miokardiale oksidatiewe substraat voorsiening is verhoog in vetsug. Hoe hierdie metaboliese omgewing en veranderde miokardiale metaboliese fenotipe in prediabetiese vetsug miokardiale funksie en iskemie/herperfusie skade beïnvloed, is onseker. Alhoewel vetsugtige individue met PPARα agoniste behandel kan word, is die resultate verkry van hierdie reseptor aktivering op miokardiale funksie en iskemiese skade teenstrydig. Doelwitte: Om te bepaal of 1) verlengde vetsug linker ventrikulêre (LV) funksie beïnvloed; 2) hierdie harte sensitief vir insulien stimulasie is; 3) insulien die hart teen iskemie/herperfusie beskadiging beskerm; en of 4) vetsug en chroniese K-111 behandeling miokardiale funksie, substraat metabolisme, mitochondriale funksie en post-iskemiese herstel in vetsugtige, insulienweerstandige rotte beïnvloed. Metodes: Manlike Wistar rotte is met gewone rotkos, of ʼn hoé kalorie dieet gevoer. 1) In vivo LV funksie in 32 week gevoerde rotte is met behulp van eggokardiografie bepaal. Ex vivo LV funksie is met of sonder insulien en/of vetsure in die perfusaat bepaal; 2) Die ex vivo insuliensensitiwiteit is in 16 weke gevoerde rotte bepaal deur miokardiale glikolise te meet. Insulien is ook voor en tydens streeksiskemie toegedien, ten einde sy effek op miokardiale beskerming te bepaal; 3) K-111 is in die drink water van rotte toegedien vir die laaste 10 weke van hul dieet (voedingsperiode van 18 weke); a) Ventrikulêre mitochondriale funksie is polarografies bepaal in die aanwesigheid van glutamaat of palmitiel-L-karnitien; b) Miokardiale koolhidraat- en lipied metabolisme, en in ʼn aparte groep rotte, infarktgrootte, is bepaal in die teenwoordigheid van fisiologiese of hoë insulien- (30 of 50μIU/ml) en vetsuurvlakke (0.7 of 1.5mM). Resultate: 1) Vetsugtige rotte het normale in vivo LV funksie gehandhaaf. Glukose geperfuseerde harte van vet rotte se LV funksie was laer as die van kontroles (Aorta omset: 32.58±1.2 vs. 46.17±0.91 ml/min; p<0.001), maar dit het verbeter in teenwoordigheid van vetsure; 2) Harte van vetsugtige rotte het verlaagde insuliengestimuleerde glikolise getoon (1.54±0.42 vs. 2.16±0.57 μmol/g ww/min, p<0.01). Alhoewel insulien infarktgrootte in die vetsugtige groep verlaag het (20.94±1.60 vs. 41.67±2.09 %, p<0.001), is sy beskermende effekte in die teenwoordigheid van vetsure verlaag; 3) deur die in vivo metaboliese omgewing van kontrole en vetsugtige rotte in die perfusaat van die harte ex vivo te simuleer, is dit aangetoon dat die verhoogde vlakke van insulien en vetsure, geassosieer met vetsugtigheid, infarktgroottes in die vetsugtige groep teenoor die kontrole groep verhoog het (47.44±3.13 vs 37.17±2.63 %, p<0.05); 4) Hoewel chroniese gebruik van K-111 die metaboliese abnormaliteite gepaardgaande met vetsug normaliseer het, het beide vetsug en die middel geen invloed op miokardiale of mitochondriale funksie of vatbaarheid vir iskemiese beskadiging gehad nie. K-111 het miokardiale palmitaatoksidasie in die vetsugtige behandelde groep verlaag. Gevolgtrekking: Verhoogde bloed vetsuurvlakke in vetsug mag n belangrike rol in die handhawing van sistoliese funksie speel. Dit blyk dat die spesifieke in vivo omgewing geassosieer met vetsug wel tot verhoogte vatbaarheid vir iskemie/herperfusie skade mag lei. K-111 is effektief om die sistemiese metaboliese abnormaliteite gepaard met vetsugtigheid te verbeter, maar het geen effek op miokardiale funksie, mitochondriale funksie of vatbaarheid vir iskemie gehad nie.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Psychosocial stressors have frequently been associated with an increased risk for developing The contributions of the cholinergic (Lobeline) and opioid (Naltrexone) systems in place preference behaviour were determined by employing a post-methamphetamine pharmacological treatment strategy. These two treatments failed to reverse the methamphetamine-induced place preference. However, administration of the drugs did lead to alterations in striatal dopamine and serotonin levels which may infer beneficial effects against the biochemical alterations induced by methamphetamine. We used both 2-D gel-based proteomics and isobaric tagging for relative and absolute quantitation (iTRAQ) to identify proteins in the frontal cortex, and nucleus accumbens shell and core of rats that were subjected to maternal separation, methamphetamine or both regimes. The proteins were associated with cytoskeletal modifications, altered energy metabolism, degenerative processes, interruptions in normal neurotransmission and enhanced intracellular signalling. We found that more proteins were quantitatively expressed in rats that were exposed to maternal separation followed by methamphetamine treatment than those animals subjected to the individual interventions independently. Additional proteins recruited by the combination of MS followed by MA which remained unchanged with independent treatments included malate dehydrogenase, V-type proton ATPase subunit E1, beta-synuclein, brevican core protein, eukaryotic translation initiation factor 4H, histidine triad nucleotide binding protein 1 and stress-induced phosphoprotein in the nucleus accumbens shell subregion. Additional proteins recruited in the core subregion with the combination treatment included thymosin beta-4, calretinin, Arpp-21 protein, alpha-synuclein, ubiquitin carboxylterminal hydrolase isozyme L1, cytochrome c, brain acid soluble protein 1, prosaposin and stress-induced phosphoprotein 1. Although, on a behavioural level via the use of CPP we found that MS did not exacerbate the rewarding effects of MA, the proteomic data does infer a role played by early life stress by the recruitment of additional proteins. We therefore propose that the molecular mechanisms by which early adverse events predispose animals to the addictive state may involve a complex assembly of cellular processes within the brain. depression, anxiety or substance abuse in adult life. Animal studies have also suggested that stressful experiences may result in altered behavioural responses to drugs of abuse as evidenced by enhanced cocaine self-administration and psychostimulant-induced hyperlocomotor activity. The main aim of our study was to establish whether adversity early in life would render individuals more vulnerable to later drug usage. We adopted maternal separation as our animal model of early life adversity and treated these animals with methamphetamine during the adolescent stage of their life. A conditioned place preference (CPP) paradigm was subsequently used to determine the rewarding effects of methamphetamine. To obtain an understanding of the underlying molecular mechanisms of methamphetamine-induced behaviour, we measured neurochemical changes on a neuroendocrine, neurotrophic, neurotransmitter and proteome level. Firstly, we established that methamphetamine-induced place preference behaviour lasted for at least 2 weeks after the last methamphetamine administration. Contrary to expectation, this behaviour was not affected by prior exposure to maternal separation. However, rats subjected to maternal separation caused a decrease in apomorphine-induced locomotor behaviour in methamphetamine-treated rats. Maternal separation therefore preferentially affected the behavioural repertoire of the dorsal striatum rather than that of the ventral striatum. A general down regulation of neuroendocrine activity (ACTH and corticosterone levels) was observed in animals subjected to maternal separation or methamphetamine treatment, as well as those subjected to the combination of the two interventions. Increased concentrations of plasma prolactin levels in maternally separated as well as normally reared animals subjected to methamphetamine-CPP were found which suggested a reduction in dopamine inhibition. Maternal separation resulted in increased NGF levels in the ventral hippocampus of methamphetamine treated rats. This suggested that the ventral hippocampus may particularly be vulnerable to the effects of early life stress. The increased neurotrophin concentrations may reflect a compensatory response to stress and drug exposure. / AFRIKAANSE OPSOMMING: Psigososiale stressors word gereeld geassosieer met ‘n verhoogde risiko vir die ontwikkeling van depressie, angs en dwelm misbruik in volwassenheid. Diere studies het ook al bewys dat vroeë lewensstres in die vorm van moederlike skeiding lei tot veranderde gedrag teenoor dwelm misbruik. Hierdie veranderde gedrag veroorsaak deur moederlike skeiding sluit die verhoodge kokaïne toediening en psigostimulant geinduseerde verhoging in lokomotoriese aktiwiteit in. Die hoofdoel van die studie was om vas te stel of vroeë lewensstres mense meer vatbaar laat vir latere dwelm misbruik. ‘n Moederlike skeidings diere model was gebruik om vroeë lewensstres voor te stel and het verder hierdie diere behandel met metamfetamiene gedurende adolesensie. Die gekondisioneerde plek voorkeur model was gebruik om die euforiese / verslawende effekte van metamfetamiene te bepaal. Om die onderliggende molekulêre meganismes van metamfetamien geinduseerde gedrag te verstaan het ons neurochemiese veranderinge op ‘n neuroendokriene, neurotrofiese, neurotransmissie en proteinvlak vasgestel. Eerstens het ons was gestel dat metamfetamien geinduseerde plek voorkeur vir ten minste twee weke na die laaste metafetamien toediening voortduur. In teenoorstelling met verwagting, het moederlike skeiding nie metamfetamien geinduseerde plek voorkeur beinvloed nie, maar eerder apo-morfien geinduseerde lokomotoriese aktiwiteit geaffekteer. Moederlike skeiding stres het by voorkeur die gedrags funksie van die dorsale striatum beinvloed eerder as die ventrale gedragsfunksie. ‘n Algemene afregulering van neuroendokriene aktiwiteit was waargeneem (adrenokortikotrofiene en kortikosteroon vlakke) in diere wat aan moederlike skeiding of metafetamien behandeling sowel as die kombinasie behandeling blootgestel was. Verhoogde plasma prolaktien vlakke was gevind in moederlike skeidings rotte sowel as kontrole diere wat verder blootgestel is aan metamfetamien behandeling wat ‘n inhibisie van die dopamiene sisteem toon. Moederlike skeiding het ook ‘n verhooging in neurotrofiene (NGF) in die ventrale hippokampus van metamfetamien behandelde rotte veroorsaak. Hierdie bevinding stel voor dat die ventrale hippokampus veral vatbaar is vir die effekte van vroeë lewensstres. ‘n Verhoging in neurotrofien konsentrasies mag ‘n kompenserende teenslag van die brein wees teen stres en dwelm blootstelling. Die bydrae van die cholinergiese (Lobeline) en opiaat (Naltrexone) sisteme in plek voorkeur gedrag was bepaal deur farmaseutiese behandeling te volg na metamftemien toediening. Lobeline en naltrexone was egter nie suksesvol om die metamfetamien geinduseerde plek voorkeur te wysig nie. Alhoewel die toediening van die twee behandelings het tot veranderinge in neurotransmissie (dopamiene en serotoniene) gelei wat moontlik tot voordelige effekte teen die biochemiese veranderinge van metamfetamien kan lei. Om veranderinge op proteinvlak in die frontale korteks en nukleus akkumbens middel en buitenste subareas vas te stel het ons gebruik gemaak van twee-dimensie gel elektroforese en isobariese merkers vir relatiewe en absolute kwantifisering (iTRAQ) gevolg deur massa spektrofotometrie. Geindentifiseerde proteine was geassosieer met sitoskeletale modifikasies, veranderde energie metabolisme, afbrekende prosesse, onderbrekings met normale neurotransmissie en intrasellulêre seintransduksie. Meer proteine was beduidend in die diere wat aan beide moederlike skeiding en metamfetamien behandeling blootgestel was. Addisionele proteine wat deur die kombinasie behandeling geaffekteer is in die buitenste subarea van die nukleus akkumbens sluit ‘malate dehydrogenase’, ‘V-type proton ATPase subunit E1’, ‘beta-synuclein’, ‘brevican core protein’, ‘eukaryotic translation initiation factor 4H’, ‘histidine triad nucleotide binding protein 1’ en ‘stress-induced phosphoprotein’ in. Additionele proteine geaffekteer in die middelste subarea van die nukleus akkumbens sluit ‘thymosin beta-4’, ‘calretinin’, ‘Arpp-21 protein’, ‘alpha-synuclein’, ‘ubiquitin carboxylterminal hydrolase isozyme L1’, ‘cytochrome c’, ‘brain acid soluble protein 1’, ‘prosaposin’ en ‘stress-induced phosphoprotein 1’ in. Vanuit ‘n gedrags benadering deur die gebruik van metamfetamien geinduseerde plek voorkeur het moederlike skeiding nie diere meer vatbaar gemaak vir die effekte van metamfetamien nie, maar die protein data wys wel dat vroeë lewens stres ‘n rol speel deur dat meer proteine geaffekteer word deur die kombinasie van moederlike skeiding gevolg deur later metamfetamien toediening. Ons stel voor dat die molekulêre meganismes waardeur vroeë lewensstres diere meer vatbaar maak vir die verslawende effekte van stimulante behels ‘n komplekse samestelling van sellulêre prosesse in die brein.

<p>Giant cell tumour of bone (GCT) is an aggressive primary bone tumour with an unclear etiology that presents with significant local osteolysis due in part to the accumulation of multinucleated osteoclast-like giant cells. However, it is the neoplastic spindle-like stromal cells within GCT that largely direct the pathogenesis of the tumour. I hypothesize that parathyroid hormone-related protein (PTHrP) is a key mediator within GCT that promotes the characteristic osteolytic phenotype by stimulating both bone resorption and giant cell formation. The work presented in this thesis collectively demonstrates that the stromal cells express PTHrP and its receptor, the parathyroid hormone type 1 receptor (PTH1R), and that PTHrP acts in an autocrine/paracrine manner within the tumour to stimulate expression of factors that promote bone resorption. Data are presented that demonstrate that PTHrP stimulates stromal cell expression of the receptor activator of nuclear factor-κB ligand (RANKL), a known essential regulator of osteoclastogenesis, which results in increased formation of multinucleated cells from murine monocytes. Moreover, the GCT stromal cells express matrix metalloproteinase (MMP)-1 and MMP-13. These results suggest that the stromal cells may participate directly in bone resorption through the degradation of type I collagen, the promotion of osteoclast activity, or through a combination of these elements. PTHrP also regulates the expression of MMP-13 by the stromal cells. Experiments with CD40 ligand show that local factors present within the tumour can influence PTHrP expression by the stromal cells and potentiate its catabolic effects by stimulation of RANKL and MMP-13 expression. Together, this thesis presents evidence that suggests PTHrP is an important factor in the pathophysiology of GCT by its actions on promoting catabolism within the tumour. The role of PTHrP in normal physiology and the mechanisms of action presented here suggest that research into the effects of PTHrP within GCT may provide invaluable information that enhances our understanding of the biology of this particularly aggressive bone tumour.</p> / Doctor of Philosophy (PhD)

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model. / AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.