Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Introduction: Obesity, which is implicated in the development of the metabolic syndrome (MS) is reaching epidemic proportions worldwide. MS significantly increases the risk of developing cardiovascular disease, which includes coronary artery disease. The current absence of animal models of diet induced obesity and the MS makes the investigation of the cardiovascular consequences of MS virtually impossible. As a result the effects of the MS on cardiac function, morphology and susceptibility to ischaemia are not well understood. Aims: We set out to: 1) develop and characterize a rodent model of dietinduced obesity and the MS, 2) investigate the susceptibility of hearts from these animals to ischaemia/reperfusion induced injury and, 3) determine whether angiotensin II (Ang II) and endothelin-1 (ET-1) plays a role in cardiac remodelling and/or the severity of ischaemia and reperfusion injury in this model. Methods: Male Wistar rats were fed a standard rat chow diet or cafeteria diet (CD) for 16 weeks. After the feeding period rats were sacrificed and blood and myocardial tissue samples were collected to document biochemical changes in these animals. Hearts were perfused on the isolated working rat heart perfusion apparatus to assess myocardial mechanical function before and after ischaemia. In a separate series of experiments, hearts underwent coronary artery ligation to determine the incidence and duration of ventricular arrhythmias during ischaemia and reperfusion, using electrocardiography. To assess a possible link between myocardial remodelling and ischaemia/reperfusion injury and myocardial Ang II and ET-1 content, we also measured these peptides under basal conditions and during ischaemia. Two-dimensional targeted Mmode echocardiography was used to assess in vivo myocardial mechanical function in control and obese rats. Results: After 16 weeks on the CD, obese rats satisfied the World Health Organization (WHO) criteria for the MS by having visceral obesity, insulin resistance, dyslipidaemia and an elevated systolic blood pressure, compared to control rats. Circulating Ang II levels, but not ET-1 levels, were elevated in CD fed rats. Obese rats had cardiac hypertrophy and ex vivo basal myocardial mechanical function was depressed in the CD fed rat hearts compared to control rat hearts. CD fed rat hearts had poorer aortic output (AO) recoveries compared to hearts from control rats. These hearts also had a higher incidence and duration of reperfusion arrhythmias. No such functional differences were seen in the in vivo experiments. No differences in basal or ischaemic myocardial Ang II and ET-1 levels were seen in either group. Conclusion: We have developed and characterized a model of diet-induced obesity and the MS. Obesity is associated with cardiac hypertrophy and an increased myocardial susceptibility to ischaemia and reperfusion injury in our model. The hearts from obese rats were also more prone to reperfusion ventricular arrhythmias. As myocardial function was only poorer in the ex vivo obese animal experiments, our data suggests that the obesity associated changes in function observed in the ex vivo studies may be related to the absence of circulating substrates or factors, which are essential for their normal mechanical function.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility. / AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid, beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer rondom die ware potensiaal van hierdie parameter weens die gebrek aan standardisering van verskillende kleuringstegnieke wat gebruik word vir die evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue® (SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp. Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate, word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer (SCA®). Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het. Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel. Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge, word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer. Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie sperme met goeie beweeglikheid.

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.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: INTRODUCTION: Obesity and its associated complications such as diabetes and cardiovascular disease are escalating worldwide. Cardiovascular mortality and the occurrence of heart failure following a myocardial infarction are increased among diabetics. A high caloric diet has been associated with specific metabolic alterations such as increased FA utilization and decreased glucose utilization. We therefore hypothesized that, in a rat model of diet-induced obesity, pathways involved in myocardial glucose utilization would be down regulated with simultaneous up regulation of FA utilization pathways and that this will lead to certain metabolic adaptations which will eventually become maladaptive. We aimed to elucidate mitochondrial oxidative capacity, biogenesis, and signaling pathways involved in substrate utilization and energy production in rats on the obesity inducing diet for a period of 8 or 16 weeks. METHODS: The diet of male Wistar rats (180-200 g) was supplemented with sucrose and condensed milk for 8 or 16 weeks (DIO) and compared to age-matched controls. We determined the fasting blood glucose and serum insulin levels, which was used to calculate the HOMA index. Furthermore, (i) A set of hearts were removed and freeze-clamped immediately. (ii) Isolated hearts were perfused with Krebs-Henseleit buffer (10 mM glucose), subjected to regional ischaemia by ligating the left anterior descending artery (35 min). After 10 min reperfusion, the infarcted and non-infarcted zones were freeze-clamped separately. Isolated mitochondria prepared from fresh tissue were used to measure oxidative capacity using either glutamate or palmitoyl-L-carnitine as substrates and exposed to anoxia (20 min) /reperfusion and used in e- transport chain complex analysis. Additionally we determined (i) ATP production (HPLC), (ii) citrate synthase activity and quantity as measure of active mitochondria per mg of protein and (iii) PDH complex expression and activity (ELISA). Levels of GLUT1, GLUT 4, PGC-1α, PPARα, PKB/Akt, GSK-3, PTEN, AMPK and PI3K activity were measured via Western blotting and Real-time PCR was used to measure the expression of PDK 4 and FAT/CD36. RESULTS: The blood glucose and serum insulin levels were significantly elevated in the diet group after 8 weeks of DIO. The PPARα, PGC-1α and PDK 4 levels were also significantly elevated in the diet group with no significant difference in the levels of any of the other proteins measured or the level of citrate synthase activity. After 16 weeks of DIO the citrate synthase, PTEN and p-PI3K activity was significantly reduced and the %recovery after anoxia/reperfusion when using palmitoyl was significantly increased in the diet group. There was no change in mitochondrial oxidative function in both groups after 8 and 16 weeks, as well as no difference in ATP production during state 3 respiration. CONCLUSION: The increased blood glucose and serum insulin levels as well as the increase in the HOMA index in the diet group after 8 weeks of DIO indicates that these obese animals were insulin resistant. An increase in the level of PPARα and PGC-1α expression indicates an early compensatory effect which facilitates enhanced fatty acid utilization. This is underscored by elevated levels of PDK 4. We could find no significant difference in the quantity of the PDH enzyme but there was a significant increase in the level of PDH activity in the diet group after 16 weeks of DIO. Mitochondria from the 16 weeks DIO animals were able to withstand anoxia/reperfusion and showed no malfunctioning of the electron transport chain, despite a reduction in PI3K & PTEN activity and the presence of insulin resistance. Mitochondrial biogenesis does not seem to play a significant role in the heart‟s adaptive response as there was no increase in the citrate synthase activity in both groups. We conclude that the hearts from these obese and insulin resistant rats are coping well and have adapted metabolically to compensate for any reduction in glucose oxidation. It is plausible that this initial metabolic adaptation may become maladaptive as obesity progresses. / AFRIKAANSE OPSOMMING: INLEIDING: Vetsug en die geassosieerde komplikasies, naamlik diabetes en kardiovaskulêre siektes is besig om wêreldwyd toe te neem. Kardiovaskulêre sterftes en die voorkoms van hartversaking ná 'n miokardiale infarksie is verhoog onder diabete. 'n Dieet met 'n hoë vetinname word ook geassosieer met spesifieke metabolise veranderings, soos verhoogde vetsuur gebruik en 'n afname in glukoseverbruik. Ons het dus vermoed dat seintransduksie paaie betrokke by miokardiale glukose verbruik afgereguleer sal wees met tesame opreguleering van seintransduksie paaie betrokke by vetsuur verbruik, en dat dit sal lei tot sekere metaboliese aanpassings wat uiteindelik wanaanpaslik sal word. Ons DOESTELLING in hierdie studie was dus om meer lig te werp op mitokondriale oksidatiewe kapasiteit, biogenese, en seintransduksie betrokke by substraatverbruik en energieproduksie in rotte op die vetsugveroorsakende-dieet (vir 'n tydperk van 8 tot 16 weke). METODE: Die dieet van die manlike Wistar rotte (180-200 g) is aangevul met sucrose en gekondenseerde melk vir 8 of 16 weke (DIO) en is dan vergelyk met kontrole rotte van dieselfde ouderdom. Die vastende bloedglukose-en insulienvlakke is bepaal en hierdie waardes is gebruik om die HOMA indeks te bereken. Verder is, (i) 'n stel harte verwyder en onmiddelik gevriesklamp (ii) geïsoleerde harte met Krebs-Henseleit buffer (10 mM glukose) geperfuseer en dan aan 35 min streeksiskemie en 10 min herperfusie blottgestel. Na 10 min herperfusie, is die infarct en nie-infarktsones apart gevriesklamp. Geïsoleerde mitokonderieë is voorberei van vars weefsel en is gebruik a) om oksidatiewe kapasiteit te meet met behulp van glutamaat of palmitoiel-L-karnitien as substrate of b) blootgestel aan 20 min anoksie gevolg deur heroksigenasie of c) is gebruik in elektronvervoerkettingkompleks analise. Verder is die volgende ook bepaal: (i) ATP-produksie (HPLC), (ii) sitraat sintase aktiwiteit en hoeveelheid, gemeet as maatstaf van die aktiewe mitkondrieë per mg van proteïen en (iii) PDH-komplekuitdrukking en aktiwiteit (ELISA). Vlakke van GLUT 1, GLUT 4, PGC-1 alpha, PPAR alpha, PKB/Akt, GSK-3, PTEN, AMPK, en PI 3 kinase is bepaal deur Western klad analise en “Real –time PCR” is gebruik om die uitdrukking van PDK 4 en FAT/CD 36 te bepaal. RESULTATE: Die bloedglukose- en serum insulinvlakke was beduidend verhoog in die dieetgroep na 8 weke van DIO. Die PPAR alpha, PGC-1 alpha en PDK 4 vlakke was ook beduidend verhoog in die dieetgroep met geen beduidende verskil in die vlakke van enige van die ander proteïene gemeet of die vlakke van sitraat-sintase aktiwiteit nie. Na 16 weke van DIO het die vlakke van sitraat-sintase, PTEN en p-PI 3 kinase beduidend verlaag en die % herstel na anoksie/heroksigenering (met die gebruik van palmitoiel-L-karnitien) het beduidend toegeneem in die dieetgroep. Geen ander mitokondriale veranderinge kon waargeneem word nie. Ons kon geen verandering vind in die mitokondriale oksidatiewe funksie tussen die twee groepe na 8 en 16 weke van DIO. Daar was ook geen verandering in die hoeveelheid ATP geproduseer gedurende staat 3 respirasie. GEVOLGTREKKING: Die verhoogde bloedglukose en serum insulienvlakke, sowel as die toename in die HOMA indeks in die dieetgroep na 8 weke van DIO, dui daarop dat hierdie oorgewig diere insulienweerstandig is. 'n Toename in die vlak van PPAR alpha en PGC-1 alpha uitdrukking dui op 'n vroeë kompenserende effek, wat die verhoogde verbruik van vetsure fasiliteer. Dit is beklemtoon deur verhoogde vlakke van PDK 4. Ons kon geen beduidende verskil in die hoeveelheid PDH-ensiem vind nie, maar daar was wel 'n beduidende toename in die vlak van PDH aktiwiteit in die dieetgroep na 16 weke van DIO. Mitokondrieë van die 16 weke DIO diere kon anoksie/heroksigenering weestaan en het geen wanfunksionering van die elektronvervoerketting getoon nie, ten spyte van 'n vermindering in PI 3 kinase en PTEN aktiwiteit en die teenwoordigheid van insulienweerstandigheid. Mitokondriale biogenese blyk nie 'n beduidende rol te speel in die hart se aanpassingreaksie nie, want daar was geen verhoging in die sitraat-sintase aktiwiteit in beide groepe nie. Na aanluiding van die resultate verkry met hierdie studie, maak ons ook die gevolgtrekking dat die harte van rotte na 16 weke se blootstelling aan 'n hoë vet omgewing, metabolies kon aanpas en goed funksioneer. Ons gevolgtrekking is dus dat die harte van diere wat lei aan vetsug en wat insulien weerstandig is die verlaaging in glukose metabolisme goed hanteer en metabolies aangepas het hierby. Dit is moontlik dat hierdie aanvanklike aanpasing wanaanpaslik kan word soos vetsug vorder.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: INTRODUCTION: Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase that was first discovered as a regulator of glycogen synthase thus playing a role in glycogen synthesis (Embi et al. 1980). GSK-3 has also been shown to down regulate the expression of SERCA-2a (a calcium ATPase pump) thus playing a role in myocardial contractility (Michael et al. 2004). However, SERCA-2a activity is regulated by phospholamban (PLM) and sarcolipin (SLN) (Asahi et al. 2003). GSK-3 is constitutively active in cells and can be acutely inactivated by insulin through phosphorylation by PKB/Akt. However, GSK-3 is known to phosphorylate and inhibit IRS-1 protein, thus disrupting insulin signaling (Eldar-Finkelman et al. 1996). In addition, abnormally high activities of GSK-3 protein has been implicated in several pathological disorders which include type 2 diabetes, neuron degenerative and affective disorders (Eldar-Finkelman et al 2009). This led to the development of new generations of inhibitors with specific clinical implications to treat these diseases (Martinez 2008). GSK-3 inhibition has been shown to improve insulin and blood glucose levels and to be cardioprotective during ischemia/reperfusion (Nikoulina et al. 2002; Kumar et al. 2007). AIMS: To determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether a specific GSK-3 inhibitor can prevent or reverse the cardiovascular pathology found in obese and insulin resistant animals. OBJECTIVES: To correlate the alterations in expression and activation of GSK-3 protein in a well characterised rat model of obesity coupled to insulin resistance with: i) myocardial contractile dysfunction and an inability of hearts to withstand ischemia/reperfusion, ii) the activation and expression of phospholamban and SERCA-2a in the sarcoplasmic reticulum, iii) the activation of intermediates (IRS-1, IRS-2 and PKB/Akt) that lie upstream in the activation pathway of GSK-3 and iv) to determine the effects of inhibition of GSK-3 on the abovementioned parameters. METHODS: Age and weight matched male Wistar rats (controls and diet induced obese (DIO) animals) were used in the present study. Controls were fed normal rat chow, while DIOs were fed a rat chow diet supplemented with sucrose and condensed milk, for 8 or 16 weeks. Half of each group of animals were treated with the GSK-3 inhibitor for 4 weeks (from 12 to 16 weeks). After the feeding and treatment period, animals were weighed, sacrificed, hearts removed and freeze clamped immediately or perfused with Krebs-Henseleit buffer and subjected to low flow ischemia (25 min) followed by 30 min reperfusion. Biometric (body weight, intraperitoneal fat, ventricular weight and tibia length) and biochemical (fasting blood glucose and insulin levels) parameters were determined. Expression of GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a and Phospholamban were determined by Western blotting. Ca2+ ATPase activity was determined spectrophotometrically. RESULTS: At both 8 and 16 weeks DIO animals were significantly bigger than control animals and this was associated with increased intraperitoneal fat in DIOs. In DIO animals: IRS-1 was downregulated at 8 weeks and both IRS-1 and IRS-2 as well as PKB/Akt at 16 weeks. There was an increased tendency of GSK-3 expression at both 8 and 16 weeks in DIO animals while SERCA-2a was severely downregulated from 8 weeks onwards and associated with lower Ca2+-ATPase activity. PLM expression was upregulated but its phosphorylation was attenuated. At 16 weeks, baseline heart rate (225 vs 275 in control, P<0.0001, n=6) and rate pressure product (21000 vs 30000 in control, P=0.019, n=6) were significantly lower in hearts from DIO animals. Functional recovery was unchanged but the time to ischemic contracture development was increased (11.6±0.4 control vs 16.2±0.5 min DIO, P<0.01, n=6). Treatment had no effect on total GSK-3 expression. However, GSK-3 phosphorylation was significantly increased in treated controls, while there was no significant difference in DIO animals. However, there was a tendency for an increased GSK-3 phosphorylation in treated DIO animals. GSK-3 inhibitor, improved hypertrophy in DIO animals, while it led to its development in control animals. GSK-3 inhibitor improved IRS-2 expression in both control and DIO animals while it had no effect on IRS-1 and SERCA-2a expression and activity. However, GSK-3 inhibition increased PKB/Akt and phospholamban phosphorylation in DIO animals. CONCLUSION: These findings show that high calorie diet as well as imbalance between energy intake and expenditure lead to the development of obesity and insulin resistance in male Wistar rats. We showed that GSK-3 and its substrate proteins are dysregulated in obesity and insulin resistance. The reduced SERCA-2a expression at baseline may have a negative impact on cardiac function. By treating the animals with GSK-3 inhibitor, we showed that GSK-3 protein may not be responsible for changes seen at baseline. The decreased IRS-1 and SERCA-2a expression may have been caused by a different mechanism other than the actions of GSK-3. However, according to this study, GSK-3 may play a role in regulation of IRS-2 expression but not in IRS-1. Increased PKB/Akt phosphorylation may contribute to the GSK-3 inhibition. In addition, GSK-3 inhibition may reverse cardiac hypertrophy in DIO animals, thus acting as a negative regulator of hypertrophy. / AFRIKAANSE OPSOMMING: Inleiding: Glikogeen sintase kinase-3 (GSK-3), 'n serien/threonien proteïen kinase, is oorspronklik ontdek as 'n rolspeler in glikogeen sintese, aangesien dit 'n reguleerder van glikogeen sintase is (Embi et al.1980). Intussen is dit ook bevind dat GSK-3 die uitdrukking van SERCA-2a ('n kalsium ATPase pomp) kan afreguleer en dus sodoende 'n rol speel in miokardiale kontraktiliteit (Michael et al. 2004). Die aktiwiteit van SERCA-2a kan egter ook gereguleer word deur fosfolamban (PLM) en sarkolipin (Asahi et al. 2003). GSK-3 is deurgaans aktief, maar kan tydelik geïnaktiveer word onder kondisies van insulien stimulasie deur PKB/Akt gemedieerde fosforilering. Aan die ander kant is dit bekend dat GSK-3 die IRS-1 proteïen kan fosforileer om dus sodoende insulien sein-transduksie af te reguleer (Eldar-Finkelman et al. 1996). Daarmee saam is abnormaal hoë vlakke van GSK-3 aktiwiteit geassosieer met verskeie patologiese versteurings, insluitend tipe 2 diabetes, neuron degeneratiewe en affektiewe versteurings (Eldar-Finkelman et al. 2009). Daar is dus nuwe generasies GSK-3 inhibitore ontwikkel met die kliniese potensiaal om hierdie patologieë te behandel (Martinez 2008). Dit is al bevind dat GSK-3 inhibisie geassosieer kan word met beide die normalisering van plasma insulien- en glukose vlakke, asook kardiobeskerming in die konteks van iskemie/herperfusie (Nikoulina et al. 2002; Kumar et al. 2007). Doelwitte: Om te bepaal of GSK-3 proteïen en sy substraat proteïene gedisreguleer is onder kondisies van obesiteit en insulien weerstandigheid, asook om vas te stel of 'n spesifieke GSK-3 inhibitor die kardiovaskulêre patologie wat gevind word in obese en insulien weerstandige diere kan verhoed of omkeer. Mikpunte: Om veranderinge in uitdrukking en aktiwiteit van GSK-3 proteïen in 'n goed gekarakteriseerde rotmodel van obesiteit, gekoppel aan insulien weerstandigheid, te korreleer met die volgende: i) miokardiale kontraktiele disfunksie en onvermoë om kardiale iskemie/herperfusie besering te weerstaan, ii) aktivering en uitdrukking van PLM en SERCA-2a in die sarkoplasmiese retikulum, iii) die aktivering van intermediêres wat proksimaal geleë is in die insulienseintransduksiepad van GSK-3 (IRS-1, IRS-2 en PKB/Akt) en iv) om die effek van behandeling met 'n spesifieke inhibitor van GSK-3 op die bogenoemde punte te bepaal. Metodes: Ouderdoms- en gewigsgepaarde manlike Wistar rotte (kontrole en dieet geïnduseerde obees (DIO) diere) is in die studie gebruik. Kontrole diere was normale rotkos gevoer, terwyl die DIO diere op 'n dieet van rotkos aangevul met sukrose en kondensmelk geplaas is vir 'n periode van 8 of 16 weke. Helfte van die diere van elke groep is behandel met die GSK-3 inhibitor vir 4 weke (vanaf week 12 tot 16). Na afloop van die voer- en behandelingsperiode is die diere geweeg, doodgemaak en die harte verwyder om dan of onmiddelik gevriesklamp te word, of retrograad geperfuseer te word met Krebs-Hensleit buffer. Ex vivo geperfuseerde harte is dan blootgestel aan 25 minute lae vloei iskemie gevolg deur 30 minute herperfusie. Biometriese (liggaamsgewig, intraperitoneale vet, ventrikulêre gewig en tibia lengte) en biochemiese (vastende bloedglukose en -insulien vlakke) parameters is telkens bepaal. Western klad tegnieke is gebruik om die uitdrukking en fosforilering van GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a en PLM te bepaal. Ca2+-ATPase aktiwiteit is spektrofotometries bepaal. Resultate: Na beide 8 en 16 weke was die DIO diere beduidend swaarder as die kontrole diere. Hierdie gewigstoename was geassosieer met meer intraperitoneale vet in die DIO diere. Verder, in die DIO diere was IRS-1 afgereguleer na 8 weke, terwyl beide IRS-1 en IRS-2 asook PKB/Akt afgereguleer was na 16 weke. GSK-3 uitdrukking het 'n neiging getoon om toe te neem na beide 8 en 16 weke in die DIO diere, terwyl SERCA-2a beduidend afgereguleer was reeds vanaf 8 weke, geassosieer met laer Ca2+-ATPase aktiwiteit. PLM uitdrukking het toegeneem en die fosforilering daarvan was verlaag. Op 16 weke was die basale harttempo (225 vs 275 in die kontrole groep, P<0.0001, n=6) en tempo druk produk (21000 vs 30000 in die kontrole groep, P=0.019, n=6) betekenisvol laer in die DIO diere. Funksionele herstel het onveranderd gebly, alhoewel die tyd tot iskemiese kontraktuur toegeneem het in die DIO groep (kontrole: 11.6±0.4 min vs DIO: 16.2±0.5 min, P<0.01, n=6). Toediening van die inhibitor het geen effek op totale GSK-3 uitdrukking gehad nie. Fosforilering van GSK-3 was egter wel beduidend verhoog in die behandelde kontrole diere, terwyl daar geen verskille in die DIO groep was nie. Die fosforilering van GSK-3 het wel geneig na 'n toename in die behandelde DIO diere. Die GSK-3 inhibitor het kontrasterende effekte op hipertrofie gehad: dit het dit omgekeer in die DIO groep, maar veroorsaak in die kontrole diere. Daarmee saam het die inhibitor die uitdrukking van IRS-2 in beide DIO en kontrole diere gestimuleer, maar geen effek op IRS-1 en SERCA-2a uitdrukking en aktiwiteit gehad nie. GSK-3 inhibisie het wel PKB/Akt en PLM fosforilering in die DIO diere verhoog. Gevolgtrekking: Hierdie bevindinge toon dat 'n hoë kalorie dieet, tesame met 'n wanbalans tussen energie inname en verbruiking, lei tot die ontwikkeling van obesiteit en insulien weerstand in manlike Wistar rotte. Die studie het ook getoon dat GSK-3 en sy substraat proteïene wel gedisreguleer is in obesiteit en insulien weerstandigheid. Die verlaagde basale uitdrukking van SERCA-2a mag dalk 'n negatiewe impak hê op kardiale funksie. Behandeling van die diere met 'n GSK-3 inhibitor het getoon dat GSK-3 moontlik nie verantwoordelik is vir die basislyn veranderinge nie. Die afname in IRS-1 en SERCA-2a uitdrukking kan moontlik toegeskryf word aan ander meganismes buiten die effekte van GSK-3. Hierdie studie toon wel dat GSK-3 moontlik 'n rol speel in die regulering van die uitdrukking van IRS-2, maar nie IRS-1 nie. Verhoogde PKB/Akt fosforilering mag dalk bydra tot die inhibisie van GSK-3. Daarmee saam blyk dit dat GSK-3 inhibisie hipertrofie kan omkeer in DIO diere, om dan sodoende op te tree as 'n negatiewe reguleerder van hipertrofie, maar in normale kontrole diere, hipertrofie in die hand werk. / South African Medical Research Council / University of Stellenbosch, Dept. of medical Physiology

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Much has been documented about the detrimental effects of adverse lifestyle factor exposure on the body. Exposure to factors, such as cigarette smoke, have proved to not only be a burden on global health and economy, but have also led to growing concerns about effects on systemic functions such as reproduction. The aim of the present study was to determine the effects of in utero and in vitro nicotine exposure on spermatozoal function and the antioxidant enzyme activity and lipid peroxidation (LPO) status of the male reproductive system. A better understanding of this process is necessary to combat the respective burdens of smoking and male infertility and for the prospective development of treatment strategies. Two experimental models were employed: Wistar rats were exposed to nicotine in utero while human and rat spermatozoa were exposed to nicotine in vitro. In utero studies were achieved by selecting healthy pregnant rats and treating them with 1 mg/kg-bodyweight/day nicotine or 1 ml/kg-bodyweight/day 0.85% physiologic saline throughout gestation and lactation. Male rat pups were selected and sacrificed at each of the following age groups (n=6): 42 days, 84 days and 168 days old. The pups were only exposed to the treatment/saline via placental uptake or lactation. Biochemical analyses of the tissue comprised of measurement of LPO and antioxidant enzyme activity. Results indicated a significant association of maternal nicotine exposure to decreased levels of primary antioxidant enzymes in rat testes. Of particular note was the observation that the treatment group, of which each of the respective antioxidant enzyme levels were significantly less than the control group, was the oldest (d168) rat group. In vitro studies were achieved by collecting sperm samples from healthy human donors (n=12), healthy rats (n=6) and obese rats (n=6). Samples were washed and exposed to different concentrations of high levels of nicotine (Control, 0.1mM, 1mM, 5mM, 10mM) in vitro. Semen parameters such as motility, viability and acrosome reaction were monitored at different time points (30min, 60min, 120min, 180min). Results revealed increasing in vitro nicotine concentrations were associated with decreased viability and acrosomal status of human spermatozoa and decreased progressive motility and viability of rat spermatozoa. Obesity was also associated with decreases in progressive motility and viability of rat spermatozoa. These results indicate that the acute in vitro exposure of spermatozoa to high levels of nicotine could adversely affect semen quality and may be an additive factor to the impediment of male fertility. In utero results reveal maternal nicotine exposure adversely affects male fertility in later life and seems to elicit more detrimental effects on the reproductive system than that of direct nicotine exposure to spermatozoa. Obesity also inhibits parameters of male fertility and these effects are exacerbated by nicotine exposure. The authors believe these adverse effects on the reproductive system to be related to an increased activation of leukocytes, excess production in reactive oxygen species (ROS) and consequent onset of oxidative stress (OS). Nevertheless this study agrees with other studies that nicotine exposure may be an additive factor to the impediment of male fertility. / AFRIKAANSE OPSOMMING: Daar is reeds baie bekend oor die moontlik newe effekte vir die liggaam wat met ‘n ongesonde lewenstyl gepaard gaan. Menslike blootstelling aan sulke faktore, soos sigaret rook, is wêreldwyd ‘n las vir gesondheid en ekonomie en het gelei tot geweldige kommer onder navorsers oor die moontlike komplikasies vir liggaamlike funksies soos voortplanting. Die doel van die betrokke projek was om die effekte van in utero en in vivo nikotien blootstelling op die antioksiderende ensiem aktiwiteit en lipied peroksidasie status van reproduktiewe weefsel en die funksionele parameters van spermatozoa te bepaal. ‘n Beter begrip van hierdie proses is noodsaaklik om die las van rook en vetsug teen te werk en vir die moontlike ontwikkeling van behandelingsstrategieë. Twee eksperimentele modelle is ontwerp: Wistar rotte is in utero blootgestel aan nikotien terwyl mens- en rot- spermatosoë ook in vitro aan nikotien blootgestel is. Vir die in utero studie is gesonde dragtige rotte gedurende swangerskap en laktasie met 1 mg/kgliggaamsgewig/ dag nikotien of 1 ml/kg-liggaamsgewig/dag 0.85% fisiologiese soutoplossing behandel. Manlike welpies is gekies en geoffer op elk van die volgende ouderdomme (n=6): 42 dae, 84 dae en 168 dae. Die welpies is slegs aan nikotien blootgestel deur plasentale opname en laktasie. Biochemiese analise van die testikulêre weefsel het ‘n beduidende assosiasie getoon tussen maternale nikotien blootstelling en verminderde vlakke van die primêre antioksiderende ensieme. Die 168 dag oue groep het ‘n merkbare vermindering getoon tussen kontrole en nikotien weefsel vir elk van die antioksiderende ensieme. Vir die in vitro studie is sperm monsters verkry vanaf gesonde mans (n=12), gesonde rotte (n=6) en vet rotte (n=6). Monsters is gewas en in vitro blootgestel aan verskeie hoë vlakke van nikotien (kontrole, 0.1mM, 1mM, 5mM, 10mM). Seminale parameters soos motiliteit, lewensvatbaarheid en akrosoom status is by verskei tydpunte gemeet (30min, 60min, 120min, 180min). Dit blyk dat verhoging in in vitro nikotien konsentrasies verband hou met verlaagde lewensvatbaarheid en akrosoom status van menslike spermatosoë en verlaagde progressiewe motilteit en lewensvatbaarheid van rot spermatosoë. Vetsug is ook geassosieer met verlagings in progressiewe beweeglikheid en lewensvatbaarheid van rot spermatosoë. In utero resultate openbaar dat maternale nikotien blootstelling manlike vrugbaarheid nadelig beïnvloed in latere lewe en blyk dat dit meer van ‘n nadelige uitwerking op die voortplantingstelsel het as dié van direkte nikotien blootstelling aan spermatosoë. In vitro blootstelling van spermatosoë aan hoë vlakke van nikotien, het wel ook semen kwaliteit nadelig beïnvloed. Vetsug inhibeer ook manlike vrugbaarheids parameters en hierdie effek word vererger deur nikotien blootstelling. Die outeure glo dat hierdie nadelige uitwerking op die voortplantingstelsel verband hou met 'n verhoogde aktivering van leukosiete, oortollige produksie van reaktiewe suurstof spesies en die gevolglike aanvang van oksidatiewe stres bevorder. Hierdie studie stem wel ooreen met ander studies wat nikotien blootstelling bestempel as ‘n bydraende faktor tot die struikelblok van manlike onvrugbaarheid. / Harry Crossley Foundation (South Africa)

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Early life trauma leads to behavioural abnormalities later in life. These include mood and anxiety disorders such as depression and posttraumatic stress disorder (PTSD). This association may be due in part to the effects of trauma on brain development. Data from basic and clinical experiments suggest that alterations in the hippocampus may be fundamental to the development of these disorders. Here we used an animal model of early life trauma to investigate its effects on the behaviour and neurochemistry of the adult rat. Adolescent rats were subjected to time-dependent sensitization stress consisting of a triple stressor (2 hours restraint, 20 min swim stress and exposure to ether vapour) on post-natal day (PND) 28, a single re-stress on PND 35 (20 min swim stress), and a second re-stress in adulthood (PND 60, 20 min swim stress). The rationale was that the frequency of exposure to situational reminders contributes to the maintenance over time of fear-related behavioural disturbances. The effects of trauma on the hypothalamus-pituitary-adrenal-axis, hippocampal and plasma neurotrophin levels, behaviour and phosphoinositide-3 kinase (PI-3 kinase) signaling proteins were initially investigated. In addition, proteomic technologies such as protein arrays and 2D-SDS PAGE combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to study trauma-induced effects on the hippocampus. Traumatized animals showed a decrease in glucocorticoid receptors in the dentate gyrus of the hippocampus and an increase in basal corticosterone levels 24 hours after adulthood re-stress. These effects were reversed by pretreatment with the serotonin selective reuptake inhibitor, escitalopram. A decrease in the neurotrophins, BDNF and NT-3 were evident 8 days, but not 24 hours after adulthood re-stress. This decrease was not accompanied by decreases in plasma neurotrophin or PI-3 kinase, protein kinase B (PKB), phosphatase and tensin homologue (PTEN), phospho-forkhead and phospho-AFX protein levels. In addition, traumatized animals showed increased rearing in both the elevated plus maze and open field. Proteomic analysis of trauma-induced changes in the hippocampus show increases in Ca2+ homeostasis / signaling proteins such as S-100B, phospho-JNK and calcineurin. Apoptotic initiator proteins, including caspase 9, -10 and -12 were increased and there was evidence of cytoskeletal protein dysregulation. Furthermore, cell cycle regulators and energy metabolism proteins were decreased. These effects indicate to a cellular state of cell cycle arrest after increased calcium influx to avoid apoptosis. Our data suggest that adolescent trauma with adulthood re-stress may affect numerous systems at different levels. These include neuroendocrine-, protein systems and behaviour, and confirmed that a systems biology approach is needed for a better understanding of the neurobiology of mental disorders.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Background: There has been a dramatic increase in the use of dietary creatine supplementation among sports men and women, and by clinicians as a therapeutic agent in muscular and neurological diseases. The effects of creatine have been studied extensively in skeletal muscle, but knowledge of its myocardial effects is limited. Objectives: To investigate the effects of dietary creatine supplementation with and without exercise on 1) basal cardiac function, 2) susceptibility to ischaemia/reperfusion injury and 3) myocardial protein expression and phosphorylation and 4) mitochondrial oxidative function. Methods: Male Wistar rats were randomly divided into control or creatine supplemented groups. Half of each group was exercise trained by swimming for a period of 8 weeks, 5 days per week. At the end of the 8 weeks the open field test was performed and blood corticosterone levels were measured by RIA to determine whether the swim training protocol had any effects on stress levels of the rats. Afterwards hearts were excised and either freeze-clamped for biochemical and molecular analysis or perfused on the isolated heart perfusion system to assess function and tolerance to ischaemia and reperfusion. Five series of experiments were performed: (i) Mechanical function was documented before and after 20 minutes global ischaemia using the work heart model, (ii) A H2O filled balloon connected to a pressure transducer was inserted into the left ventricle to measure LVDP and ischaemic contracture in the Langendorff model, (iii) The left coronary artery was ligated for 35 minutes and infarct size determined after 30 minutes of reperfusion by conventional TTC staining methods. (iv) Mitochondrial oxidative capacity was quantified. (v) High pressure liquid chromatography (HPLC) and Western Blot analysis were performed on blood and heart tissue for determination of high energy phosphates and protein expression and phosphorylation. Results: Neither the behavioural studies nor the corticosterone levels showed any evidence of stress in the groups investigated. Hearts from creatine supplemented sedentary (33.5 ± 4.5%), creatine supplemented exercised rats (18.22 ± 6.2%) as well as control exercised rats (26.1 ± 5.9%) had poorer aortic output recoveries than the sedentary control group (55.9 ± 4.35% p < 0.01) and there was also greater ischaemic contracture in the creatine supplemented exercised group compared to the sedentary control group (10.4 ± 4.23 mmHg vs 31.63 ± 4.74 mmHg). There were no differences in either infarct size or in mitochondrial oxygen consumption between the groups. HPLC analysis revealed elevated phosphocreatine content (44.51 ±14.65 vs 8.19 ±4.93 nmol/gram wet weight, p < 0.05) as well as elevated ATP levels (781.1 ±58.82 vs 482.1 ±75.86 nmol/gram wet weight, p<0.05) in blood from creatine supplemented vs control sedentary rats. These high energy phosphate elevations were not evident in heart tissue and creatine tranporter expression was not altered by creatine supplementation. GLUT4 and phosphorylated AMPK and PKB/Akt were all significantly higher in the creatine supplemented exercised hearts compared to the control sedentary hearts. Conclusion: This study suggests that creatine supplementation has no effects on basal cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise trained animals by increasing the ischaemic contracture and decreasing reperfusion aortic output. Exercise training alone also significantly decreased aortic output recovery. However, the exact mechanisms for these adverse myocardial effects are unknown and need further investigation. / AFRIKAANSE OPSOMMING: Agtergrond: Die gebruik van kreatien as dieetaanvulling het in die afgelope aantal jaar dramaties toegeneem onder sportlui, sowel as mediese praktisyns wat dit as ‘n terapeutiese middel vir die behandeling van spier- en neurologiese siektes aanwend. Die effekte van kreatien op skeletspier is reeds deeglik ondersoek, maar inligting aangaande die miokardiale effekte van die preperaat is beperk. Doelwitte: Om die effekte van kreatien dieetaanvulling met of sonder oefening ten opsigte van die volgende aspekte te ondersoek: 1) basislyn miokardiale funksie, 2) vatbaarheid vir iskemie/herperfusie besering, 3) proteïenuitdrukking en -fosforilering in die miokardium en 4) mitochondriale oksidatiewe funksie. Metodes: Manlike Wistar rotte is ewekansig in kontrole of kreatien aanvullings groepe verdeel. Helfte van elke groep is aan oefening in die vorm van swemsessies, vir ‘n periode van 8 weke, 5 dae per week blootgestel. Gedrags- en biochemiese toetse is aangewend om die moontlike effek van die swemprotokol op die rotte se stres vlakke te bepaal. In hierdie verband is die oop area toets gebruik, asook bloed kortikosteroon vlakke gemeet deur radioaktiewe immuunessais. Harte is daarna uit die rotte gedissekteer en gevriesklamp vir biochemiese en molekulêre analise, of geperfuseer op die geïsoleerde werkhart perfusiesisteem om sodoende funksie en weerstand teen iskemie en herperfusie beskadeging te bepaal. Vyf eksperimentele reekse is uitgevoer: (i) Meganiese funksie is noteer voor en na 20 minute globale isgemie in die werkhart model; (ii) ‘n Water gevulde plastiek ballon, gekoppel aan ‘n druk omsetter, is in die linker ventrikel geplaas om sodoende linker ventrikulêre ontwikkelde druk (LVDP), asook iskemiese kontraktuur te meet; (iii) Linker koronêre arterie afbinding is vir ‘n periode van 35 minute toegepas en die infarktgrootte bepaal na 30 minute herperfusie deur gebruik te maak van standaard kleuringsmetodes; (iv) Mitochondriale oksidatiewe kapasiteit is gemeet; (v) Hoë druk vloeistof chromatografie (HPLC) en Western Blot analises is uitgevoer op bloed en hartweefsel vir die bepaling van hoë energie fosfate (HEFe), sowel as proteïenuitdrukking en -fosforilering. Resultate: Beide gedragsstudies en kortikosteroonvlakke het geen teken van stres in die betrokke groepe getoon nie. Die groep blootgestel aan kreatienaanvulling en oefening se harte het na iskemie funksioneel swakker herstel as harte van die onaktiewe kontrole groep (18.22±6.2% vs 55.9±4.35%; p<0.01), asook ‘n groter ikgemiese kontraktuur in vergelyking met die onaktiewe kontrole groep ontwikkel (31.63±4.74 mmHg vs 10.4±4.23 mmHg). Daar was geen verskille in infarktgrootte of mitochondriale suurstofverbruik tussen die verskillende groepe waargeneem nie. HPLC analise het verhoogde fosfokreatien (44.51±14.65 vs 8.19±4.93 nmol/gram nat gewig, p<0.05) en adenosientrifosfaat (ATP) bloedvlakke (781.1±58.82 vs 482.1±75.86 nmol/gram nat gewig, p<0.05) in kreatien aanvullings vergelyk met die kontrole groepe getoon. Daar was egter geen meetbare veranderings in HEF vlakke in hartweefsel nie. Gepaardgaande hiermee het kreatienaanvulling geen effek gehad op die uitdrukking va die kreatien transporter nie. In vergelyking met onaktiewe kontrole harte was GLUT4, en fosforileerde AMPK en PKB/ Akt beduidend hoër in harte van geoefende rotte met kreatienaangevulling. Gevolgtrekking: Hierdie data dui daarop dat kreatienaanvulling geen effek op basislyn miokardiale funksie het nie. Kreatienaanvulling het egter die miokardium se weerstand teen iskemiese skade verlaag in harte van rotte blootgestel aan oefening: iskemiese kontraktuur is verhoog en aorta-uitset tydens herperfusie is verlaag. Die presiese meganismes hierby betrokke is egter onbekend en vereis dus verdere studie. / Division of Medical Physiology (University of Stellenbosch), The National Research Foundation and the Harry Crossley Fund for financial support.

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are conducted on macrovascular endothelial cells. The current dissertation set out to comprehensively investigate the responses of cultured primary adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory cytokine). We were interested to investigate whether this distinct endothelial cell type would develop classical features of ED, and if so, what the underlying mechanisms were. First we aimed to establish a baseline characterization of the CMECs under control conditions. Next, we developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis, hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above measurements were performed by morphological assessment (light and fluorescence microscopy), FACS analysis, western blotting and large-scale proteomic analyses. Data showed that CMECs shared many baseline features with other endothelial cell types, including morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was the high abundance of structural mitochondrial proteins, suggesting that CMECs require mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature, several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways (BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway. In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs shared many responses and mechanisms with more frequently researched endothelial cell types, the data also supplied several novel additions to the literature, particularly with the application of proteomics. We believe that this dissertation has provided more insights into endothelial heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to stimuli typically associated with cardiovascular risk. / AFRIKAANSE OPSOMMING: Blootstelling aan skadelike stimuli kan tot disfunksionaliteit van vaskulêre endoteelselle lei wat deur verlaagde biobeskikbaarheid van stikstofoksied (NO) gekenmerk word. Endoteeldisfunksie (ED) is ‘n omkeerbare voorganger van isgemiese hartsiekte (IHD), en ‘n beter begrip van die onderliggende meganismes van ED kan lei tot die ontwikkeling van kliniese strategieë vir die voorkoming/behandeling van IHD. Baie min is bekend oor die respons wat in kardiale mikrovaskulêre endoteelselle (CMECs) uitgelok word na blootstelling aan pro-ED stimuli, omdat meeste studies op makrovaskulêre endoteelselle uitgevoer word. Die huidige proefskrif het daarna gemik om die respons van primêre kulture van volwasse rot CMECs op bekende skadelike stimuli, nl. hipoksie en tumor nekrose faktor-alfa (TNF-α; pro-inflammatoriese sitokien) in diepte te ondersoek. Ons was veral geïnteresseerd om vas te stel of hierdie spesifieke endoteelseltipe die klassieke kenmerke van ED sou ontwikkel, en indien wel, wat die onderliggende meganismes sou wees. Eerstens het ons beoog om ‘n basislyn karaterisering van CMECs onder kontrole toestande daar te stel. Vervolgens het ons ‘n model van hipoksie-geïnduseerde selskade gevestig en apoptose/nekrose, intrasellulêre NO en reaktiewe suurstofspesies (ROS), sowel as die uitdrukking en aktivering van proteine betrokke by NO-biosintese, hipoksie en apoptose en differensiële regulering van proteine gemeet. Laastens het ons die respons van CMECs op behandeling met TNF-α gekarakteriseer. Ons het apoptose/nekrose, intrasellulêre NO en ROS vlakke, NO-biosintese-seintransduksieproteïene en grootskaalse differensiele regulering van proteïene gemeet. Bg. metings is uitgevoer deur gebruik te maak van morfologiese evaluasie (lig -en fluoressensiemikroskopie), vloeisitometriese analises, western blot analises en proteomiese analises. Data het getoon dat die basislyn eienskappe van CMECs grootliks met dié van ander endoteelseltipes ooreenstem, insluitende morfologiese voorkoms, LDL-opname, NO-produksie en die uitdrukking van eNOS proteïen. In ‘n nuwe waarneming, het die proteomiese data die uitdrukking van 1387 proteïene aangetoon. ‘n Ander nuwe bevinding was die voorkoms van ‘n groot aantal strukturele mitokondriale proteïene, wat daarop dui dat die CMECs mitokondria ook vir nie-respiratoriese doeleindes gebruik. ‘n Hoë uitdrukking van vesikulêre, glikolitiese en RAS-seintransduksie proteïene was ook kenmerkend van die basislyn CMECs. CMECS wat aan hipoksie blootgestel is, het reageer met ‘n verhoging in apoptose / nekrose en verhoogde uitdrukking van die hipoksie merker, HIF-1α. ‘n Interressante bevinding was dat eNOS-NO biosintese sterk toegeneem het in die hipoksiese CMECs wat met verhoogde mitokondriale ROS en verlaagde anti-oksidant sisteme (aanduidend van oksidatiewe stres) gepaardgegaan het. In ooreenstemming met die literatuur, is verskeie glikolitiese proteïene opgereguleer. ‘n Nuwe waarneming was die opregulering van proteïene wat betrokke is by proteïensintese, iets wat nie normaalweg in hipoksie-studies beskryf word nie. Die CMECs het op TNF-α behandeling gerespondeer deur tekens van ED te toon, naamlik ‘n afname in die NO afkomstig van PKB/Akt-eNOS biosintese en die ontwikkeling van uitgesproke reaksie op oksidatiewe stres soos aangedui deur die opregulering van verskeie anti-oksidant sisteme. Die data het ook aangedui dat TNF-α behandeling tot klassieke TNF-reseptor 1 bemiddelde seintransduksie gelei het, wat gekenmerk was deur die tweeledige aktivering van pro-apoptotiese seintransduksiepaaie (BID en kaspase-3) sowel as die beskermende, pro-inflammatoriese IKB-alpha-NF-KB seintransduksiepad. Ten slotte: hierdie is die eerste studie van sy soort wat die kenmerke en response van CMECs onder basislyn en pro-besering omstandighede in diepte beskryf. Alhoewel dit oor die algemeen wil voorkom asof die CMECs baie in gemeen het met ander, beter nagevorste endoteelseltipes, het die data egter ook verskeie nuwe bevindinge tot die bestaande literatuur gevoeg, spesifiek die data afkomstig van die proteomiese analises. Ons glo dat hierdie proefskrif meer insig verleen t.o.v. die heterogeniteit van vaskulêre endoteelselle asook t.o.v. die megansimes wat deur CMECs aangewend word wanneer hulle aan skadelike stimuli (geassosieer met kardiovaskulêre risiko) blootgestel word.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: HIV/AIDS is a global epidemic that accounts for a large percentage of the mortality in South Africa every year. Since the implementation of anti-retroviral treatment, HIV positive individuals have been living longer, and the cognitive impairment associated with the disease is becoming increasingly apparent. During the initial systemic infection of HIV, the virus migrates through the blood-brain barrier and inflicts axonal injury by causing upregulation of cytokines and neurotoxic proteins. HIV-associated dementia is a neuropsychological classification of cognitive impairment in HIV and a variety of symptoms have been classified as a part of the dementia complex. One of these is apathy, which is thought to be a precursor for dementia in HIV patients. Three groups of individuals have been recruited and scanned using magnetic resonance imaging (MRI) to examine changes in the brain. These are an HIV non-apathetic cohort, an HIV apathetic cohort and a healthy control cohort. Diffusion tensor imaging (DTI) is an MRI technique used to quantitatively assess white matter (WM) integrity using metrics such as fractional anisotropy (FA). Voxel-based analysis, tract-based spatial statistics (TBSS) and tractography are three established DTI analysis methods that have been applied in numerous studies. However, there are certain methodological strengths and limitations associated with each technique and therefore all three of these techniques were used to compare WM differences across groups. The frontal-subcortical pathways are known to be abnormal in apathy, and this has been demonstrated in a number of imaging studies. Most of these studies have examined apathy in the context of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s. However, to our knowledge this is the first DTI study in HIV apathetic patients. With the tractography method, the anterior thalamic radiation and the corpus callosum were reconstructed for each individual to determine whether there were any global changes in these tracts. No significant changes were found. However, a variety of regions in the WM were significantly abnormal in the HIV cohorts when comparing the data at a voxel-based level and using TBSS. This included areas such as the genu and splenium of the corpus callosum, the internal capsule and corona radiata. Changes in frontal WM for the HIV apathy group are an indication of dysfunction in the frontal-striatal circuits, and previous literature has implicated these circuits in the neuropathology of apathy in a variety of central nervous system (CNS) disorders. / AFRIKAANSE OPSOMMING: MIV/VIGS is `n wêreldwye epidemie wat verantwoordelik is vir `n hoë sterftesyfer in Suid- Afrika elke jaar. Sedert die inleiding van anti-retrovirale behandeling, het die MIV-positiewe populasie se lewensduur verleng. Tesame met langer lewensduur, het die kognitiewe verswakking wat geassosieer word met die siekte ook meer prominent na vore gekom. Gedurende die beginstadium van sistemiese infeksie in MIV is daar `n migrasie van die virus deur die bloed-breinskans. MIV kan indirek verantwoordelik wees vir aksonale beskadiging deur verhoging van neurotoksiese proteine en sitokinien te induseer. MIV-geassosieerde demensie is `n neurosielkundige klassifikasie van kognitiewe verswakking in MIV en verskeie simptome is al geïdentifiseer as deel van die demensie kompleks. Een van die simptome is apatie en daar word gespekuleer dat dit `n voorloper is vir demensie in MIV pasiënte. Drie groepe individue was gewerf vir die studie en geskandeer deur magnetiese resonansie beeldvorming (MRB) om sodoende veranderinge in die brein te ondersoek. Die groepe was onderskeidelik `n HIV nie-apatiese kohort, `n HIV apatiese kohort en `n gesonde kontrole kohort. Diffusie tensor beelding (DTB) is `n MRB tegniek wat toegepas word om witstof integriteit te meet deur gebruik te maak van maatstawwe soos fraksionele anisotropie (FA). “Voxel-based analysis”, “tract-based spatial statistics (TBSS)” en “tractography” is drie gevestigde DTB analitiese metodes wat al in talle studies toegepas was. Daar is egter sekere metodologiese voordele en beperkings verbonde aan elke tegniek en daarom is al drie tegnieke gebruik om witstof verskille tussen groepe te vergelyk. Die frontale-subkortikale roetes in die brein is bekend vir abnormaliteite in apatie en dit was ook al gedemonstreer in verskeie studies. Die meeste van die studies het apatie ondersoek in die konteks van neurodegeneratiewe siektes soos Alzheimer se siekte en Parkinson se siekte. Maar sover ons weet is hierdie die eerste DTB studie in MIV pasiënte met apatie. Met die “tractography” metode was die anterior thalamic radiation en corpus callosum herbou vir elke individu. Dit was om te bepaal of daar enige globale veranderinge is in hierdie gebiede, maar geen beduidende veranderinge is gevind nie.`n Verskeidenheid van gebiede in die witstof was beduidend abnormaal in die MIV kohorte wanneer die data vergelyk was met “TBSS” en “voxel-based analysis.” Dit het gebiede ingesluit soos die genu en splenium van die corpus callosum, die internal capsule en die corona radiata. Veranderinge in die frontale witstof vir die MIVapatie groep is `n aanduiding van disfunksie in die frontale-striatale bane. Vorige literatuur impliseer dat hierdie bane betrokke is in die neuro-patologie van apatie in verskeie sentrale senuweestelsel (SS) steurings.