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.

Bibliography / Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology (asthenoteratozoospermia) for which there is no specific therapeutic treatment. It has come to light that the modification and expression of human sperm proteins play a crucial role in sperm function. In the present study, we present proteomic data of human spermatozoa in the context of sperm dysfunction. Novel techniques have been used to successfully isolate and identify differences in protein expression on a cellular level associated with asthenoteratozoospermia. In the first part of the study, differences in protein expression within the total sperm proteome were investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=23) and separated into mature and immature sperm populations by 3-layer Percoll gradient centrifugation. Cells were washed and motility and morphology were measured by computer assisted sperm analysis (CASA). For the proteomic investigation cells were lysed and proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used to identify the differentially expressed proteins. The protein spots of interest were excised and subjected to in-gel digestion. Peptides were separated by High Pressure Liquid Chromatography (HPLC) analysis and amino acid sequences determined by mass spectrophotometry. Proteins were identified by Mascot, using the Swiss Prot database. The results show that the motility (immature; 26.1±1.75% total motile cells vs. mature; 60.93±3.24% total motile cells; p<0.001) and morphology parameters (immature; 64.1±2.75% normal head morphology vs. mature; 87.63±3.24% normal head morphology; p<0.001) of the two populations differed significantly. After 2D electrophoresis, 16 differentially expressed protein spots were identified within the total sperm proteome between the immature and mature sperm populations. 56% of the differentially expressed proteins were more abundant in the immature sperm population compared to the mature sperm population. Functions have been ascribed to these proteins of which only four proteins, namely Tubulin -3C/D chain, Tubulin -2C chain, Outer dense fibre protein 2 and A-Kinase anchoring protein 4 precursor, were directly related to sperm motility and morphology. In the second part of the study the expression of nuclear proteins in human spermatozoa was investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=156) and further separated from the seminal plasma by PureSperm® gradient centrifugation. The immature and mature sperm populations were retrieved and used during further analysis. For the proteomic analysis of nuclear proteins, cells were fractionated into four different subcellular protein fractions, instead of analyzing the whole sperm proteome. The results show that the motility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motile cells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal head morphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the two populations differ significantly. After 2D electrophoresis, 21 differentially expressed nuclear proteins were identified between the immature and mature sperm populations. 95% of the differentially expressed nuclear proteins were less abundant in the immature population compared to the mature population. Only one nuclear protein namely 78kDa Glucose regulated protein was more abundant in the immature population compared to the mature population. Functions ascribed to these individual proteins were directly related to sperm motility, morphology and energy metabolism. In conclusion,In conclusion, in the current study novel techniques have been employed to investigate protein differences between immature and mature sperm populations. From these results it is evident that protein expression in the total sperm proteome and nuclear protein fraction is significantly different and incomplete in the immature population, compared to mature population. Based on these findings, it is recommended that further studies should be done on human spermatozoa to validate the role of the individual proteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes of male infertility, as it can help to identify novel receptors (and signal transduction pathways) that can be used in the screening of drugs to alleviate sperm dysfunction. / AFRIKAANSE OPSOMMING: Manlike infertiliteit word dikwels geassosieer met verlaagde sperm motiliteit en morfologie (asthenoteratozoospermia) waarvoor daar tot dusver nog geen spesifieke terapeutiese behandeling is nie. Dit het aan die lig gekom dat die modifisering en uitdrukking van menslike sperm proteïene ‘n belangrike rol speel in spermfunksie. In die huidige studie stel ons data voor van proteiene in menslike sperme in die konteks van abnormale spermfunksie. Unieke tegnieke was gebruik om verskille in proteïen uitdrukking op sellulêre vlak suksesvol te isoleer en identifiseer wat verband hou met asthenoteratozoospermia. Tydens die eerste deel van die studie was verskille in proteïen uitdrukking binne die totale spermproteoom tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme van gesonde skenkers (n=23) is geskei in twee spermpopulasies (onvolwasse en volwasse sperme) deur middel van ‘n 3-laag Percoll gradiënt sentrifugasie tegniek. Selle is gewas en sperm motiliteit en morfologie is gemeet deur rekenaar geassisteerde sperm analise (CASA). Vir proteomiese analise is selle geliseer en proteïene geskei deur twee dimensionele gel elektroforese (2D-elektroforese). PD-Quest sagteware is gebruik om statisties beduidende proteïen verskille aan te dui. Die proteïene van belang is uitgesny en onderwerp aan in-gel vertering. Peptiede is geskei met behulp van hoë druk vloeistof chromatografie (HPLC) analise en aminosuurvolgordes is bepaal deur massa spektrofotometrie. Proteïene is geïdentifiseer met behulp van Mascot deur van die Swiss Prot databasis gebruik te maak. Die resultate toon dat die sperm motiliteit (onvolwasse; 26.1±1.75% totale motiele selle vs. volwasse; 60.93±3.24% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 64.1±2.75% normale kop morfologie vs. volwasse; 87.63±3.24% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2Delektroforese is 16 proteïen kolle geïdentifiseer wat beduidend verskil het, tussen die totale sperm proteoom van onvolwasse spermpopulasies en volwasse spermpopulasies. 56% van die proteïene wat beduidend verskil het, was meer uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse sperm populasie. Funksies is toegeskryf aan hierdie proteïene waarvan net vier proteïene naamlik Tubulin -3C/D ketting, Tubulin -2C ketting, Buite digte vesel proteïen 2 en A-Kinase anker proteïen 4 voorloper direk verband hou met sperm motiliteit en morfologie. In die tweede deel van die studie is die uitdrukking van nukluêre proteïene in menslike spermatozoa tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme was van gesonde skenkers (n=156) versamel en verder geskei van seminale plasma deur middel van ‘n PureSperm® gradiënt sentrifugasie tegniek. Vir die proteomiese analise van nukluêre proteïene is selle gefraksioneer in vier verskillende sub-sellulêre proteïen fraksies, in plaas van analise van die totale spermproteoom. Die resultate toon aan dat die sperm motiliteit (onvolwasse; 32.33±0.51% totale motiele selle vs. volwasse; 88.67±0.85% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 13.51±0.87% normale kop morfologie vs. volwasse; 20.89±1.20% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2D-elektroforese is 21 kern proteïen kolle geïdentifiseer wat betekenisvol uitgedruk was tussen onvolwasse en volwasse spermpopulasies. 95% van die nukluêre proteïene wat beduidend verskil het, was minder uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Slegs een kern proteïen naamlik 78kDa Glukose gereguleerde proteïen was meer uitgedruk in die onvolwasse spermpopulasie in vergelyking met die volwasse spermpopulasie. Funksies is toegeskryf aan hierdie proteïene wat direk verband hou met sperm motiliteit, morfologie en energie metabolisme. Ten slotte, in die huidige studie is unieke tegnieke geïmplementeer om proteïen verskille tussen onvolwasse en volwasse spermpopulasies te ondersoek. Uit hierdie resultate is dit duidelik dat proteïen uitdrukking in die totale sperm proteoom en in die kern proteïen fraksie beduidend verskil en onvolledig is in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Op grond van hierdie bevindinge word aanbeveel dat verdere studies op menslike sperme gedoen moet word ten einde die rol van individuele proteïene in sperm funksie te kan bepaal. Proteomika is ‘n ideale tegniek om die iodiopatiese oorsake van manlike infertiliteit te identifiseer, aangesien dit kan help in die identifisering van unieke reseptore (en seintransduksie paaie) wat gebruik kan word om sperm disfunksie te verbeter deur farmaseutiese behandeling.

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.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats. / AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The ultimate consequence of the interruption of blood flow to the myocardium is necrosis. In view of the prevalence of coronary artery disease in the general population, and the deleterious effects of myocardial ischaemia on myocardial tissue, it is important to develop new strategies to protect the myocardium against ischaemia. Necrosis of myocardial tissue has for a long time been considered to be the main component of the damage incurred by myocardial infarction. Recently the importance of the contribution of apoptotic cell death in the context of myocardial ischaemia/reperfusion injury has become apparent. There is a general agreement that early reperfusion is necessary to salvage myocardial tissue from cell death. Preconditioning is the phenomenon whereby brief episodes of ischaemia and reperfusion protect the heart against a subsequent longer period of ischaemia. This endogenous mechanism is the strongest form of protection against myocardial infarction that has yet been described. Apart from ischaemie preconditioning (IPC), protection can also be elicited with pharmacologic agents, such as activation of the beta-adrenergic receptor with isoproterenol. Ischaemie preconditioning protects the myocardium against necrosis, arrhythmias and apoptosis, and increases functional recovery upon reperfusion. Betaadrenergic receptor stimulated preconditioning (PPC) has been shown to improve post-ischaemie functional recovery, but it is not known whether it also protects against myocardial infarction and apoptosis. The signaling pathways involved in preconditioning have been extensively studied. A distinction is usually made between factors that act as triggers, or as mediators of protection. Triggers activate cellular responses before the onset of sustained ischaemia, and its involvement is demonstrated by showing that inhibitors of the trigger bracketing the preconditioning protocol can block its protective effect, or that transient administration with washout before sustained ischaemia can activate a protective effect. A mediator operates during sustained ischaemia, and its involvement is demonstrated by showing that infusion of an inhibitor of its action immediately prior to sustained ischaemia (without washout) can block its protective effect. Another approach to demonstrate a mediator role is to attempt to activate signal transduction pathways during sustained ischaemia. As it is not possible to infuse substances during ischaemia, activators are infused immediately prior to ischaemia without washout of the agent and subsequently its effect on protection is observed. It is clear that the evolutionary conserved stress activated pathways are involved in preconditioning. There are three pathways i.e., the extracellular receptor activated pathways (ERK), c-jun terminal activated kinases (JNK) and p38 mitogen-activated protein kinases (MAPK). The precise role of the p38 MAPK pathway has not been elucidated. Experimental evidence has suggested a role for the activation of p38 MAPK as a trigger, as well as a mediator of the protective effect of preconditioning. There is however also strong evidence that the attenuation of p38 MAPK activation during sustained ischaemia, rather than its activation, is responsible for the protection that is observed. Furthermore, the role of p38 MAPK has only been investigated in relation to its protection against necrosis, but not apoptosis. AIMS: The aim of this study was to: (I) Establish a model of preconditioning in neonatal cardiomyocyte cell culture. The reason was that such a model could potentially enable one to rapidly elucidate the signal transduction pathways in an environment without the influence of non-cardiac cells. (II) Investigate whether IPC and ~PC protect against necrosis and apoptosis. (III) Elucidate the role of the stress-activated kinase, p38 MAPK, in preconditioning. METHODS: 1. Neonatal rat cardiomyocyte cell culture model A viability assay with 3-[4,5- Dimethylthaizol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was first developed using different concentrations - a concentration of 0.25% was found to be optimal to determine viability. Neonatal cardiomyocyte cell cultures were subjected to sustained simulated "ischaemia" by using either 5 mM KCN plus deoxyglucose (DOG) for 5 min or potassium cyanide (KCN) for 45 min. Some cell cultures were preconditioned with either chemical ischaemia (5 mM KCN for 5 min) or isoproterenol (10-7 M) for 5 min and 60 min reoxygenation before being exposed to sustained simulated ischaemia. 2. Isolated adult rat cardiomyocyte model Isolated cardiac myocytes were exposed to 2 hours of hypoxia, which was induced by pelletting the cells by centrifugation, and covering them with a thin layer of mineral oil. Some groups were preconditioned with either hypoxia for 10 min at 37° C or isoproterenol (10-7 M) for 5 min, followed by reoxygenation for 20 minutes. The trypan blue exclusion method and MTT method developed in the neonatal cardiomyocytes were used to assess viability. 3. Isolated perfused rat heart model 3.1 Infarct size was determined in a model of regional ischaemia by using tetrazolium staining and determining the area of necrosis (exclusion of tetrazolium) as a percentage of area at risk. These hearts were subjected to 35 min global ischaemia and 30 min reperfusion. Some groups were preconditioned by three cycles of 5 min global ischaemia or addition of isoproterenol (10-7 M) for 5 min, followed by 5 min reperfusion before the onset of sustained regional ischaemia. 3.2 p38 MAPK activation and markers of apoptosis: p38 MAPK activation was determined using antibodies against dual phosphorylated p38 MAPK (i.e. activated p38 MAPK). Apoptosis was measured by using antibodies against activated caspase-3, and against a fragment of PARP (PARP cleavage). For these experiments isolated rat hearts were exposed to global ischaemia for 25 min followed by 30 min reperfusion. Some groups were preconditioned with three cycles of 5 min global ischaemia. A global ischaemia model was used in order to have sufficient tissue available for the Western blot determinations. This necessitated a shorter period of sustained ischaemia, as the globally ischaemie heart does not recover sufficiently after a longer period of ischaemia such as is necessary in regional ischaemia experiments. 3.3 The role of p38 MAPK in ischaemie preconditioning was investigated by administration of SB 203580 (1IJM),a selective inhibitor of p38 MAPK, either bracketing the preconditioning (i.e. to determine its role as a trigger) or for 10 min immediately prior to sustained ischaemia (i.e. to determine its role as a mediator). The second approach was to use anisomycin, an activator of p38 MAPK, as a trigger (infusion for 10 min followed by wash out) or as a mediator (10 min immediately prior to sustained ischaemia) in the same model as used for determination of p38 MAPK activity. The infusion of anisomycin for 10 min has been shown to elicit activation of p38 MAPK to a similar extent as has been observed with an ischaemie preconditioning protocol. The endpoints used were infarct size and markers of apoptosis. RESULTS: 1. Neonatal rat cardiomyocyte cell culture model It was not possible to establish a model of preconditioning of neonatal cardiomyocytes that was consistently successful. It was therefore decided to abandon the attempts and to use a different cell model. 2. Isolated adult rat cardiomyocyte model Isolated adult cardiomyocytes were preconditioned successfully, but produced too little material to perform simultaneous determinations of cell viability and Western blots (p38 MAPK activation and markers of apoptosis). It was therefore decided to use the isolated perfused adult rat heart. 3. Isolated perfused adult rat heart model 3.1 Both IPC and PPCprotect against infarction and apoptosis: Using two models of preconditioning i.e., IPC and PPC, the protective effects of preconditioning were demonstrated convincingly against infarction (necrosis). IPC and PPC both caused a significant reduction in infarct size (12.2±1.4 and 15.2±2.6%) versus Non-PC hearts (29.6±2.9%) (p < 0.001). Both forms of preconditioning also protected against apoptosis, by significantly reducing the markers of apoptosis, caspase-3 activation and PARP cleavage. The protection afforded by both forms of preconditioning was accompanied by a marked decrease in activation of p38 MAPK upon reperfusion. The relationship between p38 MAPK and the protection that was elicited by preconditioning was then investigated, namely whether p38 MAPK acted as a trigger, or as a mediator of protection. To investigate the role of p38 MAPK as a mediator or a trigger in preconditioning, use was made of (i) a specific inhibitor of p38 MAPK activation i.e., SB 203580 and (ii) a known activator of p38 MAPK i.e., anisomycin. 3.2 p38 MAPK as a trigger of protection: Administration of SB 203580 during the IPC protocol and washed out before sustained ischaemia did not abolish the protective effect of ischaemie preconditioning, and resulted in a small, but significant increase in caspase-3 activation and PARP cleavage. On the other hand, activation of p38 MAPK with anisomycin for 10 min followed by washout also resulted in a significant reduction in necrosis (infarct size 14.9±2.2 versus 29.6±2.9% in Non-PC hearts) (p < 0.001) and both markers of apoptosis. The latter results suggested that p38 MAPK was a trigger of preconditioning. If this was the case, why didn't SB 203580 abolish the protection of IPC? The most likely explanation was that multiple protective mechanisms were activated during a multi-cycle protocol of ischaemic preconditioning, of which activation of p38 MAPK was only one. Inhibition of p38 MAPK with SB 203580 would therefore not be expected to block the activation of those mechanisms that were independent of p38 MAPK, but were still capable of protecting against necrosis or apoptosis. It is very interesting that a small increase in apoptosis was observed when SB 203580 was used in this situation, as it may indicate that the protection against apoptosis was more dependent on the activation of p38 MAPK than the protection against necrosis, as no effect was seen on infarct size. Another explanation could be that infarct size determination was not sensitive enough to detect such small effects. 3.3 p38 MAPK as a mediator of protection: Inhibition of p38 MAPK activation with SB 203580 administered 10 min before sustained ischaemia caused a significant decrease in infarct size compared to Non-PC hearts (12.6±1.9 vs 29.6±2.9%) (p < 0.001) equivalent to that of hearts preconditioned with ischaemia. This was accompanied by a similar pattern of protection against apoptosis, with significantly reduced activation of caspase-3 activation and PARP cleavage. These results strongly supported a role for the attenuation of p38 MAPK activation as a mediator of preconditioning against ischaemia/reperfusion-mediated necrosis and apoptosis. However, the results of the experiments with anisomycin were at first glance not compatible with such a conclusion. The administration of the activator of p38 MAPK, anisomycin, for 10 min immediately prior to sustained ischaemia resulted in significant protection against necrosis (infarct size 16.6±2.4% vs 29.6±2.9% in Non-PC hearts) (p < 0.01) and reduced caspase-3 activation and PARP cleavage indicating less apoptosis. The reason for these findings were probably that this method of administration of anisomycin did in fact not activate p38 MAPK during sustained ischaemia, but actually served as a trigger to protect against ischaemia - similarly as if it had been infused with washout of the drug. Support for this notion was found in the fact that p38 MAPK activation was decreased upon reperfusion. These results suggested that the logistical problem of not being able to infuse a drug into the myocardium during ischaemia could not be overcome by immediate prior infusion, and that the administration of anisomycin in this way had activated downstream effectors of the p38 MAPK signal transduction pathway. An important contender for such an effector would be heat shock protein 27 (HSP27), which has been shown to play an important role in protection against apoptosis, and stabilisation of actin, and thus the cytoskeleton. Another possibility was that anisomycin had activated the JNK stress activated kinases. The elucidation of a role of this signal transduction pathway would necessitate the use of anisomycin in the presence of an agent such as curcumin, an inhibitor of JNK. Final conclusion: The work in this thesis showed that the stress activated kinase, p38 MAPK, was involved in the protective effect of ischaemie preconditioning. The results suggested a role for the activation of p38 MAPK as a trigger of protection, and the attenuation of p38 MAPK as a mediator of protection, which was observed in the reduction of both necrosis (infarct size) and apoptosis as determined with caspase- 3 activation and PARP cleavage. / AFRIKAANSE OPSOMMING: Die afsluiting van bloedvloei na die miokardium gee aanleiding tot nekrose. In die lig van die voorkoms van koronêre bloedvatsiekte onder die algemene populasie, en die nadelige effekte van miokardiale isgemie op miokardiale weefsel, is dit belangrik om nuwe strategieë te ontwikkel wat die miokardium teen isgemie beskerm. Nekrose van miokardiale weefsel word tradisioneel as die belangrikste komponent van die skade aangerig deur miokardiale infarksie beskou. Die belang van apoptotiese seldood in die konteks van miokardiale isgemie/herperfusie (I/R) het onlangs na vore getree. Dit word algeneem aanvaar dat vroeë vroegtydige herperfusie noodsaaklik is om miokardiale weefsel te beskerm teen seldood. Prekondisionering is 'n verskynsel waartydens kort episodes van IIR die hart teen 'n daaropvolgende langer periode van isgemie beskerm. Hierdie endogene meganisme is die kragtigste vorm van beskerming teen miokardiale infarksie tot dusver beskryf. Afgesien van isgemiese prekondisionering (IPC), kan beskerming ook deur farmakologiese middels, soos byvoorbeeld die aktivering van die beta-adrenerge reseptore met isoproterenol, ontlok word. IPC beskerm die miokardium teen nekrose, arritmieë en apoptose, en verhoog funksionele herstel na herperfusie. Daar is reeds aangetoon dat betaadrenerge prekonsionering (~PC) post-isgemiese funksionele herstel verbeter, maar dit is nog onbekend of beskerming ook teen miokardiale infarksie en apoptose verleen word. Die seintransduksie paaie betrokke tydens prekondisionering is reeds in detail bestudeer. Daar word gewoonlik tussen faktore wat optree as snellers, of as mediators van beskerming, onderskei. Snellers aktiveer sellulêre response voor die aanvang van volgehoue isgemie, en hul betrokkenheid word aangetoon deurdat inhibisie van snellers tydens die prekondisionering protokol, beskerming ophef. Snellers se effekete kan ook ontlok word deur hulle tydelike toe te dien en dan net voor volgehoue isgemie weer uit te was. Mediators oefen hulle effek tydens volgehoue isgemie uit, en hulle betrokkenheid word gedemonstreer deurdat toediening van inhibitors net voor volgehoue isgemie (sonder uitwas) hulle beskermende effekte ophef. Mediators se rol kan ook aangetoon word deur te poog om seintransduksie paaie tydens volgehoue isgemie te aktiveer. Aangesien dit ontmoontlik is om middels tydens isgemie te infuseer, word aktiveerders onmiddelik voor die aanvang van isgemie toegedien sonder om hulle uit te was, sodat hulle effekte op beskerming vervolgens bestudeer kan word. Dit is duidelik dat die evolusionêr-behoue stres geaktiveerde paaie tydens prekondisionering betrokke is. Daar is drie paaie nl. die ekstrasellulêre reseptor geaktiveerde pad (ERK), c-jun terminaal geaktiveerde kinases (JNK) en p38 mitogeen geaktiveerde proteïen kinases (MAPK). Die spesifieke rol van die p38 MAPK pad is nog nie ontrafel nie. Eksperimentele bewyse stel 'n rol vir die aktivering van p38 MAPK as 'n sneller, sowel as 'n mediator van die beskermende effek van prekondisionering, voor. Daar is egter ook sterk bewyse dat 'n afname in p38 MAPK aktivering tydens volgehoue isgemie, eerder as sy aktivering, verantwoordelik is vir die waargenome beskermende effek. Verder is die rol van p38 MAPK slegs in die konteks van beskerming teen nekrose, maar nie teen apoptose nie, bestudeer. DOELWITTE: Die doelwit van hierdie studie was: (I) Die vestiging van 'n prekondisionering model in neonatale kardiomiosiet in selkultuur. Hierdie model sou potensieel 'n spoedige ontrafeling van die seintransduksie paaie sonder die invloed van nie-kardiale selle bewerkstellig. (II Om ondersoek in te stelof IPC en PPCteen nekrose en apoptose beskerm. (III) Die ontrafeling van die rol van die stres geaktiveerde kinase, p38 MAPK, tydens prekondisionering. METODES: 1. Neonatale rot kardiomiosiet weefselkultuur model 'n Lewensvatbaarheids essai is ontwikkel deur van verskillende konsentrasies van 3-[4,5-dimetielthiazol-2-yl]-2,5-difeniel-tetrazolium bromied (MTT) gebruik te maak - 'n konsentrasie van 0.25% was optimaalom lewensvatbaarheid te bepaal. Neonatale kardiomiosiet weefselkulture is onderwerp aan volgehoue gesimuleerde "isgemie" deur gebruik te maak van 5 mM KCN plus deoksiglukose (DOG) vir 5 minute of 45 min KCN. Sommige weefselkulture is geprekondisioneer deur middel van chemiese isgemie (5 mM KCN vir 5 min) of van isoproterenol (10-7 M) vir 5 minute en 60 minute reoksigenasie alvorens dit bloot gestel is aan volgehoue gesimuleerde isgemie. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde kardiomiosiete is aan twee uur hipoksie blootgestel deur selle in 'n pellet te sentrifugeer en met 'n dun lagie mineraalolie te bedek. Sommige groepe is geprekondisioneer deur middel van 10 minute hipoksie by 37°C, of toediening van isoproterenol (10-7 M) vir 5 minute gevolg deur 20 minute reoksigenasie. Die tripaanblou uitsluitings metode en MTT metode soos ontwikkel in die neonatale kardiomiosiet model is gebruik om lewensvatbaarheid te bepaal. 3. Geïsoleerde geperfuseerde volwasse rot hart model 3.1 Infarkgrootte is bepaal met 'n model van streeks isgemie deur van tetrazolium kleuring gebruik te maak, waarna die area van nekrose (uitsluiting van tetrazolium) as 'n presentasie van die risiko area bepaal is. Hierdie harte was onderwerp aan 35 minute globale isgemie en 30 minute herperfusie. Sommige groepe is geprekondisioneer met 3 siklusse van 5 minute globale isgemie, of die toevoeging van isoproterenol (10-7 M) vir 5 minute, gevolg deur 5 minute herperfusie voor die aanvang van volgehoue streeks isgemie. 3.2 p38 MAPK aktivering en merkers van apoptose: p38 MAPK aktivering is bepaal deur gebruik te maak van anti-liggame teen tweeledige gefosforileerde p38 MAPK (d.w.s. geaktiveerde p38 MAPK). Apoptose is bepaal deur gebruik te maak van anti-liggame teen geaktiveerde kaspase-3, en teen 'n fragment van PARP (PARP kliewing). Tydens hierdie eksperimente is geïsoleerde rotharte bloot gestel aan 25 minute globale isgemie gevolg deur 30 minute herperfusie. Sommige groepe is geprekondisioneer met drie siklusse van 5 minute globale isgemie. Om voldoende weefsel vir Westerse klad tegnieke te verkry, is gebruik gemaak van 'n globale isgemie model. As gevolg hiervan was 'n kort periode van volgehoue isgemie genoodsaak, aangesien die globale isgemiese hart nie voldoende herstel na 'n langer periode van isgemie nie, soos wat benodig word in streeks isgemiese eksperimente. 3.3 Die rol van p38 MAPK tydens IPC is bepaal deur die toediening van 'n 1IJM konsentrasie van SB 203580, 'n selektiewe inhibitor van p38 MAPK, hetsy tydens prekondisionering (d.w.s. om die rol as 'n sneller te bepaal), óf vir 10 minute direk voor die aanvang van volgehoue isgemie (d.w.s. om dus sy rol as mediator te bepaal). Die tweede benadering was om anisomisien, 'n aktiveerder van p38 MAPK, as sneller (toediening vir 10 minute gevolg deur uitwassing) of as mediator (10 minute direk voor aanvang van volgehoue isgemie) in dieselfde model as in die geval van p38 MAPK aktiviteit bepaling, te gebuik. Die toediening van anisomisien vir 10 minute het aangetoon dat dit p38 MAPK aktivering kan ontlok tot dieselfde maate as die IPC protokol. Die eindpunte was infarkgrootte en merkers van apoptose. RESULTATE: 1. Neonatale rot kardiomiosiet weefselkultuur model Dit was nie moontlik om 'n suksesvolle model met konsekwente resultate vir die prekondisionering van neonatale kardiomiosiete te vestig nie. Daar is dus besluit om af te sien van hierdie pogings en eerder 'n alternatiewe selmodel te gebruik. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde volwasse kardiomiosiete is suksesvol geprekondisioneer, maar het te min materiaalopgelewer vir die gelyktydige bepaling van sellewensvatbaarheid, p38 MAPK aktivering en merkers vir apoptose. Daar is dus besluit om die geïsoleerde geperfuseerde volwasse rothart te gebruik. 3. Geïsoleerde geperfuseerde volwasse rothart model 3.1 Beide IPC en PPCbeskerm teen infarksie en apoptose: Deur gebruik te maak van twee prekondisionering modelle d.w.s. IPC en PPC, is die beskermende effekte van prekondisionering teen infraksie (nekrose) oortuigend gedemonstreer. Beide IPC en PPC het In betekenisvolle afname in infarkgrootle veroorsaak (12.2 ± 1.4 en 15.2 ± 2.6% respektiewelik), vs Nie-PC harte (29.6 ± 2.9%)(p < 0.001). Beide vorme van prekondisionering het ook teen apoptose beskerm deur die apoptose merkers, kaspase-3 aktivering en PARP kliewing te verlaag. Die beskerming verkry deur beide vorms van prekondisionering is geassosieer met In merkbare afname in die aktivering van p38 MAPK na herperfusie. Die verband tussen p38 MAPK en die beskerming ontlok deur prekondisionering is gevolglik ondersoek, naamlik of p38 MAPK optree as 'n sneller of as 'n mediator van beskerming. Om die rol van p38 MAPK as 'n mediator of sneller tydens prekondisionering te ondersoek is daar gebruik gemaak van (I) 'n spesifieke inhibitor van p38 MAPK aktivering nl. SB 203580 en (II) 'n bekende aktiveerder van p38 MAPK nl. anisomisien. 3.2 p38 MAPK as 'n sneller vir beskerming: Toediening van SB 203580 tydens die IPC protokol en uitwassing daarvan voor die aanvang van volgehoue isgemie het nie die beskermende effek van IPC opgehef nie, en het gelei tot 'n klein maar betekenisvolle verhoging in kaspase-3 aktivering en PARP kliewing. Andersins het die aktivering van p38 MAPK met anisomisien vir 10 minute gevolg deur In uitwas ook tot In betekenisvolle afname in nekrose (infarkgrootte 14.9 ± 2.2 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.001) in beide merkers van apoptose gelei. Laasgenoemde resultate dui daarop dat p38 MAPK inderdaad 'n mediator van prekondisionering is. Indien dit die geval is, waarom het SB 203580 nie die beskermende effek van IPC opgehef nie? Die mees waarskynlike verklaring is dat veelvuldige beskermingsmeganismes tydens 'n multi-siklus protokol van IPC geaktiveer word, waarvan p38 MAPK aktivering slegs een is. Dit is dus onwaarskynlik dat die inhibisie van p38 MAPK met SB 203580 die aktivering van daardie meganismes onafhanklik van p38 MAPK sal blokkeer en steeds in staat sal wees tot beskerming teen nekrose en apoptose. Dit is interessant dat In klein verhoging in apoptose waargeneem is toe SB 203580 gebruik is onder hierdie toestande, aangesien dit daarop kan dui dat die beskerming teen apoptose meer afhanklik was van die aktivering van p38 MAPK as die beskerming teen nekrose, siende dat geen effek op infarkgrootte waargeneem is nie. 'n Verdere verklaring kan wees dat die bepaling van infarkgrootte nie sensitief genoeg is om sulke klein effekte waar te neem nie. 3.3 p38 MAPK as 'n mediator vir beskerming: Inhibisie van p38 MAPK aktivering deur SB 203580 toediening 10 minute voor volgehoue isgemie het 'n betekenisvolle verlaging in infarkgrootte in vergelyking met Nie-PC harte veroorsaak (12.6 ± 1.9 vs 29.6 ± 2.9%) (p < 0.001) soortgelyk aan dié van harte geprekondisioneer met isgemie. Dit is geassosieer met In soortgelyke patroon van beskerming teen apoptose, met betekenisvolle verlaagde kaspase-3 aktivering en PARP kliewing. Hierdie resultate ondersteun die rol van die afname van p38 MAPK aktivering as 'n mediator van prekondisionering teen I/R-gemedieerde nekrose en apoptose. Die resultate van die anisomisien eksperimente was met die eerste oogopslag nie in oorstemming met hierdie gevolgtrekking nie. Die toedienning van die p38 MAPK aktiveerder, anisomisien, vir 10 minute voor volgehoue isgemie het tot 'n betekenisvolle beskerming teen nekrose aanleiding gegee (infarkgrootte 16.6 ± 2.4 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.01) en verlaagde kaspase-3 aktivering en PARP kliewing wat dui op verlaagde apoptose. Die rede vir hierdie bevindings is moontlik dat die metode van anisomisien toediening nie p38 MAPK geaktiveer het tydens volgehoue isgemie nie, maar eintlik gedien het as 'n sneller vir beskerming teen isgemie - amper asof dit toegedien sou word sonder om uitgewas te word. Ondersteuning vir hierdie aanname word gevind in die feit dat p38 MAPK aktivering verlaag is na herperfusie. Hierdie resultate stel voor dat die logistiese probleem dat In middel nie tydens isgemie toegedien kan word nie, nie oorkom kan word deur onmiddelike voortydige infusie nie, en dat die toediening van anisomisien op hierdie manier gelei het tot die aktivering van stroom-af effektors van die p38 MAPK seintransduksie pad. 'n Belangrike kandidaat vir so 'n effektor is "heat shock protein 27" (HSP27), wat reeds aangetoon is om 'n belangrike rol in die beskerming teen apoptose en destabilisering, en dus die sitoskelet, te speel. 'n Ander moontlikheid is dat anisomisien die JNK stres geaktiveerde kinases geaktiveer het. Die ontrafeling van die rol van hierdie seintransduksie pad noodsaak die gebruik van anisomisien in die teenwoordigheid van 'n agent soos curcumin, 'n JNK inhibitor. Finale gevolgtrekking: Die werk soos vervat in hierdie tesis toon aan dat die stres geaktiveerde kinase, p38 MAPK, betrokke is in die beskermings effek van isgemiese prekondisionering. Die resultate dui op 'n rol vir die aktivering van p38 MAPK as 'n sneller vir beskerming, en die afname in p38 MAPK as 'n mediator vir beskerming, soos waargeneem in die vermindering van veranderlikes van beide nekrose (infarkgrootte) en apoptose soos bepaal deur kaspase-3 aktivering en PARP kliewing.