Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008. / Cells are equipped with an array of adaptive mechanisms to contest the undesirable effects of ischemia and the associated hypoxia. Indeed, many studies have suggested that there is an increase in the PI3K/Akt pathway activation during hypoxia and ischemia. Damaged muscle can be regenerated by recruiting myogenic satellite cells which undergo differentiation and ultimately lead to the regeneration of myofibres. The C2C12 murine myogenic cell line is popular for studying myogenesis in vitro, and has been used in many studies of ischemic microenvironments. PI3K/Akt pathway activity is increased during C2C12 myogenesis and this is known to produce an apoptosis resistant phenotype. In this study, we provide evidence that high basal levels of PI3K activity exist in C2C12 myotubes on day ten post-differentiation. Ischemia is characterized by depleted oxygen and other vital nutrients, and ischemic cell death is believed to be associated with an increasingly harsh environment where pH levels decrease and potassium levels increase. By employing a model that mimics these changes in skeletal muscle culture, we show that both acute simulated ischemia and acute hypoxia cause decreases in endogenous levels of the p85 and p110 subunits of PI3K and a consequent reduction in PI3K activity. Supplementing skeletal muscle cultures with inhibitors of the PI3K pathway provides evidence that the protective effect of PI3K/Akt is subsequently lost in these conditions. Using Western blot analysis, a PI3K ELISA assay as well as known inhibitors of the PI3K pathway in conjunction with the MTT assay we are able to demonstrate that the activation of downstream effectors of PI3K, including Akt, are concurrently decreased during acute simulated ischemia and acute hypoxia in a manner that is independent of PDK-1 and PTEN and that the decreases in the PI3K/Akt pathway activity produce a knock-on effect to the downstream signalling of transcription factors, such as Fox01 and Fox04, in our model. We proceed to provide compelling evidence that the apoptotic resistance of C2C12s is at least partially lost due to these decreases in PI3K/Akt pathway activity, by showing increased caspase-3 and PARP cleavage. Then, using vital staining techniques and a DNA fragmentation assay, we demonstrate increased cell membrane impairment, cell death and apoptosis after three hours of simulated ischemia and hypoxia in cultured C2C12 myotubes. In addition to the main findings, we produce evidence of decreased flux through the mTOR pathway, by showing decreased Akt-dependant phosphorylation at the level of TSC2 and mTOR during simulated ischemia and hypoxia. Finally, we present preliminary findings indicating increased levels of HIF1α and REDD-1, representing a possible oxygen sensing mechanism in our model. Therefore, we show that there is in fact a rapid decrease in PI3K/Akt activity during severe, acute simulated ischemia and hypoxia in C2C12 myotubes on day ten post-differentiation, and this causes a concomitant down regulation in cell survival pathways and increased activity of cell death machinery. Thereafter, we propose a possible mechanism of action and provide a platform for future studies.

Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008. / Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by a reduction in muscle fibre cross-sectional area as well as protein content, reduced force, elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated process and signs of this condition are often seen in inflammatory conditions such as cancer, AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an imbalance between protein degradation (increase) and protein synthesis (decrease) both contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are different, the loss of muscle mass in each case involves a common phenomenon that induces muscle proteolysis. It is becoming evident that interactions among known proteolytic systems (ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of clinical settings but the mechanisms mediating its effects are not completely understood. It is also now apparent that the transcription factor NF-κB is a key intracellular signal transducer in muscle catabolic conditions. This study investigated the various proposed signalling pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in order to synthesize our current understanding of the molecular regulation of muscle atrophy. Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a maximum of 8 days. During the last 2 days, myotubes were incubated with increasing concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24 and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT assay and Trypan Blue exclusion technique. Morphological assessment of cell death was conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for the measurement of cell size. In order to determine the source and amount of ROS production, MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the quantity of ROS production in TNF-α-induced muscle atrophy. Results and Discussion: Incubation of L6 myotubes with increasing concentrations of recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF- α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1 and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as significant reductions in differentiation markers. Western blot analysis also showed that certain MAPKs are activated in response to TNF-α. No profound changes were observed with ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS production. These findings suggest that TNF-α may elicit strong catabolic effects on L6 myotubes in a dose and time dependent manner. Conclusion: These observations suggest that TNF-α might have beneficial effects in skeletal muscle in certain circumstances. This beneficial effect however is limited by several aspects which include the concentration of TNF-α, cell type, time of exposure, culture conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The effect of TNF-α can be positive or negative depending on the concentration and time points analysed. This action is mediated by various signal transduction pathways that are thought to cooperate with each other. More understanding of these pathways as well as their subsequent upstream and downstream constituents is obligatory to clarify the central mechanism/s that control physiological and pathophysiological effects of TNF-α in skeletal muscle.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Bi-directional communication exists between HPA-axis activation and interleukin-6 (IL-6). However, the relative contribution of centrally versus peripherally secreted IL- 6 remains unclear, especially under psychological stress conditions. We hypothesised that the HPA response to mild psychological stress is dependent on IL- 6, both centrally and peripherally. 120 male Wistar rats were divided into four groups, depending on whether they received an anti-IL-6 antibody (Ab) (2μg/ml/kg body weight) or a placebo (sterile saline) injection and whether or not they were subjected to 1 hour of restraint stress for 1, 2 or 3 days. Rats were euthanized 24 hours after stress exposure. Plasma corticosteroid (GC) levels remained significantly increased 24 hours after a single stress exposure (control placebo (CP) versus stress placebo (SP): p < 0.05). The undetectable plasma IL-6 levels evident across all groups may be explained by the short half-life of IL-6. Plasma IL-1β levels decreased when IL-6 was blocked in unstressed animals (CP versus CAb: p < 0.05), suggesting a role for IL-6 in the maintenance of IL-1β levels under tonic physiological conditions. At tissue level, pituitary gland mass increased significantly at time point 2, independently of stress when blocking IL-6 (CAb: p < 0.05). This suggests that when normal homeostasis is threatened, immediate adaption or at least compensation may occur. It was observed that GR, IL-1β, IL-1βR, IL-6, IL-6R and GABAARα1 showed no response to stress alone in the pituitary. It is therefore more likely that resistance to adaptation exists centrally. IL-1β and IL-1βR (p < 0.05) and GABAARα1 (p < 0.005) expression increased in the CAb group in the pituitary, again suggesting a role for IL-6 under control conditions. In terms of the adrenal, blocking IL-6 resulted in decreased glandular mass at time point 1, independent of stress (CAb and SAb: p < 0.005). The up-regulation in GR expression seen in CAb and SAb (p < 0.05) may be the effect of a compensatory mechanism to increase IL-6 dependent bioactivity of GCs. The fact that expression of IL-6, IL-6R, IL-1β and IL- 1βR consistently increased in the Ab groups, and mostly in the zona fasciculata and zona reticularis, suggests that lack of local direct negative cytokine feedback occurred in response to very low plasma IL-6 levels and that this contributes more than GCs in the down-regulation of inflammatory cytokine release. In conclusion, consistent effects of the Ab were apparent in the tissues investigated, even in control conditions, suggesting that IL-6 plays a role in the maintenance of basal homeostasis, including its regulation of the response to psychological stress. We found differential regulation in terms of cytokines and GCs when comparing peripheral versus central effects of stress and Ab, as well as the levels of cytokines in the blood compartment, compared to within tissues. / AFRIKAANSE OPSOMMING: Daar bestaan twee-rigting kommunikasie tussen HPA-as aktivering en interleukin-6 (IL-6), allhoewel die relatiewe bydrae van sentraal versus perifeer afgeskeide IL-6 nog onduidelik is, veral gedurende sielkundige strestoestande. Ons hipotese is dat die HPA reaksie tot sielkundige stres afhanklik van IL-6 is, beide sentraal en in die periferie. 120 manlike Wistar rotte is in vier groepe verdeel, afhangende van of hulle ‘n anti-IL- 6 teenliggaampie (Ab) (2μg/ml/kg liggaamsgewig) of ‘n plasebo (steriele soutoplossing) inspuiting gekry het, en of hulle onderworpe was aan 1 uur van vaskeer-stres vir 1, 2 of 3 dae. Rotte is 24 uur na blootstelling aan stres aan genadedood onderwerp. Bloed kortikosteroïed (GC) vlakke het beduidend toegeneem binne 24 uur na ‘n eenmalige stres blootstelling (kontrole plasebo (CP) versus stres plasebo (SP): p < 0.05). Die onmeetbaar lae vlakke van IL-6 regoor al die groepe, kan verduidelik word na aanleiding van die kort half-leeftyd van IL-6. Bloed IL-1β vlakke het afgeneem in kontrole rotte wanneer IL-6 geblok is (CP versus CAb: p < 0.05). Dit kan beteken dat IL-6 noodsaaklik is vir die onderhoud van IL-1β vlakke gedurende basale toestande. Op weefselvlak het die hipofise massa toegeneem by tydpunt 2 toe IL-6 geblok is, onafhanklik van stres (CAb: p < 0.05). Dit dui aan dat wanneer normale homeostase bedreig word, daar onmiddelike aanpassing of kompensasie plaasvind. Dit is opvallend dat GR, IL-1β, IL-1βR, IL-6, IL-6R en GABAARα1 geen respons in terme van stres alleen in die hipofise getoon het nie. Na aanleiding daarvan is dit meer waarskynlik dat weerstand tot aanpassing sentraal bestaan. IL-1β and IL-1βR (p <0.05) en GABAARα1 (p < 0.005) uitdrukking in die hipofise het toegeneem in die CAb groep, wat weereens ‘n rol vir IL-6 onder kontrole toestande uitwys. In terme van die bynier, het die blok van IL-6 ‘n afname in massa veroorsaak by tydpunt 1, wat weer onafhanklik van stres was (CAb en SAb: p < 0.005). Die opregulering in die CAb en SAb groepe (p < 0.05), kan wees as gevolg van ‘n kompensasie meganisme om IL-6 afhanklike GC aktiwiteit te verhoog. Die feit dat die uitdrukking van IL-6, IL-6R, IL-1β and IL-1βR in die Ab groepe deurlopend verhoog was, en meeste in die zona fasciculata en zona reticularis, stel voor dat daar ‘n tekort aan plaaslike, direkte sitokien negatiewe terugvoering was, as gevolg van die merkwaardige lae bloed IL-6 vlakke en dat dit meer bydra as GCs in die afregulering van inflammatoriese sitokien vrystelling.

Thesis MSc (Physiological Sciences))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Ischaemic heart disease is a leading cause of death worldwide and is also largely contributing to deaths in Africa. Better treatment or even prevention of ischaemia/reperfusion injury in the heart, necessitates a better understanding of the molecular pathways and mechanisms of cell death. Three types of cell death can occur in the diseased myocardium. Type I, better known as apoptotic cell death, is characterised by cell shrinkage and chromatin condensation, type II, known as autophagic cell death, is characterised by intracellular accumulation of double membranes vacuoles and type III, necrotic cell death, is characterised by cellular swelling and loss of membrane integrity. Many signaling pathways are activated during ischaemia/reperfusion injury which include the mitogen activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK) and p38 MAPK. These kinases are dephosphorylated by appropriate phosphatases. MAPK phosphatase-1 (MKP-1), a dual specificity phosphatase, inactivates the MAPKs by dephosphorylating specific Thr/Tyr residues. Upregulation of MKP-1 during ischaemia/reperfusion injury has been shown to be cardioprotective, however no knowledge regarding a role of MKP-1 in autophagy exists. Therefore the aim of this study is to investigate the role of MKP-1 in autophagy, apoptosis and necrosis during simulated ischaemia/reperfusion injury in the heart.METHOD: H9C2 cells (rat cardiomyocytes) were cultured under standard conditions. Upon reaching 75-80% confluency, cells were treated for 30 min during normoxic conditions with dexamethasone, to induce MKP-1 expression, or sanguinarine, to inhibit MKP-1 induction. Thereafter, they were exposed to 3 hrs simulated ischaemia (induced by an ischaemic buffer and 5% CO2/1% O2) in the presence of the above mentioned treatments. Cells were then allowed to reperfuse for 30 min in the presence of dexamethasone or sanguinarine. Samples were analysed after simulated ischaemia and after reperfusion. Cell viability was measured by MTT assay. Propidium iodide and Hoechst staining were used to assess morphological markers of apoptosis and necrosis. LDH release during reperfusion was assessed as indicator of necrotic cell death. LysoTracker®Red was used to visualise the autophagic flux occurring during ischaemia/reperfusion in the cell. Flow cytometry was used to quantify cells stained with acridine orange as indicator for autophagy. Autophagic and apoptotic protein markers as well as MAPK and MKP-1 activity were analysed by Western Blotting. RESULTS: Our results indicate a clear relationship between MKP-1 induction, autophagy and cell survival during simulated ischaemia/reperfusion (SI/R). MKP-1 inhibition during SI/R resulted in decreased autophagy activity accompanied by significant apoptotic and necrotic cell death. Increased MKP-1 induction, on the other hand, during SI/R resulted in increased levels of autophagy activity and subsequent attenuation of apoptotic and necrotic cell death. p38 MAPK phosphorylation was significantly higher while MKP-1 was inhibited and significantly lower while MKP-1 was induced. This strongly indicates that upregulation of MKP-1, known to attenuate ischaemia/reperfusion injury, has an important role in cell survival during ischaemia/reperfusion injury in the heart, through its involvement in the regulation of autophagic activity as a stress response against apoptotic or necrotic cell death. / AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die grootste oorsake van sterftes wêreldwyd en dra ook beduidend by tot sterftes in Afrika. Om iskemiese hartsiektes te behandel of selfs te voorkom, is 'n goeie begrip van die molekulêre paaie wat betrokke is tydens iskemie/herperfusie, noodsaaklik. Drie tipes seldood kom tydens patologiese toestande in die hart voor. Tipe I, ook bekend as apoptotiese seldood, word gekenmerk deur selkrimping en kromatien kondensasie, tipe II, ook bekend as autofagiese seldood word gekenmerk deur intrasellulêre opeenhoping van dubbelmembraan vakuole en tipe III, bekend as nekrotiese seldood, word deur sellulêre swelling en verlies van membraan integriteit gekenmerk. Iskemie/herperfusie lei tot die aktivering van seintransduksiepaaie wat die MAPKs, soos p38, ERK en JNK insluit. Hierdie kinases word deur die gepaste fosfatases gedefosforileer. MKP-1, 'n dubbele spesifieke fosfatase, deaktiveer MAPKs deur hul Thr/Tyr eenhede te defosforileer. Alhoewel daar al voorheen getoon is dat verhoogte MKP-1 ‘n beskermende funksie in die hart tydens iskemie/herperfusie het, is daar nog geen bewyse vir ‘n rol van MKP-1 tydens autofagie nie. Die doel van hierdie studie is dus om die rol van MKP-1 in autofagie, apoptose en nekrose te ondersoek tydens gesimuleerde iskemie/herperfusie in die hart. METODE: H9C2 selle (rot ventrikulêre hartselle) is onder standaard toestande gekweek. Wanneer die selle 75-80% konfluensie bereik het, is dit behandel met dexamethasone of sanguinarine onder standaard toestande vir 30 min. Daarna is selle blootgestel aan 3 ure iskemie, in die teenwoordigheid van dexamethasone of sanguinarine. Selle is dan toegelaat om vir 30 min te herperfuseer, weer in die teenwoordigheid van dexamethasone of sanguinarine. Monsters is na iskemie en herperfusie geneem vir analise. Selvatbaarheid is gekwantifiseer deur ‘n MTT bepaling. Morfologiese merkers van seldood is bepaal met behulp van propidium iodide en Hoechst kleuringsmetodes. Laktaatdehidrogenase (LDH) vrystelling tydens herperfusie is as merker van nekrose gebruik. Autofagie is gevisualiseer deur gebruik te maak van LysoTracker®Red kleuring tydens iskemie en herperfusie. Akridienoranje is gebruik om suur kompartemente te kleur. Vloeisitometrie is as kwantifiseringstegniek vir autofagie gebruik. Western Blotting is gebruik om uitdrukking van merkerproteïene van autofagie en apoptose sowel as MAPK en MKP-1 aktiwiteit tydens iskemie/reperfisie te bepaal. RESULTATE: Ons resultate toon ‘n verband tussen MKP-1 induksie, autofagie en seloorlewing gedurende gesimuleerde iskemie/herperfusie (SI/R) aan. MKP- 1 inhibisie gedurende SI/R het tot ‘n afname in autofagie gelei tesame met ‘n beduidende toename in apoptotiese en nekrotiese seldood. Verhoogde MKP-1 induksie gedurende SI/R, daarteenoor, het autofagiese aktiwiteit verhoog, gepaardgaande met ‘n verlaging in apoptose en nekrose. p38 MAPK fosforilasie was beduidend hoër tydens MKP-1 inhibisie en laer met MKP-1 induksie. Hierdie resultate toon dat MKP-1 ‘n belangrike rol in seloorlewing speel tydens iskemie/herperfusiesskade in die hart, deur sy deelname in die regulering van autofagiese aktiwiteit as ‘n stres reaksie teen apoptotiese en nekrotiese seldood.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Background: IL-6 belongs to a cytokine super-family known to affect cell proliferation, although other family members are better characterized. Proliferation promoting factors (IL-6) compete with differentiation promoting factors (myogenic regulatory factors: MyoD and myogenin) to affect cell cycle. Cell cycle progression is assessed by determining the proportion of cells shifting from arrest to chromatin synthesis and mitosis phases (G0/G1 and S and G2/M respectively). Methods: This study assessed the effects of IL-6 on cell cycle progression and proliferation vs. differentiation of C2C12 skeletal myoblasts. Physiological doses (10 or 100 pg/ml) were compared to a high dose (10 ng/ml), with exposure lasting 48 hours (addition of IL-6 dose to proliferation medium at 0 and 24 hours). Acute signaling downstream of the IL-6 gp130 receptor was assessed after the first exposure. Results: Propidium iodide analysis of nuclear material using flow cytometry indicated shifts in forward scatter. Both Low and Medium doses shifted a greater proportion (p<0.05) of cells from G0/G1 to S and G2M phases at 24 hours and all doses resulted in the same shift (p<0.05) at the 48 hour time point. However, the High dose significantly (p<0.05) increased myogenin expression at the 48 hour time point. Microscopy indicated that confluence was prevented by low seeding density and did not influence the result. Cells harvested at 5 minutes post stimulation indicated that all doses significantly increased STAT3 phosphorylation. 10 minutes post stimulation the High dose group sustained elevated levels of STAT3 phosphorylation. Conclusions: Low and medium doses of IL-6 increase proliferation in a muscle satellite cell line by activating cell division and allowing myoblasts to remain in the active cell cycle. High doses of IL-6 increase differentiation by mediating upregulation of myogenic regulatory factors and this is thought to be due to prolonged STAT3 activation. Physiological control of myoblast behaviour by cytokines is evident and such control would be influenced by the severity of the endogenous cytokine response to various stimuli. / AFRIKAANSE OPSOMMING: Agtergrond: IL-6 behoort aan n sitokien super-familie bekend vir die affektering van sel verspreiding, alhoewel ander familie lede beter gekenmerk is. Bevordering van verspreiding faktore (IL-6) kompeteer met bevordering van differensiasie fatore (myogenic regulatory factors: MyoD en myogenin) om die sel siklus te affekteer. Sel siklus progressie word geassesseer deur die bepaling van die proporsie selle wat verskuif van arrestasie na chromatien sintese en mitose fases (G0/G1 en S en G2/M onderskeidelik). Metodes: Hierdie studie het die effekte van IL-6 op die progressie van die sel siklus geassesseer asook die proliferasie vs. differensiasie van C2C12 skelet spier satelliet selle. Fisiologiese dosisse (10 en 100 pg/ml) was vergelyk tot n hoog dose (10 ng/ml), met blywende blootstelling van 48 uur (byvoeging van IL-6 dose tot verspreidings medium op 0 and 24 uur). Akute sein stroomaf van die IL-6 gp130 reseptor was ook geassesseer na die eerste blootstelling. Resultate: Propidium iodide analise van kern materiaal deur vloei sitometrie het voorwaarts verskuiwing aangedui. Beide Laag and Medium doses het n groter proporsie (p<0.05) selle verskuif van die G0/G1 tot die S en G2M fases na 24 uur en alle dosisse het gelei in die selfde verskuiwing (p<0.05) by die 48 huur tyd punt. Alhoewel die Hoog dose myogenin uitdrukking aansienlik (p<0.05) verhoog het na 48 uur. Mikroskopie het aangedui dat samevloeiing voorkom was deur n lae loting digtheid en dit het nie resultate geaffekteer nie. Selle wat geoes was 5 minute na stimulasie het aangedui dat alle dosisse STAT3 fosforilasie laat toeneem het. 10 minute na stimulasie het die Hoog dose groep volgehoue vlakke van STAT3 fosforilasie besit. Gevolgtrekkings: Laag en Medium dosisse van IL-6 verhoog verspreiding in n spier satelliet sel lyn deur die aktivering van sel deling en deur selle toe te laat om in die aktiewe sel siklus te bly. Hoog dosisse van IL-6 verhoog differensiase deur bemiddelende opstoot van myogenic regulatory factors en die gedagte is dat dit bewerkstellig word deur aanhoudende aktivering van STAT3. Fisiologies beheer van satelliet selle deur sitokiene is duidelik en die beheer sal beinvloed word deur die erns van die endogene sitokien reaksie op verskillende stimuli.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The hypothalamic-pituitary-adrenal-(HPA) axis has long been closely associated with psychological stress-induced activation of the adrenal cortex and subsequent glucocorticoid production. Another, less known peripheral limb of the psychological stress response, is the sympatho adrenal medullary pathway. We hypothesized that the sympatho-adrenal medullary system constitutes the primary response to acute psychological stress, with the HPA-axis functioning as a secondary response. We tested our hypothesis by manipulating a model of acute mild psychological stress (restraint) by blocking IL-6, a valuable constituent of the sympatho-adrenal medullary system. Serum corticosterone concentration increased in response to stress (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), a response attenuated when IL-6 was blocked (17 ± 7 ng/ml). Stress increased pituitary mass only when IL-6 was blocked (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stress increased left adrenal mass only in the presence of IL-6 (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stress did not influence the circulating levels of TNF-α, IL-1β or IL-6 significantly. IL-1β and TNF-α concentrations in the unstressed rats were lower when IL-6 was blocked. We then manipulated the stress model by administering S. frutescens extract to elucidate both the central and peripheral effects of acute S. frutescens administration on the psychological stress response. Restraint caused decreases in hippocampal GR levels when compared to respective controls. S. frutescens administration and exposure to restraint synergistically decreased hippocampal GABAAR levels. In addition, exposure to both stress and S. frutescens led to a noteworthy increase in pituitary mass (P = 0.078), as well as pituitary ACTH levels (P < 0.01). Similarly, differences in circulating ACTH levels showed an effect of stress on ACTH secretion only in the presence S. frutescens (P < 0.05). Adrenal mass was significantly increased in S. frutescenstreated animals that were also exposed to restraint (P < 0.05). Adrenal levels of ACTH showed a reciprocal trend to pituitary and circulating ACTH levels. No statistically significant differences were seen in adrenal IL-6 content. However, marked increases in IL-6 levels were seen at this level with administration of S. frutescens stress exposure and a cumulative increase seen with both S. frutescens-treatment and stress exposure. Hippocampal GABAAR, pituitary mass, pituitary ACTH and circulating ACTH levels showed a similar trend towards a synergistic effect of S. frutescens and restraint in activation of the psychological stress response, while adrenal ACTH levels showed an inverse trend. Hippocampal GR did not show any effect of stress or S. frutescens-treatment. The results from these two experiments indicate that the sympatho-adrenal medullary system constitutes the primary response to acute mild psychological stress and that the HPA-axis is only activated during an exacerbated stress response or when the sympatho-adrenal medullary contribution is inadequate. Furthermore, the acute administration of S. frutescens possibly led to a functional shift in GABAergic function, resulting in activation of the stress response. The anecdotal reports of a “docile” effect of S. frutescens most likely results from activation of the mesolimbic dopaminergic system by the hippocampus and amygdala. These results have dramatic consequence in GABA-based anxiety-treatments. / AFRIKAANSE OPSOMMING: Die hipotalamo-pituïtêre-adrenale (HPA)-as is lank bekend as ‘n primêre rolspeler in die respons op emosionele stres en daaropvolgende glukokortikoïed produksie. ‘n Ander, minder bekende arm van die sielkundige stres respons is die simpatiese bynier-medulla-sisteem. Ons hipotese was dat die laasgenoemde simpatiese bynier-medulla-sisteem die primêre respons tot sielkundige stres behartig terwyl die HPA-as ‘n sekondêre respons bied. Ons het ons hipotese getoets deur die manupilering van ‘n beproefde stres model waar ons IL-6, ‘n waardevolle rolspeler in die simpatiese bynier-medulla-sisteem, onderdruk het. In respons op stress, het serum kortikosteroon konsentrasies toegeneem slegs in die teenwoordigheid van IL-6 (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), maar nie wanneer IL-6 onderdruk is nie (17 ± 7 ng/ml). Stres het ‘n verhoging in hipofise massa teweeggebring slegs tydens die onderdrukking van IL-6 (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stres het ook linker-byniermassa verhoog slegs wanneer voldoende IL-6 beskikbaar was (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stres alleen het geen invloed gehad op serum IL-1β, IL-6 of TNF-α nie, maar die onderdrukking van IL-6 het wel ‘n inhiberende effek op basale IL-1β en TNF-α gehad. Daarna het ons weer eens die stresmodel manipuleer deur die rotte ‘n S. frutescens ekstrak te gee in ‘n poging om beide die sentrale en perifere effekte daarvan op die sielkundige stres respons te evalueer. Stres alleen het gelei tot ‘n afname in GR terwyl ‘n kombinasie van stres en S. frutescens administrasie tot ‘n afname in GABAARα1 in die hippokampus gelei het. Hierdie kombinasie het ook tot ‘n merkwaardige toename in hipofise massa (P = 0.078) sowel as ACTH-inhoud van die hipofise (P < 0.01) gelei. ‘n Soortgelyke patroon is waargeneem betreffende sirkulerende ACTH en byniermassa met P < 0.05 vir elk. Bynier ACTH inhoud, aan die ander kant, het ‘n omgekeerd eweredige verhouding met ACTH in die hipofise en in sirkulasie getoon. Bynier IL- 6 inhoud het geen statisties beduidende verskille getoon nie, maar ‘n merkwaardige verhoging is weereens gesien met ‘n kombinasie van stres en S. frutescens administrasie. Die soortgelyke tendens wat waargeneem word in GABAAR in die hippokampus, asook hipofise- en sirkulerende ACTH vlakke, en dui op ‘n samewerkende rol van stres en S. frutescens in die aktivering van die sielkundige stres respons. GR in die hippokampus toon geen veranderinge nie. Die resultate van die twee eksperimente dui op ‘n primêre rol van die simpatiese bynier-medulla-sisteem in die respons op ‘n akute stressor en dat die HPA-as net geaktiveer word tydens ‘n ooreiste stres reaksie of indien die simpatiese bynier-medulla-sisteem onderdruk word. Die waargenome “verdowings”-effek van S. frutescens word moontlik deur aktivering van die mesolimbiese dopamien pad deur die hippokampus en amigdala bewerkstellig. Die resultate mag ook lei tot die heroorweging van GABA-gebaseerde angs medikasies.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy. / AFRIKAANSE OPSOMMING: Inleiding: Die antrasikliene (AC’s), daunorubisien (DNR) en doksorubisien (DKS), is twee van die mees effektiewe AC wat bekend is vir die behandeling van sistemiese neoplasmas en soliede tumore. Hulle kliniese gebruik word egter deur dosis afhanklike kumulatiewe kardiotoksisiteit benadeel, wat tot onomkeerbare en dodelike kongestiewe hartversaking kan lei. Die meganisme waardeur AC’s hartversaking kan veroorsaak, word nog nie ten volle verstaan nie. Onlangse navorsing het aangetoon dat DKS autofagie en die ubikwitienproteosoom-bemiddelde degradasie van spesifieke transkripsie faktore aktiveer. Daar is egter geen literatuur wat die effek van AC’s op die E3-ubikwitienligases, MuRF-1 en MAFbx beskryfnie. Die doel van hierdie eerste afdeling van die studie is om die effek van DNR behandeling op die proteïen- en organel degradasie sisteme in die hart te ondersoek en om van die betrokke seinmeganismes te bepaal. Alhoewel hierdie model ideaal is om sommige seinweë wat deur DNR geaffekteer word, te ondersoek, kon seinoordragpaaie wat potensieël voordelig in hierdie konteks is, nie in bg. model gemanipuleer word nie. Die in vitro model is gebruik om die hipotese dat verhoogde outofagie AC-geïnduseerde kardiotoksisiteit verlaag en sodoende seldood verminder, te bevestig. Die doel van hierdie afdeling van die studie was: (i) om die effek van DKS op H9C2 selle te karakteriseer, (ii) om te bepaal of die induksie/inhibisie van outofagie in kombinasie met DKS kardiotoksisiteit verbeter (iii) om die invloed van verhoogde/verlaagde outofagie in kombinasie met DKS op reaktiwe suurstof species (ROS), mitokondriale funksie, endoplasmiese retikulum (ER) stress en die ubikwitienproteosoompad te ondersoek. In die finale deel van hierdie studie, is ‘n in vivo model gebruik om die moontlike voordelige effek van verhoogde outofagie in ‘n GFP-LC-3 tumor-draende muismodel met akute DKSgeïnduseerde kardiotoksisiteit, ondersoek. Materiaal en Metodes: Volwasse rotte is in twee groepe verdeel waar een groep ses intraperitoneale inspuitings van 2 mg/kg DNR op afwissellende dae ontvang het en die andergroep as ‘n kontrole, ‘n soutoplossing gekry het. Die harte is verwyder en geperfuseer op ‘n werkende hartsisteem een dag na die laaste inspuiting en gevriesklamp vir biochemiese analises. H9C2 selle is vir 6 uurgekweek en behandel met Bafilomisien A1 (10 nM, ‘n autofagie inhibitor), 24 uur met Rapamisien (50 μM, ‘n autofagie induseerder), 24 uur met DKS (3 μM) of ‘n kombinasie van hierdie middels. Na behandeling is selle ge-oes vir analises in seldood, proteolitiese aktiwiteit en oksidatiewe stress deur van westelike kladtegniek, fluoresensie mikroskopie en vloeisitometrie gebruik te maak. In die finale fase van hierdie studie is vier en twintig, agt weke oue wyfie muise ingespuit met ‘n muisborskankersellyn (E0771) en is tumorgroei waargeneem; die diere is of behandel met een rapamisien inspuiting (i.p) (4 mg/kg), of twee DKS inspuitings (i.p.) (10 mg/kg) of ‘n kombinasie van die twee middels. Na die eksperimentele protokol, is die muise van kant gemaak en hulle harte vinnig verwyder. Die harte is in twee verdeel en gebruik vir biochemiese- en histologiese analises. Resultate en Bespreking: DNR behandeling het kardiale funksie betekenisvol verswak en apoptose in die hart verhoog. DNR-geïnduseerde kardiotoksisiteit is geassosieer met die opregulering van E3-ligases, MuRF-1 en MAFbx en het ook ‘n betekenisvolle toename in twee outofagie merkers, beclin-1 en LC-3 veroorsaak. Hierdie veranderinge wat in die hart waargeneem is, is ook geassosieer met ‘n onderdrukking van die PI3-kinase/Akt seinweg. Die toename in outofagie met rapamisien voor DKS behandeling het seldood in die vorm van apoptose betekenisvol verlaag. Daarmee saam het verhoogde outofagie ‘n noodsaaklike oorlewings meganisme vir akute DKS-geïnduseerde kardiotoksisiteit gedemonstreer. Die rede hiervoor is dat dit ROS produksie verlaag het, mitokondriale funksie verbeter het en DKS translokasie vanuit die sitoplasma tot binne die nukleus verhoed het. Hierdie veranderinge in kardiomiosiete is ook met ‘n afname in die ubikwitienproteosoomseinweg (EPS) geassosieer. In die finale deel van hierdie studie, is ‘n nuwe tumor-draende muismodel ontwikkel om die resultate wat in die in vitro studie gekry is, te bevestig. Daar is bewys dat akute DKS-geïnduseerde kardiomiotoksisiteit aanleiding gegee het tot verhoogde apoptose, outofagie inhibisie en verhoogde proteolise via die EPS. Hierdie bevindinge is geassosieer met ‘n verlaging in liggaamsgewig en kardiomiosiet dwarssnit area. Die kardiotoksiese effekte van DKS is insiggewend verminder as autofagiege ïnduseer is met rapamisien. Om saam te vat: Ons data bevestig dat dit moontlik is om die kardiotoksiese effekte van DKS in kanker pasiënte te verminder deur outofagie vlakke te monitor en te kontroleer deur middel van rapamisien behandeling as bykomende terapie.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Genetic screening to improve obesity treatment outcomes is available despite the lack of conclusive evidence, specifically for Caucasian South Africans, in this regard. The aim of this study was to investigate the association between genotype (seven polymorphisms) and body mass index (BMI), health and lifestyle indicators in a cross-sectional sample of overweight/obese Caucasian adults (n=133), as well as the association between genotype and weight loss outcomes following an intervention (n=88) using a quasi experimental study design (time-series). The intervention consisted of a 24-week conservative weight loss programme that included dietary, physical activity and behavioural components. The primary null hypothesis for the cross-sectional sample, namely that there is no association between genotype and BMI, has not been rejected. A number of the secondary/exploratory hypotheses were rejected of which the most plausible associations (based on support by the literature and a physiological basis for the findng) are: 1) the mutant TT homozygotes of the GNB3 C825T polymorphism may have a higher risk to develop the metabolic syndrome (MetS) as they had significantly higher fasting triglyceride and glucose levels, a higher number of traits that met the diagnostic cut-off criteria for MetS and higher number of these subjects was diagnosed with MetS compared to the wild-type C-allele carriers; and 2) subjects with mutant alleles of either the FTO rs1421085 or rs17817449 polymorphisms may have poorer eating behaviours (a higher rigid control, habitual and emotional disinhibition, perceived hunger and internal locus for hunger) and higher intake of high-fat foods. The primary null hypothesis for the intervention sample, namely that there is no association between genotype and weight loss outcome, was not rejected for the FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg and GNB3 C825T polymorphisms. However, it was rejected in some instances indicating the following associations: 1) The wild-type TT homozygotes of the FTO rs17817449 polymorphism lost significantly more weight during the first two months of the program compared to the mutant allele carriers (this is a novel finding); 2) The wild-type Arg16Arg homozygotes of the ADRB2 Arg16Gly polymorphism lost significantly more weight during the first month of the program compared to the mutant allele carriers (this finding is supported by one other intervention study); 3) Subjects with a mutant C-allele of the INSIG2 rs7566605 polymorphism and a mutant Gly16-allele of the ADRB2 Arg16Gly polymorphism lost significantly less weight over the six month intervention period (this is a novel genegene interaction finding). A number of secondary/exploratory hypotheses were rejected, of which the most plausible finding include that the improvement in emotional disinhibition in the wild-type TT subjects of the FTO rs1421085 polymorphism was associated with a more pronounced decrease in BMI over the six month weight loss period. The integration of the results from this study with the literature indicates that there is insufficient evidence at this stage for genetic screening of the polymorphisms investigated in this study and the provision of evidence-based personalized recommendations for weight loss in obese individuals. It is recommended that these associations should be viewed as priority in future research. / AFRIKAANSE OPSOMMING: Genetiese sifting om die resultate van vetsug behandeling te verbeter is beskikbaar ten spyte van ‘n tekort aan genoegsame bewyse, spesifiek ten opsigte van Kaukasiërs van Suid-Afrika. Die doel van hierdie studie was om die assosiasie tussen genotipe (sewe polimorfismes) en liggaamsmassa indeks (LMI), gesondheid en lewenstyl indikatore in ‘n dwarssnit (cross-sectional) steekproef van oorgewig/vetsugtige Kaukasiër volwassenes (n=133) te ondersoek, asook die assosiasie tussen genotipe en gewigsverlies uitkomste na afloop van ‘n intervensie (n=88) in ‘n kwasi-eksperimentele studie ontwerp (tydreeks). Die intervensie het bestaan uit ‘n 24-week konserwatiewe gewigsverlies program met dieet, fisieke aktiwiteit en gedragskomponente. Die primêre nul hipotese vir die dwarsnit steekproef, naamlik dat daar geen assosiasie tussen genotipe en LMI is nie, is nie verwerp nie. ‘n Aantal sekondêre/spekulatiewe hipotesis is verwerp waarvan die mees geloofwaardige assosiasies (gebasseer op ondersteuning van die literatuur en ‘n fisiologiese basis vir die bevinding) die volgende insluit: 1) die mutante TT homosigote van die GNB3 C825T polimorfisme het moontlik ‘n hoër risiko vir die ontwikkeling van die metaboliese sindroom (MetS) aangesien hulle betekenisvolle hoër vastende trigliseriede en glukose vlakke gehad het, ‘n grooter aantal kenmerke gehad het wat aan die diagnostiese afsnykriteria vir MetS voldoen en ‘n grooter aantal van hierdie persone was met MetS gediagnoseer in vergelyking met die wilde-tipe C-alleel draers; en 2) persone met die mutante allele van die FTO rs1421085 of rs17817449 polimorfismes het moontlik ‘n swakker eetgedrag (‘n hoër rigiede kontrole, gewoonte en emosionele disinhibisie, waarneembare honger en interne lokus van honger) en ‘n hoër inname van hoë-vet voedsel. Die primêre nul hipotese vir die intervensie steekproef, naamlik dat daar geen assosiasie tussen genotipe en gewigsverlies uitkomste is nie, is nie vir die FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg en GNB3 C825T polimorfismes verwerp nie. Dit was egter in sommige gevalle vir die volgende assosiasies verwerp: 1) Die wilde-tipe TT homosigote van die FTO rs17817449 polimorfisme het betekenisvol meer gewig in die eerste twee maande van die program verloor in vergelyking met die mutante alleel draers (dit is ‘n nuwe bevinding); 2) Die wilde-tipe Arg16Arg homosigote van die ADRB2 Arg16Gly polimorfisme het betekenisvol meer gewig gedurende die eerste maand van die program verloor in vergelyking met die mutante alleel draers (hierdie bevinding word ondersteun deur een ander intervensie studie); 3) Persone met ‘n mutante C-alleel van die INSIG2 rs7566605 polimorfisme en ‘n mutante Gly16-allele van die ADRB2 Arg16Gly polimorfisme het minder gewig tydens die ses maande intervensie periode verloor (dit is ‘n nuwe geen-geen interaksie bevinding). ‘n Aantal sekondêre/ spekulatiewe hipoteses is verwerp, waarvan die mees geloofwaardigste bevinding insluit dat ‘n verbetering in emosionele disinhibisie van die wild-tipe TT persone van die FTO rs1421085 polimorfisme geassosieer was met ‘n meer prominente daling in LMI oor die ses maande gewigsverlies periode. Die integrasie van die resultate van hierdie navorsing met die literatuur dui aan dat daar op hierdie stadium onvoldoende bewyse vir genetiese sifting en die voorsiening van bewys-gebasseerde persoonlike aanbevelings vir gewigsverlies in vetsugtig individue bestaan vir die polimorfismes wat ondersoek is. Dit word aanbeveel dat hierdie assosiasies as prioriteit in toekomstige navorsing beskou moet word.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Muscle injuries are associated with changes in skeletal muscle as well as the immune system. All studies investigating possible treatment modalities have found both positive and negative effects on muscle recovery. Since no universally accepted treatment modality exists, this thesis aims to determine whether a plant-derived antioxidant, proanthocyanidolic oligomer (PCO), might prove beneficial as treatment for sports injuries in order for athletes to return to the sports field quicker. The difference in recovery of muscle following both chronic (supplementation started 14 days prior to injury and continued thereafter) and acute supplementation (supplementation started two hours after injury) were also investigated. Both chronic and acute PCO supplementation in a rat hindlimb contusion injury model resulted in earlier muscle recovery, verified by an earlier satellite cell response compared to the placebo group. This effect was most prominent already at the four hour time point following injury, compared to day seven and three after chronic and acute placebo treatment respectively. PCO supplementation also resulted in quicker foetal myosin heavy chain (MHCf) expression compared to placebo treatment. Chronic supplementation specifically resulted in a blunted circulatory pro-inflammatory cytokine response, whilst allowing for a significant increase in IL-10, an anti-inflammatory cytokine, on day three (in the PCO group only). At tissue level, the response of the muscle pro-inflammatory cytokines, TNF- and IL- 6, coincided with the satellite cell response. Macrophage infiltration into the injured muscle also followed a similar pattern to that seen for the pro-inflammatory cytokines. Macrophages invaded the injured area quicker when supplemented with PCO chronically, however, macrophage infiltration could not explain the cytokine response seen with acute supplementation. Both chronic and acute supplementation with PCO was responsible for a severely blunted neutrophil response, a novel finding of this particular antioxidant. The main findings of the in vivo rodent study were that PCO was able to blunt the neutrophil response, whilst allowing for earlier macrophage infiltration. To establish possible mechanisms by which PCO might exert these beneficial effects, further analysis included determining macrophage phenotypes and neutrophil numbers in circulation. An in vitro neutrophil migration assay was also employed to further elucidate PCO’s ability to blunt neutrophil infiltration into the injured area. For this study, conditioned plasma were harvested from experimental animals and added together with neutrophils from control rats and granulocyte colony stimulating factor (G-CSF) to the insert of the migration chamber. A chemotactic factor, N-formyl methionine-leucine-phenylalanine (fMLP), was added to the bottom well and neutrophils were allowed to migrate for two hours. Results from this study indicated that neutrophil migration was attenuated in vitro in the presence of conditioned plasma from PCO supplemented rats only. The studies in this thesis on the effect of PCO on parameters of muscle and the immune system led to the following main conclusions: a) PCO supplementation resulted in earlier muscle recovery as a result of earlier satellite cell activation and MHCf synthesis; b) PCO favours an anti-inflammatory cytokine reaction, whilst blunting the pro-inflammatory cytokine response; and c) PCO blunted the neutrophil response whilst facilitating earlier macrophage infiltration into the injured area. The specific mechanism of action of PCO to blunt the neutrophil response specifically, possibly includes the ability to suppress adhesion molecule expression on the neutrophils themselves. However, this warrants further investigation. / AFRIKAANSE OPSOMMING: Spier beserings word geassosiëer met veranderinge in skeletspier sowel as die immuunstelsel. Meeste studies wat moontlike behandelingsopsies ondersoek, het beide positiewe en negatiewe spierherstel gerapporteer. Omrede daar geen universele behandelingsmoontlikheid bestaan nie, is die doel van hierdie tesis om die effek van ‘n plantgebaseerde anti-oksidant, pro-antosianiedoliese oligomeer (PSO), as ‘n voordelige behandelingstrategie vir sportbeserings te toets. Die verskil in spierherstel na beide kroniese (supplementering wat 14 dae voor besering begin is, en volgehou is daarna) en akute supplementering (supplementering het twee uur na besering begin), is ook ondersoek. Beide kroniese en akute PSO supplementering, in ‘n rot agterbeen-kneusbeseringmodel, het gelei tot vroeë spierherstel. Die bevindinge is geverifiëer deur ‘n vroeë satelietselrespons in vergelyking met die plasebo groep. Hierdie effek was reeds opvallend vier uur na besering, in vergelyking met die dag sewe en dag drie tydpunt tydens kroniese en acute plasebo behandeling onderskeidelik. In vergelyking met die kontrole groep, het PSO supplementering ook gelei to vininger uitdrukking van miosienswaarketting (MHCf). Kroniese supplementering het spesifiek gelei to ‘n onderdrukte sirkulatoriese pro-inflammatoriese sitokien response, terwyl ‘n betekenisvolle toename in IL-10 op dag drie (in die PSO groep alleenlik) waargeneem is. Op weefselvlak, het die pro-inflammatoriese sitokiene, IL-6 en TNF- , dieselfde patron gevolg as die van satelietselle. Makrofaaginfiltrasie binne die beseerde spier het ook ‘n soorgelyke patroon gevolg. Makrofage het die beseerde area vinniger geïnfiltreer in die kronies PSO-gesupplementeerde groep, maar kon nie die sitokienrespons, wat waargeneem is met akute supplementasie, verklaar nie. Beide kroniese en akute PSO supplementering was verantwoordelik vir ‘n onderdrukte neutrofiel respons, wat ‘n nuwe bevinding is vir hierdie spesifieke anti-oksidant. Die hoof bevindinge in die in vivo rotstudies, is dat PSO instaat is om die neutrofielrespons te onderdruk, en sodoende vroeë makrofaaginfiltrasie teweeg te bring. Om meganismes waarby PSO hierdie voordelige effekte veroorsaak te ondersoek, is verdere analises gedoen om makrofaagfenotipe en neutrofielgetalle in die sirkulasie te bepaal. ‘n In vitro neutrofielmigrasie studie is ook aangewend om PSO se vermoë om neutrofielinfiltrasie in die beseerde area te onderdruk, te ondersoek. Neutrofiele van kontrole rotte, tesame met gekondisioneerde plasma van eksperimentele diere en granulosiet-kolonie stimulerende faktor (G-KSF), is toegelaat om vir twee ure in die teenwoordigheid van ‘n chemotaktiese faktor, N-formiel metionien-leusien-fenielalanien (fMLP) te migreer. Resultate van hierdie studie het aangetoon dat neutrofielmigrasie, in vitro, alleenlik onderdruk word in die teenwoordigheid van gekondisioneerde plasma van PSO-gesupplementeerde rotte. Die studies in hierdie tesis oor die effek van PSO op parameters van spier en die immuunsisteem, het tot die volgende hoofgevolgtrekkings gelei: a) PSO supplementering het vroeë spierherstel, as gevolge van vroeë satelietselaktivering en MHCf sintese, teweeg gebring; b) PSO verkies ‘n anti-inflammatoriese sitokien reaksie, terwyl dit die proinflammatoriese sitokienrespons onderdruk; en c) PSO onderdruk die neutrofielrespons, terwyl vroeë makrofaaginfiltrasie in die beseerde area gefasiliteer word. Die spesifieke meganisme van aksie van PSO, om die neutrofielrespons te onderdruk, kan moontlik die vermoë van neutrofiele om adhesie molekule uit te druk, insluit. Hierdie aanname moet egter verder ondersoek word.

Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Skeletal muscle adapts to stimuli by modifying structural and metabolic protein expression. Furthermore, a muscle group may vary within itself to accommodate specialisation in regions. Structural and metabolic characteristics of an individual are regulated partly by genotype, but contraction duration and intensity may play a greater role in muscle phenotype. The aims of this dissertation were to investigate: structural and metabolic regionalisation in a muscle group, possible relationships between training volume and intensity and hybrid fibres, muscle characteristics of athletes from two different ethnic groups, and muscle adaptation in already well-trained athletes subjected to high intensity interval training. Myosin heavy chain (MHC) isoform content and citrate synthase (CS) activities were measured in the Quadriceps femoris (QF) muscle of 18 female rats. Muscle was divided into superficial, middle and deep, distal, central and proximal parts. MHC IIb and IIx were more abundant in superficial regions (P < 0.05) with low CS activities compared to deeper parts. Isoform content varied along the length of deep regions. This study showed that the QF has regional specialisation. Therefore, standardisation of sampling site is important. Hybrid fibre proportions in muscle biopsies of 12 middle distance runners and 12 non-runners were investigated. MHC IIa/IIx correlated with training volume/week in runners (r = -0.66, P < 0.05) and MHC IIa/IIx correlated with exercise hours/week in non-runners (r = -0.72, P < 0.01). Average preferred racing distance (PRDA) correlated better with MHC IIa/IIx in runners (r = -0.85, P < 0.001). MHC IIa/IIx may therefore be more closely related to exercise intensity than previously thought. Fibre type characteristics and performance markers were investigated in 13 Xhosa and 13 Caucasian distance runners, matched for performance, training volume and PRDA. Xhosa runners had less MHC I and more MHC IIa fibres in muscle biopsies than Caucasian runners (P < 0.05). Xhosa runners had lower plasma lactate at 80% peak treadmill speed (PTS) (P < 0.05), but higher lactate dehydrogenase (LDH) (P < 0.01) and phosphofructokinase (P = 0.07) activities in homogenate muscle samples. LDH activities in MHC I (P = 0.05) and IIa (P < 0.05) fibre pools were higher in Xhosa runners. Xhosa athletes may thus have a genetic advantage or they may have adapted to running at a higher intensity. Six weeks of individually standardised high intensity interval treadmill training (HIIT) were investigated in 15 well-trained runners. PTS increased after HIIT (P < 0.01), while maximum oxygen consumption (VO2max) only showed a tendency to have increased as a result of HIIT (P = 0.06). Sub-maximal tests showed lower plasma lactate at 64% PTS (P = 0.06), with lower heart rates at workloads from 64% to 80% PTS (P < 0.01) after HIIT. No changes were observed for cross-sectional area, capillary supply and enzyme activities in homogenates muscle samples. LDH activity showed a trend (P = 0.06) to have increased in MHC IIa pools after HIIT. Higher HIIT speed was related to decreases in MHC I fibres, but increases in MHC IIa/IIx fibres (r = -0.70 and r = 0.68, respectively, P < 0.05). Therefore, HIIT may alter muscle fibre composition in well-trained runners, with a concomitant improvement in performance markers. / AFRIKAANSE OPSOMMING: Skeletspier kan adapteer deur strukturele en metaboliese protein ekspressie te verander as gevolg van stimulante. ‘n Spiergroep kan ook intern verskil om spesialisering in spierdele toe te laat. Strukturele en metaboliese karaktereienskappe van ‘n individu word deels gereguleer deur gene, maar kontraksie tydperk en intensiteit mag ‘n groter rol speel in spierfenotipe. Die doelwitte van hierdie tesis was om ondersoek in te stel in: strukturele en metaboliese eienskappe in spiergroepstreke, moontlike verhoudings tussen oefeningsvolume of intensiteit en baster vesels, spier eienskappe in atlete van twee etniese groepe, en spier adaptasie in goed geoefende atlete blootgestel aan hoë intensiteit interval oefening. Miosien swaar ketting (MSK) isovorm inhoud en sitraat sintase (SS) aktiwiteite is gemeet in die Quadriceps femoris (QF) spier van 18 wyfie rotte. Spiere was opgedeel in oppervlakkig, middel en diep, asook distaal, sentraal en proksimale dele. MSK IIb en IIx was meer oorvloedig in oppervlakkige dele (P < 0.05) met lae SS aktiwiteite in vergelyking met dieper dele. Isovorm inhoud het ook verskil oor die lengte van diep dele. Dus bevat die QF gespesialiseerde streke en is die area van monsterneming belangrik. Baster vesel proporsies is ondersoek in spiermonsters van 12 middel afstand hardlopers en 12 niehardlopers. MSK IIa/IIx van hardlopers het met oefeningsvolume/week gekorreleer (r = -0.66, P < 0.05), asook MSK IIa/IIx van nie-hardlopers met oefeningsure/week (r = -0.72, P < 0.01). Gemiddelde voorkeur wedloop afstand (VWAG) het beter met MSK IIa/IIx gekorreleer in hardlopers (r = -0.85, P < 0.001). MSK IIa/IIx mag dus meer verwant wees aan oefeningsintensiteit. Veseltipe eienskappe en prestasie merkers was ondersoek in 13 Xhosa en 13 Caucasian langafstand atlete, geëweknie vir prestasie, oefeningsvolume en VMAG. Xhosa hardlopers het minder tipe I en meer tipe IIA vesels in hul spiermonsters gehad as die Caucasian hardlopers (P < 0.05). Xhosa hardlopers het laer plasma laktaat by 80% van hul maksimale trapmeul spoed (MTS) (P < 0.05), maar hoër laktaat dihidrogenase (LDH) (P < 0.01) en fosfofruktokinase (P = 0.07) aktiwiteite in homogene spiermonsters gehad. LDH aktiwiteite in MSK I (P = 0.05) en IIa (P < 0.05) veselbondels was hoër in Xhosa hardlopers. Xhosa atlete mag dus ‘n genetiese voorsprong geniet, of hulle het geadapteer om by hoër intensiteite te hardloop. Ses weke van geïndividualiseerde gestandardiseerde hoë intensiteit interval trapmeul oefening (HIIT) was ondersoek in 15 goed geoefende hardlopers. MTS het verhoog na HIIT (P < 0.01), en maksimale surrstof verbruik (VO2max) het ‘n neiging getoon om te verhoog het na HIIT (P = 0.07). Submaksimale toetse het laer plasma laktaat by 64% MTS getoon (P = 0.06), met laer harttempos by werkladings 64% tot 80% MTS (P < 0.01). Geen veranderings was gemerk vir deursnit area, kapillêre toevoer en ensiem aktiwiteite in homogene spiermonsters nie. LDH aktiwiteit het ‘n neiging getoon om te verhoog het (P = 0.06) in MSK IIa veselbondels na HIIT. Hoër HIIT snelhede was verwant aan ‘n daling in MSK I vesels, maar ‘n verhoging in MSK IIa/IIx vesels (r = -0.70 en r = 0.68, respektiwelik, P < 0.05). HIIT mag dus spier veseltipe verander in goed geoefende hardlopers, met gevolglike verbetering in prestasie merkers.