Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Both, the cytokine Tumor Necrosis Factor-α (TNF-α) and the enzyme cytosolic phospholipase A2 (cPLA2) are crucial driving forces in mediating the cellular inflammatory response and are involved in ischaemic injury. During an ischaemic insult, TNF-α is endogenously generated. Apart from the recognized effects of TNF- α, such as the induction of apoptosis, proliferation and differentiation, if present in low dosages, it also mediates cytoprotective effects. Upon activation, cPLA2 contributes to the ischaemic challenge with the generation of mediators of cellular injury and apoptosis. Upon stimulation, this calcium dependent enzyme translocates to the phospholipid compartment of the cell membrane and induces the hydrolysis of sn-2 ester bonds in phospholipids. It governs the release of free fatty acids and lysophospholipids and generates role players of inflammation. We suggest a role for cPLA2 in the TNF-α mediated cytoprotection, with a distinct phosphorylation and translocation pattern. Aims The involvement of cPLA2 in TNF-α mediated cytoprotection in the C2C12 murine skeletal muscle cell line in tolerance to ischaemia was examined. To investigate the nature of the cPLA2 phosphorylation pattern, the mitogen activated protein kinases (MAPKs) p38 and extracellular regulated kinase (ERK) as contributors to cPLA2 phosphorylation and activation, were examined at appropriate time points. To dissect out the cPLA2 interplay and dependencies with these MAPKs within the pathway context, the selective cPLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) was employed and its effect on cell viability was examined. Fluorescence microscopy was used to substantiate cPLA2 activation, by assessing its cellular distribution, translocation and cell organelle target preference, using co-localization and z-stack techniques. In addition, the induction of the apoptotic pathway through analysis of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage was examined. The role of caspase-3 in cPLA2 turnover was addressed employing the caspase inhibitor, Z-DEVD-FMK. Methods Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions, until 50%-70% confluent. Using DMEM supplemented with 1% horse serum, cell differentiation into myotubes was induced. Differentiated cells were preconditioned for 30 min classically, with 0.5 ng/ml TNF-α or the cPLA2 selective inhibitor AACOCF3 (10 μM) respectively. Followed by a 60 min washout period the cells were subjected to 8 hrs simulated ischaemia. Cellular viability; and cPLA2 phosphorylation- and translocation events were assessed using Western blots and advanced immunocytochemistry and imaging techniques. Results Preconditioning with TNF-α, ischaemic preconditioning; and the use of the cPLA2 inhibitor AACOCF3, attenuated the decrease is cell viability brough about by ischaemia. Western blot analysis indicates the induction of the apoptotic pathway with caspase-3 and PARP cleavage. A significantly reduced translocation of pcPLA2 to the nuclear region in the TNF-α preconditioned group compared to the ischaemic group, as reflected by reduced mean nuclear fluorescence intensity, was observed. A z-stack analysis confirmed that the nuclear and endonuclear region was the target organelle for cPLA2. 3-dimensional co-localazation analysis of pcPLA2 with the nuclear marker nucleoporin p62 mirrored these results. Discussion and conclusion Our results provide evidence that there is a role for cPLA2 in TNF-α mediated cytoprotection. Although we do not observe a differential activation pattern in terms of cPLA2 phosphorylation at various time points within the ischaemic event, and no differential inactivation of cPLA2 via caspase-mediated cPLA2 cleavage, we describe a differential cPLA2 translocation pattern, similar to that in IPC. Through inhibition of cPLA2 translocation, a functional cPLA2 inhibition might be achieved. This would imply inhibition of the inflammatory pathway and a subsequent reduction in the generation of inflammatory mediators. In addition we describe an effect of TNF-α preconditioning on the efficacy of the caspase inhibitor Z-DEVD-FMK. Our results shed light on the survival mechanisms employed by the ischaemically challenged cell in a setting of TNF-α mediated cytoprotection. This might lead to novel approaches in the context of inflammation treatment, through agents that control differential cPLA2 trafficking within the cell. / AFRIKAANSE OPSOMMING: Beide, die sitokien “Tumor Necrosis Factor-α (TNF-α)” en die ensiem, sitosoliese fosfolipase A2 (cPLA2) is uiters belangrike bemiddelaars van die sellulêre inflammatoriese respons en is verder ook betrokke by isgemiese selskade. TNF-α word endogeen gegenereer tydens ‘n isgemiese intervensie. Afgesien van ‘n verskeidenheid effekte, soos die inisiëring van apoptose, sel-proliferasie en - differensiasie, bemiddel dit ook selbeskermende meganismes indien dit in lae konsentrasies in die sel teenwoordig is. Na aktivering dra cPLA2 by tot die isgemiese intervensie deur die vorming van bemiddelaars van selskade en apoptose. Hierdie kalsium-afhanklike ensiem translokeer na die fosfolipied membraankomponent na stimulering en induseer die hidrolise van die sn-2 esterbinding in die fosfolipied. Die vrystelling van vry vetsure en lisofosfolipiede word sodoende bewerkstellig wat verder gemetaboliseer kan word tot inflammatoriese bemiddelaars. Ons stel voor dat cPLA2 ‘n rol in TNF-α bemiddelde selbeskerming speel en dat dit gepaardgaan met kenmerkende fosforilerings- en translokeringspatrone. Doelwitte Die rol van cPLA2 tydens TNF-α bemiddelde selbeskerming is in ‘n C2C12 skeletspiersellyn na blootstelling aan isgemie ondersoek. Die rol van die MAPKs, p38 en ERK, is ondersoek om vas te stel of hulle betrokke is by die aktivering van cPLA2. Die selektiewe cPLA2 inhibitor, AACOCF3, is gebruik om te bepaal of die fosforilering van MAPKs ook cPLA2-afhanklik is. Die sellulêre cPLA2 verspreiding, translokering en teiken selorganelle is ook ondersoek met behulp van fluoresensie mikroskopie deur gebruik te maak van ko-lokalisering en z-plaat tegnieke. Verder, is die indusering van die apoptotiese paaie ondersoek deur tegnieke wat kaspase- en PARP kliewing meet. Die kaspase inhibitor, Z-DEVD-FMK, is gebruik om vas te stel of kaspase-3 ‘n rol speel in cPLA2 kliewing in ons selmodel. Metodes Selle is gekweek in Dulbecco’s gemodifiseerde Eagles Medium (DMEM) waarby 10% fetale kalf serum (FBS) gevoeg is, en wat geïnkubeer is in 5% CO2 totdat dit 50%-70% konfluent was. Die selle is verder gedifferensieer in miobuise deur gebruik te maak van DMEM waarby 1% perdeserum gevoeg is. Gedifferensieerde selle is vir 30 min klassiek geprekondisioneer asook respektiewelik met 0.5 ng/ml TNF-α en die cPLA2 selektiewe inhibitor, AACOCF3 (10 μM). Na ‘n 60 minute uitwas periode is die selle blootgestel aan 8 h gesimuleerde isgemie. Sellulêre lewensvatbaarheid, cPLA2 fosforilering- and translokering is ondersoek deur onderskeidelik gebruik te maak van die “Western” klad metode en gesofistikeerde immunositochemiese beeld tegnieke. Resultate Prekondisionering met TNF-α, isgemiese prekondisionering asook inhibisie van as cPLA2 met die inhibitor, AACOCF3, het ‘n beduidende toename in sellewensvatbaarheid tot gevolg gehad. Daar is ook dmv die “Western” klad tegniek bewys dat apoptose geïduseer word deur middel van kaspase-3- en PARP kliewing. Daar is insiggewend minder translokasie van cPLA2 na die nukluêre fraksie in die isgemiese groep in vergelyking met die TNF-α geprekondisioneerde groep waargeneem (die gemiddelde nukluêre fluoreserende intensiteit is bepaal om voorafgaande feit te staaf). Die cPLA2 teiken organel is geverifieer as die nukleus en die endonukluêre gebied deur middel van z-plaat analises. Drie-dimensionele kolokaliserings analises van pcPLA2 met die nukluêre merker, nucleoporin p62 het hierdie resultate bevestig. Bespreking en Gevolgtrekking Ons resultate verskaf bewyse vir ‘n rol vir cPLA2 in TNF-α bemiddelde selbeskerming. Alhoewel daar nie ‘n differensiële aktiveringspatroon in terme van cPLA2 fosforilering tydens verskeie tydspunte in die isgemiese intervensie waargeneem is nie, en ook geen kaspase-3 bemiddelde kliewing van cPLA2 nie, word ‘n differensiële translokeringspatroon soorgelyk aan die isgemiese prekondisioneringsgroep, waargeneem. Funsksionele cPLA2 inhibisie kan dus moontlik bewerkstellig word deur inhibisie van cPLA2 translokasie. Die inflammatoriese respons kan dus moontlik so inhibieer word en die vorming van minder inflammatoriese bemiddelaars tot gevolg hê. Verder het TNF-α prekondisionering ook ‘n effek op die effektiwiteit van die kaspase-inhibitor, ZDEVD- FMK. Ons resultate werp ook lig op die meganismes wat deur selle onder isgemiese toestande uitgeoefen word tydens TNF-α bemiddelde selbeskerming. Hierdie resultate mag lei tot nuwe benaderings in die konteks van behandeling teen inflammasie deur gebruik te maak van middels wat cPLA2 translokering in die sel beheer.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2006. / Early adverse life events appear to increase the susceptibility of developing psychiatric disorders later in life. The molecular mechanisms involved in the development of pathological behaviour remain unclear. Dysregulation of the hypothalamic-pituitary-adrenal axis and alterations in neurotrophic factors have been implicated.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The human body has developed an integrated antioxidant defence system to protect against free radical damage. Acute exercise may result in the increased generation of free radicals, including reactive oxygen species, and this may overwhelm antioxidant defence systems resulting in oxidative stress. However, it has been shown that individuals who undergo regular exercise training may have improved antioxidant capacity when compared to sedentary controls. Results from research regarding the association between antioxidant capacity and exercise training are however not conclusive and further investigation is required. Therefore, the aim of this study was to investigate the association between the total plasma antioxidant status and selected plasma indicators of antioxidant status and the MnSOD Ala-9Val (-28C®T) polymorphism in trained male athletes (rugby players) and sedentary male students while controlling for dietary intake of the major antioxidants using a validated dietary assessment method. In order to address the potential confounding effect of dietary antioxidant intake on antioxidant status in the main study, a FFQ that measures vitamin C, vitamin E, carotenoid and flavonoid intake was developed. The reproducibility was assessed by the repeat administration of the FFQ (n = 38), while the va lidity was assessed using a 28-day closeended dietary record and repeated plasma vitamin C values (n = 18). Several statistical tests were conducted to compare the values obtained from the FFQ with values obtained from the various reference methods. While results from Bland-Altman plots suggested that the reproducibility and validity of FFQ was not completely satisfactory, similar mean values, moderate to strong correlation coefficients, and a high percentage of individuals classified correctly according to quartiles of intake indicated satisfactory reproducibility and validity of the FFQ in assessing antioxidant intake. Furthermore, moderate to strong validity coefficients obtained from the method of triads also indicated satisfactory validity for the FFQ. The main study involved a cross-sectional study that compared plasma vitamin C and carotenoid levels as well as total plasma antioxidant status in trained rugby players (n = 76) and sedentary male subjects (n = 39) with different MnSOD genotypes, while controlling for dietary antioxidant intake. Rugby players had significantly higher plasma vitamin C and carotenoid levels compared to sedentary students, which indicated more satisfactory plasma antioxidant status. This was also reflected in the tendency for total plasma antioxidant status (ORAC assay) to be higher in rugby players than sedentary students. MnSOD genotype did not influence plasma vitamin C and carotenoid levels or plasma total antioxidant status, with or without control for dietary antioxidant intake. Dietary vitamin C, vitamin E, carotenoid an flavonoid intake (from foods + supplements) was similar for rugby players and sedentary students and was adequate for both groups. Thus the association between antioxidant status and MnSOD genotype in rugby players and sedentary students seemed not to be influenced by dietary antioxidant intake. In conclusion therefore, rugby players undergoing regular exercise training had a more satisfactory antioxidant status compared to sedentary students. Based on this conclusion, the widespread use of antioxidant supplements by athletes is questioned. / AFRIKAANSE OPSOMMING: Die menslike liggaam beskik oor ‘n geintegreerde antioksidantmeganisme om dit teen vryradikaalskade te beskerm. Akute oefening kan bydra tot ‘n verhoogde produksie van vry radikale, insluitend reaktiewe suurstofspesies, wat kan veroorsaak dat die antioksidantbeskermingsmeganisme oorlaai word, wat dan kan aanleiding gee tot die ontstaan van oksidatiewe stress. Dit is aangetoon dat persone wat gereeld oefening doen verbeterde antioksidantkapasiteit toon in vergelyking met persone wat geen oefening doen nie. Die resultate van navorsingstudies wat die verband tussen antioksidantkapasiteit en oefening ondersoek is egter teenstrydig en verdere navorsing op hierdie gebied is essensieël om uitsluitsel te kry oor kontensieuse vraagstukke. Die doel van hierdie studie was dus om ondersoek in te stel na die verband tussen plasma antioksidant status, die MnSOD Ala-9Val (-28C T) polimorfisme en geselekteerde plasma antioksidantmerkers in geoefende manlike atlete (rugby spelers) en ‘n onaktiewe manlike kontrolegroep terwyl gekontroleer word vir die dieetinname van die vernaamste antioksidante. Om vir die potensiële invloed van dieetantioksidantinname op die antioksidantstatus van proefpersone in die hoofstudie te kontroleer, is ‘n voedsel frekwensievraelys wat vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname meet, ontwikkel. Die herhaalbaarheid (betroubaarheid) van die vraelys is getoets deur herhaalde voltooiing daarvan deur ‘n toetsgroep (n=38), terwyl die geldighied getoets is deur gebruik te maak van ‘n 28-dag geslote dieetrekord en herhaalde plasma vitamien C bepalings as verwysingswaardes (n=18). Verskeie statistiese toetse is uitgevoer om die frekwensievraelys waardes met die verskillende verwysingswaardes te vergelyk. Alhoewel die Bland -Altman grafieke nie dui op bevredigende herhaalbaarheid en geldigheid van die voedselfrekwensie vraelys nie, dui gelyke gemiddelde waardes, matig tot sterk en betekenisvolle korrelasiekoeffisiënte en ‘n hoë persentasie individue korrek geklassifiseer volgens kwartiele van inname, wel op bevredigende herhaalbaarheid en geldigheid. Matige tot sterk geldigheidskoeffisiënte is ook verkry met die toepassing van “The method of Triads”, wat verdere steun bied vir bevredigende geldigheid. In die hoofstudie is plasma vitamien C, karotenoïedvlakke en totale plasma antioksidantstatus in manlike rugby spelers (n=76) vergelyk met dié van onaktiewe manlike kontroles (n=39). Vergelykings tussen MnSOD genotipes binne die aktiwiteitsgroepe is ook getref. Al genoemde analises is gekontroleer vir dieet antioksidantinname. Resultate dui daarop dat die plasma vitamien C en karotenoïedvlakke van rugby spelers betekenisvol hoër was as dié van die kontrolegroep, wat dui op ‘n meer bevredigende antioksidantstatus. Hierdie resultaat is ook weerspieël in die feit dat totale plasma antioksidantstatus (ORAC) in die rugby spelers oog geneig was om hoër te wees as dié van die kontrole groep. Dit het ook geblyk dat MnSOD genotipe nie ‘n effek gehad het op plasma vitamien C-, karotenoïed- of totale antioksidantstatus nie, met of sonder kontrole vir dieet antioksidantinname. Die dieet vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname (vanaf voedsel en supplemente) was dieselfde vir rugby spelers en kontrole en was toereikend vir beide groepe. Dit blyk dus dat dat die verband tussen antioksidantstatus en MnSOD genotipe in die twee groepe nie beinvloed is deur antioksidantinname nie. Ten slotte kan die gevolgtrekking gemaak word dat manlike rugby spelers ‘n meer bevredigende antioksidant status het as onaktiwe manlike kontroles. Op grond van hierdie gevolgtrekking word die algemene gebruik van antioksidant supplemente deur atlete bevraagteken.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical (HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter protein function and contribute to the onset of myocardial insulin resistance and impaired contractile function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation, and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4 translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose. Although our results were not conclusive, the data suggest a potential novel link between dexamethasone exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and related metabolic derangements and diseases), these results provide new targets for potential therapeutic agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier (HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie. Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak, die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê. Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4) translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4 translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie. Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.

Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCb) is induced in a glucose-dependent manner. ACCb produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake. Previous studies show that increased flux through the hexosamine biosynthetic pathway (HBP) under hyperglycaemic conditions may contribute to the development of insulin resistance. In light of this, we hypothesised that increased HBP flux induces cardiac ACCb gene expression thereby contributing to the onset of insulin resistance. We tested our hypothesis by transiently transfecting cardiac-derived rat H9c2 myoblasts with a 1,317 bp human ACCb promoter-luciferase construct (pPIIb-1317) and an expression construct encoding the rate-limiting step of the HBP i.e. glutamine: fructose 6-phosphate amidotransferase (GFAT). Overexpression of GFAT increased ACCb gene promoter activity by 75 ± 23% versus controls (n=6, p<0.001). When cotransfection experiments were repeated in the presence of varying concentrations of L-glutamine (0 mM, 4 mM, 8 mM), a substrate for the HBP, ACCb promoter activity was dose-dependently increased. To further corroborate these findings, we employed two inhibitors of GFAT, i.e. 40 μM azaserine and 40 μM 6-diazo-5-oxo-Lnorleucine were administered to transfected cells for a period of 24 hours. Here both azaserine and 6-diazo-5-oxonorleucine attenuated ACCb gene promoter activity. In agreement, co-transfections with two dominant negative GFAT constructs also diminished ACCb gene promoter activity. We next inhibited two enzymes of the HBP acting downstream of GFAT, i.e. O-GlcNAc transferase and O-GlcNAcase using alloxan (0.1 mM, 1 mM and 2 mM) and streptozotocin (5 mM and 10 mM), respectively, for a period of 24 hours. Addition of alloxan attenuated ACCb gene promoter activity by 35.6 ± 1.9% (n=16, p<0.001) and streptozotocin increased activity by 32 ± 12% (n=12, p<0.001). We also investigated USF1 and USF2 as transcriptional regulatory candidates for HBP-induced ACCβ promoter regulation. Our data implicates USF2 as an important transcriptional regulator of HBP-induced ACCβ promoter regulation. In summary, this study demonstrates that increased flux through the hexosamine biosynthetic pathway induces ACCb gene promoter activity. We further propose that such an induction would reduce cardiac fatty acid oxidation, thereby leading to intracellular lipid accumulation due to a mismatch between sarcolemmal FA uptake and mitochondrial FA oxidation in the insulin resistant setting (i.e. hyperlipidaemia). / AFRIKAANSE OPSOMMING: Geen uitdrukking van die kardiale isoform asetiel-KoA karboksilase (ACCb) word in ‘n glukose afhanklike wyse geïnduseer. ACCb produseer maloniel-KoA, ‘n kragtige inhibeerder van mitochondriale vetsuuropname. Vorige studies toon aan dat verhoogde fluks deur die heksosamien biosintestiese weg (HBW) onder hiperglukemiese toestande bydra tot die ontwikkeling van insulienweerstand. In die lig hiervan, word daar gehipotetiseer dat verhoogde HBP fluks kardiale ACCb geenuitdrukking induseer en so bydra tot die ontstaan van insulienweerstand. Ons hipotese is getoets deur die kardiale afkomstige rot H9c2 mioblaste met ‘n 1.317 bp mens ACCb-lusiferase promotor konstruk (pPII-1317) te transfekteer en ‘n uitdrukking te konstrueer wat die tempo bepalende stap van HBP i.e. glutamien: fruktose-6-fosfaat amidotransferase (GFAT) kodeer. Ooruitdrukking van GFAT verhoog ACCb geenpromotor aktiviteit deur 75 ± 23% teenoor kontrole (n=6, p<0.001). Die herhaling van ko-transfeksie eksperimente is herhaal in die teenwoordigheid van variëerbare L-glutamienkonsentrasies (0 mM, 4 mM, 8 mM), ’n substraat vir die HBP, ACCb promotor aktiwiteit is dosisafhanglik verhoog. Om die bevindinge verder te staaf, is twee inhibeerders van GFAT, i.e. 40 μM azaserien en 40 μM 6-diazo-5-oxo-L-norleusien aan transfeksie selle toegedien vir ’n tydperk van 24 uur. Beide azaserien en 6-diazo-5-oxo-L-norleusien verlaag ACCb geenpromotor aktiwiteit. In ooreenstemming met die bogenoemde het ko-transfeksies met twee dominante negatiewe GFAT konstrukte ook ACCb geenpromoter aktiwiteit verminder. Die volgende stap is om twee ensieme van die HBP wat stroomaf van GFAT aktief is, vir ‘n periode van 24 uur te inhibeer i.e. O-GlcNAc transferase en O-GlcNAcase deur alloxan (0.1 mM, 1 mM en 2 mM) and streptozotosien (5 mM en 10 mM) onderskeidelik vir ‘n 24 uur periode te gebruik. Toevoeging van alloxan het die ACCb geenpromotor aktiwiteit by 35.6 ± 1.9% (n=16, p<0.001) verlaag en streptozotosien aktiwiteit verhoog by 32 ± 12% (n=12, p<0.001). Ons het ook die USF1 en USF2 as transkripsie regulerings kandidate vir HBP-geïnduseerde ACCβ promotor regulering ondersoek. Ons data impliseer dat USF2 as ‘n belangrike transkripsie reguleerder van HBP-geïndiseerde ACCβ promotor regulering is. Samevattend het hierdie studie demonstreer dat verhoogde fluks deur die hexosamien biosintetiese weg ACCb geenpromotor aktiwiteit induseer. Ons stel verder voor dat hierdie induksie die kardiale vetsuuroksidasie verlaag wat daartoe lei dat intrasellulêre lipied akkumulasie as gevolg van onparing tussen sarkolemma vetsuuropname en mitochondriale vetsuuroksidasie in ’n insulien weerstandige situasie (i.e. hiperlipidaemia).

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Human studies on the response of muscle to contusion injury are limited, probably due to the large variability in injury severity and the non-specificity of clinical symptoms reported. To circumvent this problem, several experimental animal models have been designed to study muscle damage and regeneration after contusion injuries. However, the majority of techniques currently used to induce contusion injury are very invasive and therefore not optimal. Furthermore, published studies regarding clinical treatment of such injuries are limited. The main aims of this study were therefore: a) to establish and characterise an in vivo model of non-invasive contusion injury, and b) to assess the effect of pre-injury chronic administration of the antioxidant supplement Oxiprovin™ - a natural grape seed extract (GSE) - on skeletal muscle recovery after experimentallyinduced injury. Two groups of male Wistar rats were subjected to 14 days of oral administration of isovolaemic placebo (sterile isotonic saline) or GSE (20 mg/kg/day) prior to induced contusion. Contusion injury was induced with the mass-drop technique, and recovery parameters assessed for up to 14 days post-injury. Placebotreated rats on average exhibited a 56 % higher creatine kinase (CK) activity when compared to the GSE-treated rats when area under the curve (AUC) was calculated for 14 days post-injury (p < 0.001). In the placebo group, plasma oxygen radical absorbance capacity (ORAC) was unchanged over time, but muscle ORAC was significantly increased by day 7 post-injury (p < 0.001). In the GSE group, a significant decrease in both plasma (p < 0.01) and muscle ORAC (p < 0.001) was evident 4 hr after injury, followed by a significant increase by day 3 (p < 0.05 and p < 0.001 respectively). CD34+ satellite cell (SC) numbers (quiescent and activated) peaked earlier in GSE-treated rats when compared to placebo-treated rats (4 hours vs. day 7 post-injury). Total satellite cell number (CD56+) also peaked earlier in GSE-treated rats than in placebo-treated rats (4 hours vs. 3 days post-injury), while M-cadherin+ SC numbers (quiescent, activated or proliferating) in both treatment groups were significantly increased 4 hours post-injury (p < 0.001), but more so in the placebo group. In GSE-treated rats when compared to placebo-treated rats, newly generated muscle fibres (displaying central nuclei and MHCf +) both appeared (day 3 vs. day 7 post-injury) and peaked in number (day 3 vs. day 7 post-injury; increase from baseline p < 0.001 for both) earlier. The results of this study demonstrate that we have successfully established an in vivo model for non-invasive contusion injury in rats. Furthermore, we have shown that Oxiprovin™: a) increased the ability to scavenge reactive species generated after injury and b) resulted in the activation of satellite cells and formation of newly generated muscle fibres at an earlier time point, thus accelerating the recovery of skeletal muscle after a standardised contusion injury. / AFRIKAANSE OPSOMMING: Eksperimente aangaande die reaksie van spier op kneusings in mense is beperk, waarskynlik as gevolg van die groot verskeidenheid simptome wat mag voorkom en die verskille in die ernstigheid van beserings. Ten einde hierdie problem te oorbrug, is verskeie eksperimentele diermodelle opgestel om kneusings en die herstel van spier daarna te ondersoek. Die tegnieke wat grootendeels vandag gebruik word om kneusings te veroorsaak, maak inbraak op die spier deur die spier te ontbloot voor besering, en is dus nie ideaal nie. Daar is ook nie baie bewyse aangaande die mees geskikte manier om so ‘n besering klinies te behandel nie. Die doel van hierdie studie was dus om: a) ‘n in vivo model van kneusings op te stel en te omskryf, en b) die effek van chroniese toediening van die antioksidant Oxiprovin™ - ‘n natuurlike druifsaad ekstrak (DSE) – op die herstel van skeletspier na ‘n kneusing te ondersoek. Twee groepe manlike Wistar rotte is onderwerp aan mondelikse toediening van isovolemiese plasebo (steriele isotoniese soutoplossing) of DSE (20 mg/kg/dag) vir ‘n tydperk van 14 dae voor kneusing. Kneusing is geïnduseer met die “massdrop” tegniek, en parameters van herstel is ondersoek tot en met 14 dae na besering. Plasebo-behandelde rotte het gemiddeld 56 % hoër kreatien kinase (KK) aktiwiteit in vergelyking met DSE-behandelde rotte (p < 0.001), toe die oppervlak onder die kurwe (OOK) tot en met 14 dae na besering bereken is. Geen verskil oor tyd is in die plasebo groep opgemerk toe plasma suurstof radikaal absorpsie kapasiteit (SRAK) bepaal is nie, maar ‘n betekenisvolle toename in spier SRAK teen dag 7 (p < 0.001) is waargeneem. ‘n Betekenisvolle afname in beide plasma (p < 0.01) en spier (p < 0.001) SRAK van die DSE is teen 4 hr waargeneem, gevolg deur ‘n betekenisvolle toename teen dag 3 na besering (p < 0.05 en p < 0.001 onderskeidelik). Die aantal CD34+ satelliet selle (SS – rustend en geaktiveerd) het beduidend vroeër in die DSE groep gestyg in vergelyking met die plasebo groep (4 uur vs. 7 dae na besering). Die totale aantal SS (CD56+) het ook vroeër in die DSE-behandelde rotte as die plasebobehandelde rotte gestyg (4 uur vs. 3 dae na besering), terwyl die aantal Mcadherin+ SS (rustend, geaktiveerd of prolifererend) betenisvol gestyg het in beide groepe teen 4 uur (p < 0.001) na besering, maar hoër in die plasebo groep was. Die aantal nuutgevormde spiervesels (met sentraal geleë nukleï en MHCf +) het beide vroeër verskyn en gepiek in die DSE-behandelde rotte in vergelyking met die plasebo-behandelde rotte (dag 3 vs. dag 7 na besering). Die resultate van hierdie studie dui aan dat ons instaat was om ‘n in vivo model van nie-indringende kneusing in rotte op te stel. Verder, het ons ook bewys dat Oxiprovin™ toediening die vermoë verleen het om: a) reaktiewe spesies wat na beserings gevorm word, meer doeltreffend te verwyder en b) satelliet selle vroeër te aktiveer en die vorming van nuwe skeletspiervesels te vervroeg, om sodoende die herstel van skeletspier na ‘n gestandardiseerde kneusing vinniger te bewerkstellig.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2006. / This study aimed to assess the effect of mild psychological stress in male Wistar rats using incremental, intermittent stress on parameters of atrophy, including body mass, soleus and extensor digitorum longus (EDL) muscle mass, and mechanisms possibly contributing to atrophy. Serum corticosterone concentrations, 20s proteasome activity, glutamine synthetase (GS) and tyrosine amino-transferase (TAT) activities were determined. I also assessed whether Sutherlandia (Su) or resistance exercise was able to reverse the effects of stress on any of these parameters.

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.