Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Cryotherapy is one of the popular treatments used to alleviate muscle soreness, especially in the competitive sports arena. However, the therapeutic use of cryotherapy is unsubstantiated because of a lack of proper investigations in the literature, especially a hypothesised effect on muscle recovery. Thus, our aims were to characterise satellite cell (SC) activity in human subjects with delayed onset muscle soreness (DOMS) and to shed light on the effect of cryotherapy on SC activity. DOMS was induced in six male subjects (24 ± 3 years) by completion of a downhill-run (DHR) protocol (5 x 8 min bouts, 2 min rest between bouts) at 70 or 80% of their individual peak treadmill speed. Ice application was applied to only one leg per subject for 3 days: 30 min every 2 hours, 5 times per day. In total 5 muscle biopsies were obtained from each subject: 1 baseline and 4 post-DHR. Post-DHR biopsies: 1 from each leg on day 1 and 7 (1st group, n=3) and 1 from each leg on day 2 and 9 (2nd group, n=3). DOMS was successfully induced as indicated by significant increases in muscle soreness at days 1 and 2 post-DHR (P < 0.01), and creatine kinase activity at day 1 post-DHR (P < 0.01). No difference in muscle soreness was found between treated and untreated legs. SC quiescence and activation were characterised by their expression of the cell surface markers CD34 and CD56 respectively. No significant change in quiescent SC was observed in the untreated or treated legs over time. However, at day 1 post-DHR the number of quiescent SC was significantly lower in the untreated compared with the treated leg (P < 0.05). There was a significant increase in activated SC numbers at day 2 post-DHR in the untreated leg, which was sustained up to day 9 post-DHR (P < 0.01). However, no such increase was found in biopsies taken on days 1 and 7. Also, no change was found in the treated leg, however a significant difference between the number of activated SC in untreated and treated legs on days 2 and 9 post-DHR (P < 0.01) was seen. No significant effect of DOMS or ice treatment was observed for the expression of the myogenic regulatory factors, MyoD and myogenin. C2C12 cell cultures induced to differentiate, however, did stain using these antibodies. This is the first study to report an effect of cryotherapy at the tissue level. In conclusion, this study highlights many unanswered questions on the SC response to DOMS at tissue level, and lays a good foundation for future studies. / AFRIKAANSE OPSOMMING: Kreoterapie is een van die gewilde behandelings wat gebruik word om spierseerheid te verlig, veral in die kompeterende sport arena, maar die gebruik van kreoterapie is onbevestig as gevolg van ‘n gebrek aan voldoende ondersoeke in die literatuur, veral ‘n hipotese oor die effek op spier-herstel. Ons doelstellings was dus om satellietsel (SC) aktiwiteit te ondersoek in mens proefpersone met vertraagde aanvang spierseerheid (DOMS) en ook om lig te werp op die effek van kreoterapie op SC aktiwiteit. DOMS was in ses mans proefpersone (24 ± 3 jare) geїnduseer deur voltooїng van ‘n afdraend-hardloop (DHR) protokol (5 x 8 min rondtes, 2 min rus tussen rondtes) teen 70 of 80% van elkeen se individuele maksimum trapmeul-spoed. Ys was vir 3 dae op net een been per proefpersoon aangewend: 30 min elke 2 ure, 5 keer per dag. 5 spierbiopsies in totaal was van elke proefpersoon verkry: 1 basislyn en 4 post-DHR. Post-DHR biopsies: 1 van elke been op dae 1 en 7 (1ste groep, n=3) en 1 van elke been op dae 2 en 9 (2de groep, n=3). DOMS was suksesvol geїnduseer soos aangedui deur die betekenisvolle verhogings in spierseerheid op dae 1 en 2 post-HR (P < 0.01) en kreatien kinase aktiwiteit op dag 1 post-DHR (P < 0.01). Geen verskil in spierseerheid is gevind tussen die onbehandelde en behandelde bene nie. SC dormansie en aktivering was gekarakteriseer deur die onderskeidelike uitdrukking van die sel oppervlak merkers CD34 en CD56. Geen betekenisvolle verandering is in SC dormansie in die onbehandelde en behandelde bene waargeneem nie, maar op dag 1 post-DHR was die getal dormante SC betekenisvol laer in die onbehandelde been as in die behandelde been (P < 0.05). Daar was ‘n betekenisvolle verhoging in die getalle geaktiveerde SC op dag 2 post-DHR in die onbehandelde been wat volgehou was tot op dag 9 post-DHR (P < 0.01), maar so ‘n verhoging was nie in biopsies wat op dae 1 en 7 geneem is gevind nie. Daar is ook geen verandering in die behandelde been gevind nie, maar ‘n betekenisvolle verskil in die getal geaktiveerde SC is tussen die onbehandelde en behandelde bene op dae 2 en 9 post-DHR gevind(P < 0.01). Geen betekenisvolle effek van DOMS en ys-aanwending vir die uitdrukking van die miogeniese (myogenic) regulatoriese faktore, MyoD en myogenin, is waargeneem nie. C2C12 sel kulture wat geїnduseer is om te differensieer het wel gekleur vir hierdie antiliggame. Dit is die eerste studie wat ‘n effek van kreoterapie op weefselvlak rapporteer. Ten slotte, hierdie studie beklemtoon baie onbeantwoorde vrae oor die SC respons op DOMS op weefselvlak en dit lê ‘n goeie grondslag neer vir toekomstige studies.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection against the consequences of ischaemia/reperfusion injury. Insulin-induced improvements in cardiac functions are widely investigated in models of ischaemia and reperfusion. It has been shown that many signalling pathways may be involved in the cardioprotection properties of insulin under those conditions. These pathways include PI3-K, PKB/Akt, p70S6k, ERK and many others. However, little data exists on the effects of insulin on the heart under normoxic condition. Some evidence has been presented that insulin has a positive inotropic effect on the normoxic perfused rat heart, but no precise cellular mechanism has been investigated or described in this regard. We believe that an investigation into the effects of insulin on cardiac function and pathways involved under normoxic conditions may help us to better understand the mechanisms of insulin-induced cardioprotection. Aims: To determine a suitable dose of insulin at which a positive inotropic response could be detectable under normoxic conditions, to investigate the possible mechanisms involved in insulin-induced increases in contractility with specific reference to the vasculature and the coronary flow and to investigate a possible involvement of PI3-K and its downstream effectors on the insulin effects on cardiac functions under normoxic conditions. Materials and methods: Isolated rat hearts were perfused retrogradely using the Langendorff technique. After 10 minutes of stabilization hearts were perfused for 30 minutes either with standard perfusion solution i.e. Krebs-Henseleit buffer + glucose gassed with 95%O2, 5%CO2 (control hearts), or with standard perfusion solution plus insulin alone or insulin together with the nitric oxide synthase inhibitor L-NAME or the PI3-K inhibitor wortmannin. Left ventricular developed pressure (LVDevP), heart rate (HR) and coronary flow (CF) as well as phosphorylated PI3-K and PKB/Akt in heart were measured. Results: Administration of insulin alone at physiological concentrations showed improved cardiac function compared to hearts in the control group. Hearts that received insulin+L-NAME showed a significant decrease in function compared to the control hearts and the hearts that received insulin alone (p<0.05). Phosphorylated PKB/Akt (Thr308) was increased in hearts that received insulin alone and insulin+L-NAME compared to the control hearts. Phosphorylated PI3-K tended to be higher in hearts where insulin was administered alone compared to the hearts that received insulin+L-NAME or insulin+wortmannin. Conclusion: This study confirmed that physiological concentrations of insulin exert positive inotropic effects on cardiac function in normoxic perfused rat hearts as seen with the improved LVDevP. Inhibition of PI3-K by wortmannin induced a decrease in phosphorylated PKB/Akt in hearts that received insulin+wortmannin and administration of L-NAME impaired the beneficial effects of insulin on cardiac functions. Therefore these results may indicate that nitric oxide may have a role in the positive effect of insulin on cardiac function in the healthy heart perfused under normoxic conditions. L-NAME as well as wortmannin reversed the positive inotropic effects of insulin. Both inhibitors also unmasked effects of insulin via nitric oxide and PI3-K on heart rate and coronary flow. / AFRIKAANSE OPSOMMING: Inleiding: Dit is welbekend dat toediening van insulien die hart beskerm teen ischemie/reperfusie-beserings, wat lei tot verbeterde hartfunksie. Hierdie effek word wyd ondersoek in modelle van ischemie en reperfusie. Dit is bewys dat ‘n verskeidenheid seintransduksie paaie, insluitend PI3-K, PKB/Akt, p70S6k en ERK, betrokke is by hierdie beskermende effek van insulien op die hart. Baie min data is egter beskikbaar rakende die effek van insulien tydens normoksiese toestande. Alhoewel dit bekend is dat insulien ’n inotropiese effek op die normale geperfuseerde hart het, is die presiese sellulêre meganismes wat dit bewerkstellig nog nie nagevors nie. Om dus ‘n beter begrip van hierdie meganismes te verkry is dit dus noodsaaklik om die effekte van insulien onder normoksiese perfusie toestande na te vors. Doelstellings: Om ‘n geskikte dosis, waarby insulien sy positiewe inotropiese effek onder normale toestande het, vas te stel, om die moontlike meganismes betrokke by insulien-geïnduseerde verbetering in hartsametrekbaarheid te bestudeer, met spesifieke verwysing na die bloedvoorsiening en koronêre vloei, en om die moontlike betrokkenheid van die PI3-K pad en sy teiken effektore onder normale suurstof-toestande te ondersoek. Materiaal en metodes: Geïsoleerde rotharte is geperfuseer deur gebruik te maak van die Langendorff tegniek. Na ‘n stabilisasie periode van 10 minute is rotharte blootgestel aan 30 minute perfusie met een van vier oplossings: ‘n standaard perfusie oplossing (Krebs-Henseleit buffer met glukose onder spesifieke gaskondisies van 95% O2, 5% CO2 – kontrole harte); standaard perfusie oplossing en insulien; standaard perfusie oplossing met insulien en die stikstofoksied sintase inhibitor L-NAME, of standaard perfusie oplossing, met insulien en die PI3-K inhibitor wortmannin. Met verloop van die perfusie protokol, is ontwikkelde linker ventrikulêre druk (LVDevP), harttempo (HR) en koronêre vloei (CF), sowel as PI3-K en PKB/Akt fosforilasie, gemeet. Resultate: Toediening van insulien teen fisiologiese konsentrasies het ‘n verbeterde hartfunksie tot gevolg, in vergelyking met harte in die kontrole groep. In teenstelling hiermee het harte wat insulien+L-NAME ontvang het ‘n betekenisvolle verlaagde funksie getoon in vergelyking met die kontrole harte en harte wat slegs insulien ontvang het (p<0.05). Harte wat slegs insulien, of insulien+L-NAME ontvang het, het ‘n verhoging in gefosforileerde PKB/Akt (Thr308) getoon in vergelyking met kontrole harte. Gefosforileerde PI3-K het ook geneig om hoër te wees in harte wat insulien+L-NAME of insulien+wortmannin ontvang het, as in harte wat slegs insulien ontvang het. Gevolgtrekking: Hierdie studie bewys dat fisiologiese konsentrasies van insulien, onder normale suurstof-toestande, ‘n positiewe inotropiese effek op hartfunksie uitoefen, soos gesien in die verbeterde LVDevP. Wortmannin-geïnduseerde inhibering van die PI3-K pad het ‘n verlaagde PKB/Akt fosforilasie tot gevolg gehad in harte wat insulien+wortmannin ontvang het, terwyl die toediening van L-NAME die voordelige effekte van insulien op hartfunksie onderdruk het. Hierdie resultate dui dus aan dat stikstofoksied ‘n rolspeler is in die positiewe inotropsiese effek van insulien op hartfunksie tydens normoksiese toestande, aangesien beide inhibitore hierdie effek onderdruk het. Beide inhibitore het ook die betrokkenheid van stikstofoksied en die PI3-K pad by die effek van insulien op harttempo en koronêre vloei onthul.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Premature luteal regression occurs on average in 30% of superovulated sheep ewes. This phenomenon occurs early in the cycle before the embryo’s can be collected and is a major contributor to failure in embryo transfer programs. This research was done to understand the physiological mechanisms involved. Chapter two provides a general background of the physiology of natural luteolysis and the maternal recognition of pregnancy. The chapter introduces some new concepts on the topic of cell death and provides a recent literature review on research done on the phenomenon of premature luteal regression. This chapter forms the base of ideas and arguments that follows in the two studies containing new original work in this field. The research contained in this thesis comprises of two in vivo studies. The first study (Chapter 3) compare premature luteal regression to Prostaglandin F2α (PGF2α) induced regression with emphasis on the changes in levels of the steroid hormones progesterone (P4) and estradiol - 17β (E2-17β) and changes in structure and ultra structure. The following conclusions were made: 1. Premature luteal regression is not merely inadequate luteal support, but indeed early luteal regression, since seasonal influences could merely be nutritional influences, and a definitive increase in P4 were recorded in animals exhibiting the phenomena. 2. Nutritional influences could play a role, but the type and quality of nutrients and mechanism involved, is still unclear. 3. PGF2α-induced regression differs from premature luteal regression in that: a) The progression of functional and structural regression in PGF2α -induced regression is slower than in premature luteal regression. b) Regressed corpora lutea do not occur with normal functioning corpora lutea. 4. There is a distinct second E2-17β peak preceding the decline in P4 in animals that exhibits signs of premature luteal regression. A threshold initiating premature luteal regression was not established. The second study (Chapter 4) compares the changes in the ovine β estradiol - 17 β receptor (oERβ) between premature luteal regression and PGF2α induced regression. The study concludes that there could be a potential role for oERβ in premature luteal regression. The findings of these two studies raise some questions about the conventional perception that early release of PGF2α is the cause of premature luteal regression. The thesis concludes in a hypothesis (Chapter 4) explaining the phenomenon. / AFRIKAANSE OPSOMMING: Premature luteale regressie kom gemiddeld in 30% van gesuperovuleerde skaap-ooie voor. Die verskynsel kom vroeg in die siklus voor, voor die embrios gekollekteer kan word, en is een van die belangrikste oorsaake van mislukkings in ‘n embrio-oorplaasingsprogram. Die huidige navorsing poog om die fisiologiese meganismes betrokke by premature luteale regressie te verstaan. Hoofstuk twee verskaf ‘n algemene agtergrond van die fisiologiese aspekte betrokke by natuurlike luteale regressie en maternale herkenning van swangerskap. Die hoofstuk stel nuwe konsepte voor oor sel afsterwing en verskaf ‘n opgedateerde literatuuroorsig met betrekking tot die navorsing wat in die veld oor die verskynsel gedoen is. Die hoofstuk vorm die basis vir die idees en argumente, wat volg in die twee studies en wat oorspronklike nuwe navorsing bevat oor die onderwerp. Die navorsing in die tesis bestaan uit twee in vivo studies. Die eerste studie (Hoofstuk 3) vergelyk premature luteale regressie en prostaglandien F2α (PGF2α) ge-induseerde regressie met ‘n klem op die vlakke van die steröiedhormone progesteroon (P4) en estradiol - 17β (E2-17β) en veranderinge in die mikroskopiese struktuur en ultra struktuur van die corpus luteum. Die studie bevind: 1. Premature luteale regressie is nie slegs onvoldoende luteale funksie nie, maar vroë luteale regressie aangesien seisoenale invloede eitlik voedings invloede kan wees en P4 gestyg het in diere waar die verskynsel voorgekom het. 2. Voeding kan ‘n rol speel maar die tiepe en gehalte van die voedingstowwe en die meganismes betrokke is nie duidelik nie. 3. PGF2α - ge-induseerde regressie verskil van premature regressie in dat: a) Die verloop van funksionele en strukturele regressie is stadiger in PGF2α - ge-induseerde regressie in vergelyking met premature luteale regressie. b) Corpora lutea wat regressie ondergaan het kom nie voor saam met corpora lutea wat normal voorkom nie. 4. Daar die ‘n duidelike tweede piek van E2-17β gaan die afname in P4 vooraf in diere waar premature regressie voorkom. 5. Daar is nie geslaag om ‘n drempel vas te stel waar premature regressie ge-inisieer word nie. Die tweede studie vergelyk die veranderinge in estradiol-17β reseptore (oERβ) in die skaap tydens premature luteale regressie en PGF2α geinduseerde regressie. Die studie bevind dat daar ‘n moontlike rol is vir ERβ in premature luteale regressie. Die bevindinge van die twee studies bevraagteken die konvensionele opvatting dat vroë vrystelling van PGF2α verantwoordelik is vir premature luteale regressie. Die tesis sluit af met ‘n nuwe hipotese om die verskynsel te verduidelik.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background:Obesity is a growing global burden; current studies have projected the prevalence of obese / overweight individuals to increase to ~1.35 billion by 2030. A number of factors contribute to cardiovascular diseases, of which the focus of this study is what effect an increased level of free fatty acids has on the flux through the hexosamine biosynthetic pathway (HBP). It has been widely proven that an increased flux through the HBP causes an increase in protein O-GlcNAcylation, which leads to increased reactive oxygen species (ROS) production as well as an increase in cell death (apoptosis). Methods: For the purpose of this study a cell model was used. H9c2 cardiomyoblasts were cultured in 5ml Dulbecco‟s Modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin. The cells were then exposed to 0.25mM monounsaturated fatty acid (oleic acid) for 24, 48 and 72 hours respectively. The cultured cells were then evaluated to assess the degree ROS production, overall O-GlcNAcylation and cell death (apoptosis and necrosis), using flow cytometry and immunofluorescence microscopy. Results: We found that oleic acid causes a significant decrease in ROS production at the 48 hour time point when analysed on the flow cytometer, which indicates that oleic acid is metabolized by the cells in a independent manner. Oleic acid also caused a significant decrease in cell death at all the time intervals. With regard to the HBP, oleic acid activates this pathway but causes downstream cardioprotective effects that do not necessarily occur along this pathway. Conclusion: This study explored whether a monounsaturated fatty acid, oleic acid, is able to act as a novel cardioprotective agent. The in vitro data supports this concept and we showed that it is able to blunt oxidative stress and cell death. It was also found that although oleic acid activated the HBP, it did not mediate its protective effects via this pathway only. / AFRIKAANSE OPSOMMING: Agtergrond: Vetsug is 'n groeiende wêreldlas; huidige studies voorspel dat die voorkoms van vetsugtige / oorgewig individue toe sal neem tot ~1.35 biljoen teen 2030. Alhoewel verskeie faktore tot kardiovaskulêre siektes bydra is die fokus van hierdie studie om die effek van verhoogde vryvetsuurvlakke op die fluks deur die heksosamienbiosintestiese weg (HBW) te ondersoek. Dit is reeds bewys dat verhoogde fluks deur die HBW 'n verhoging in proteïen O-GlcNAsilering lei, wat verder tot verhoogde reaktiewe suusrtofspesies (ROS) vorming aanleiding gee en ook seldood (apoptose) verhoog. Metodes:'n Selmodel is vir die doel van hierdie studie gebruik. H9c2 kardiomioblaste is in 5ml Dulbecco's Modified Eagles Medium (DMEM) gekweek en gesupplementeer met 10% fetale beesserum en 1% penisillien-streptomysien. Die selle is blootgestel aan 'n 0.25mM mono onversadigde vetsuur (oleïensuur ) vir 24, 48 en 72 uur onderskeidelik. Die gekweekte selle is gevolglik ondersoek vir die graad van ROS ontwikkeling, algehele O-GlcNAsilering en seldood (apoptosis en nekrose), deur van vloeisitometrie en immunofluoresensie mikroskopie gebruik te maak. Resultate: Ons het bevind dat oleïensuur 'n betekenisvolle verlaging in ROS ontwikkeling teen 48 uur soos bepaal deur die vloeisitometer, veroorsaak. Dit wys daarop dat oleïensuur deur die selle op 'n onafhanklike wyse gemetaboliseer is. Oleïensuur het ook 'n betekenisvolle verlaging in seldood by alle tydsintervalle veroorsaak. Met betrekking tot die HBW het oleïensuur hierdie weg geaktiveer maar afstroom kardiobeskermings effekte versoorsaak wat nie noodwendig langs hierdie weg onstaan nie. Gevolgtrekking:Hierdie studie het die moontlikheid van 'n mono-onversadige vetsuur, oleïensuur, om op te tree as 'n nuwe kardiobeskermingsmiddel ondersoek. Die in vitro data ondersteun hierdie konsep en hier is aangetoon dat dit wel oksidatiewe stres en seldood onderdruk. Daar is verder bevind dat alhoewel oleïensuur die HBW aktiveer dit nie die beskermings effekte alleenlik via hierdie weg medieer nie.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Introduction Cardiovascular disease is one of the leading causes of death in the world. Formation of harmful reactive oxygen species (ROS) is associated with several pathological conditions, and contributes to ischaemia/reperfusion injury. Antioxidants can be added to the diet in an attempt to decrease the prevalence of cardiovascular disease by decreasing the harmful effects of ischaemia/reperfusion injury. Red Palm Oil (RPO) consists of saturated, monounsaturated and polyunsaturated fatty acids and is rich in antioxidants such as -carotene, tocopherols and tocotrienols. It has previously been shown that RPO-supplementation improved reperfusion mechanical function. In these studies it was found that RPO might exert its beneficial effects during reperfusion through increased PKB/Akt pathway activity, which may lead to inhibition of apoptosis and improved mechanical function. Aims The aims of this study were: 1) to determine whether RPO-supplementation protected against ischaemia/reperfusion injury in the isolated perfused rat heart, 2) to confirm RPO-supplementation’s effect on the PKB/Akt pathway activity and, 3) to elucidate the regulators in the PKB/Akt pathway that RPOsupplementation influenced. Methods Male Wistar rats were divided into 4 groups, 2 control groups and 2 experimental groups. The 2 control groups were fed a standard rat chow (SRC) for 4 weeks. The two experimental groups received SRC and RPOsupplementation for 4 weeks. Hearts were excised and transferred to a Langendorff perfusion apparatus and perfused with Krebs-Henseleit buffer. Mechanical functional recovery was measured after 25 min of total global noflow ischaemia. The following parameters were also measured during various time points in the protocol: left ventricular develop pressure, heart rate, coronary flow, rate pressure product. Hearts were also freeze-clamped for biochemical analysis at 10 min during reperfusion. The biochemical analysis was aimed at determining PKB/Akt involvement. In a second protocol, hearts were subjected to the same perfusion protocol, but wortmannin was also added to the perfusion fluid, in order to inhibit PI3- kinase. Results Hearts from the RPO-supplemented rats showed an improved RPP recovery (92.26 ± 5.89 % vs 63.86 ± 7.74 %) after 10 min of reperfusion. This finding corroborated the findings of previous studies. Hearts of the RPOsupplemented rats perfused with wortmannin, showed increased RPP recoveries at several time points. Biochemical results showed that wortmannin did indeed inhibit PI3-K phosphorylation in the RPO-supplemented group, as was expected. The RPO-supplemented group that was perfused with wortmannin had an increased PKB/Akt (Ser473) phosphoyrylation, when compared to the wortmannin control group. It was also found that the combination of RPO and wortmannin had prosurvival effects. Discussion This study showed that RPO-supplementation offered protection against ischaemia/reperfusion injury in the Langendorff-perfusion apparatus at 10 min into reperfusion. Thereafter the significance of the protection was lost. This protection has been confirmed in several previous studies and several mechanisms have been proposed for this protection. Since no conclusive evidence exists on the precise mechanism of protection, our investigation focused on the regulators of the pro-survival PKB/Akt pathway. An improved functional recovery was also seen in the RPO-supplemented group that was perfused with wortmannin. This was an unexpected finding, because Wortmannin is a known PI3-kinase inhibitor (as was confirmed by our biochemical data). PI3-kinase phosphorylation leads to PKB/Akt phosphorylation and therefore, activation of a pro-survival pathway. It would be expected that wortmannin would inhibit PKB/Akt and thus decrease the survival of the cells. The RPO-supplementation thus reversed wortmannin’s detrimental effect to such an extent that the functional recovery was far better than RPO-supplementation alone. In the RPO + wortmannin group, PKB/Akt (Ser473) phosphorylation was increased, contrary to previous findings. This is an indication that RPO may have the ability to override wortmannin’s inhibitory effect on PI3-kinase, or that PKB/Akt (Ser473) may be phosphorylated independently of PI3-kinase. / AFRIKAANSE OPSOMMING: Inleiding Kardiovaskulêre siektes is een van die hoof oorsake van sterftes in die wêreld. Die vorming van skadelike reaktiewe suurstof spesies word geassosieer met verskeie patologiese kondisies en dra ook by tot isgemie/reperfusie skade. ‘n Moontlike manier om die voorkoms van isgemie/herperfusie skade asook kardiovaskulêre siektes te voorkom, is om antioksidante by die dieet te voeg. Rooi Palm Olie (RPO) bevat versadigde, mono-onversadigde en polionversadigde vetsure. RPO bevat ook ‘n oorvloed van antioksidante soos β- karoteen en tokoferole en tokotriënole. Dit is bewys in vorige studies dat RPO-aanvulling verbeter funksionele herstel. Hierdie voordelige effekte mag dalk wees agv verhoogde PKB/Akt pad aktiwiteit. Die PKB/Akt pad word geassosieer met die inhibisie van apoptose en verhoogde meganiese funksie. Doelwitte Die doelwitte van hierdie studie was om te bepaal of 1) RPO-aanvulling beskermende effekte teen isgemie/herperfusie skade in die geisoleerde rotharte het, 2) Bevestig of RPO-aanvulling wel die PKB/Akt pad beïnvloed 3). om die effekte wat RPO-aanvulling het op die reguleerders van die PKB/Akt pad te onthul. Metodes Manlike Wistar rotte is in 4 groepe verdeel. 2 Groepe kontrole rotte is ‘n standaard rotkosmengsel gevoer vir 4 weke. Die 2 eksperimentele groepe het ook ‘n standaard rotkosmengsel gekry plus ‘n RPO-aanvulling vir 4 weke. Harte is uitgesny en op ‘n Langendorff perfusie sisteem gemonteer en met Krebs-Henseleit buffer geperfuseer. Meganiese funksie herstel is gemeet na 25 min totale globale geen-vloei isgemie. Linker ventrikulêre ontwikkelde druk, harttempo, koronêre vloei en tempo druk produk is gemeet by verskillende tydpunte. Sommige harte is na 10 min herperfusie vir biochemiese analiese gevriesklamp. Die biochemiese analisiese was beoog om die PKB/Akt pad betrokkenheid te bepaal. ‘n Tweede stel harte is aan dieselfde perfusie protokol blootgestel, maar wortmannin (PI3-kinase inhibitor) is ook bygevoeg by die perfusie vloeistof. Resultate Die groep wat met RPO aangevul is, het na 10 min herperfusie, ‘n verbeterde tempo druk produk herstel getoon (92.26 ± 5.89 % vs 63.86 ± 7.74. Hierdie bevinding is ook met ander studies bevestig. ‘n Interessante bevinding was dat die groep wat met RPO aangevul is en met wortmannin geperfuseer is, ‘n verbeterde meganiese funksionele herstel getoon het. Biochemiese resultate het getoon dat wortmannin wel PI3-K fosforilering geinhibeer het. Die harte van die rotte in die groep wat aangevul is met RPO en daarna met wortmannin geperfuseer is, het ‘n toename in PKB/Akt (Ser473) fosforilering getoon, relatief tot die wortmannin geperfuseerde harte van die rotte in die kontrole groep. Hierdie groep (RPO-aanvulling en wortmannin perfusie) het beskermende effekte getoon. Bespreking Hierdie studie het getoon dat RPO-aanvulling beskerming gebied het teen isgemie/herperfusie skade in die Langendorff geperfuseerde rothart na 10 min herperfusie. Daarna is die beduidenheid van die beskerming verloor. Hierdie bevindings ondersteun die resultate van vorige studies. Verskeie moontlike meganismes is voorgestel vir die beskerming, maar die presiese meganisme is nog nie duidelik nie. In hierdie studie is daar gekyk na die reguleerders van die PKB/Akt pad. Geen vorige studies het al gefokus op RPO-aanvulling en sy effek op die reguleerders van die PKB/Akt pad nie. ‘n Onverwagte bevinding is dat harte van die rotte in die RPO + wortmannin groep ‘n verbeterde funksionele herstel getoon het. Wortmannin is ‘n PI3- kinase inhibitor. PI3-K fosforilering lei tot PKB/Akt fosforilering, wat tot sel beskerming lei. Dus, aangesien wortmannin PI3-K inhibeer, sou dit verwag word dat wortmannin sel beskerming sal verminder. Die RPO het egter die wortmannin se nadelige effekte tot so ‘n mate oorskrei dat die funksionele herstel baie beter was as die RPO-aanvulling alleen. Die verhoogde PKB/Akt (Ser473) fosforilering, wat gesien is in die RPO + wortmannin groep kan toegeskryf word aan RPO se vermoë om wortmannin se nadelige effekte te oorskrei. ‘n Moontlike verduideliking vir hierdie bevinding mag wees dat rooi palm olie PKB/Akt (Ser473) op ‘n PI3-K onafhanklike manier fosforileer.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2006. / Despite a growing interest into the mechanisms of the repeated bout effect, little is known about the consequences of chronic eccentrically biased training and the possible advantageous such training may offer to athletes as well as patients with muscle-debilitating disease. This study investigated the role of satellite cells in the muscle adaptation in response to either downhill or uphill high intensity training (HIT). Welltrained endurance runners were divided into two training groups matched for training volume and 10 km running times (n = 6, uphill training, UP; n = 6, downhill training, DH) and subjects in both groups completed 10 HIT sessions over a period of 4 weeks. Running performance was tested before and after the training intervention by a 10 km road race and peak treadmill speed (PTS) in horizontal and inclined (+5%) laboratory incremental tests to exhaustion. Skeletal muscle biopsies were sampled at baseline, after 2 HIT sessions, and after 4 weeks of HIT. Muscle was analysed immunohistochemically for satellite cell frequency as identified by CD56 and M-cadherin (Mcad) expression. Myogenin protein contents of muscle homogenates were determined by western blotting. Myosin heavy chain (MyHC) isoform proportions and mean fibre crosssectional area was measured. During the HIT intervention, UP exercised at a higher percentage of their HRmax than DH (mean ± SD, 97 ± 1 vs. 92 ± 3 %HRmax, p < 0.005), but at a similar rate of perceived exertion (RPE). DH completed more intervals per session and covered greater distance per session than their UP counterparts. Both training groups increased their training intensity but decreased their training volume during the 4 weeks of HIT. The combined group of 12 athletes improved their PTSgradient (mean ± SD, 16.7 ± 0.8 vs. 17.3 ± 1.0 km/h, p < 0.05). No significant differences between groups were found for PTS, VO2max or 10 km performance. Satellite cell frequency in this cohort of trained runners (48.9 ± 10.3 km/week) at baseline was similar to healthy young males (CD56+ cells/fibre, 0.19 ± 0.10). Satellite cell frequency increased significantly in DH after 4 weeks (Mcad, 123%; CD56, 138%) and non-significantly in UP (Mcad, 45%, CD56, 39%). No significant differences were found after two training sessions or at any time between groups. Mcad and CD56 expression correlated well (r = 0.95, p < 0.0001). Muscle myogenin content increased for both groups (UP: 56%; DH: 60%) after 4 weeks. No notable changes were seen after two training sessions. However, myogenin levels 2 days after session 1 correlated well (r= 0.99, p<0.005) with muscle pain experienced on the same day, as measured by the visual analogue scale. No changes were seen in the MyHC proportions or the fibre cross-sectional area after the training intervention. It was concluded that the training intervention was too short to induce changes in MyHC distribution or fibre area. Is seems likely that satellite cell proliferation was initiated as an early response to DOMS, but the response was maintained for 4 weeks. However, due to the lack of change in fibre morphology and myonuclear number, the role of satellite cell proliferation in fibre type transformation or muscle hypertrophy could not be established. Similarly, various possible roles for increased myogenin protein are offered, but since the origin of myogenin expression (satellite cells vs. myonuclei) was not determined, no definite conclusion regarding the precise function can be made. In conclusion, this study is the first to definitively indicate satellite cell proliferation in well-trained endurance runners in response to a change in training, including specifically downhill HIT. This response was early and sustained. This study asks several questions about the role of satellite cells during muscle adaptation to repetitive downhill training, and lays a foundation for further research into this unexplored field.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Breast cancer is currently the primary cause of cancer-related death in women worldwide. Conventional treatments such as radiation and chemotherapy have many deleterious and long lasting side-effects, some of which are permanent, such as infertility. As certain tumour cells can also acquire resistance to chemotherapy, the need for the development of a less severe, yet more effective, targeted anti-cancer treatment exists. Curcumin, a plant polyphenol from Curcuma longa, has long been thought to possess antitumour, antioxidant, anti-arthritic, anti-amyloid, anti-ischemic and anti-inflammatory properties. Numerous studies conducted over the past sixty years confirm this. We aimed at examining the effect of curcumin on cell viability and the different modes of cell death, namely apoptosis, necrosis and autophagy, in the MCF-12A (non-tumorigenic mammary epithelial) and MCF-7 (mammary adenocarcinoma) cell lines. Cells were incubated with different doses of curcumin to evaluate the dose response through a MTT assay. Thereafter, cells were incubated with 200 μM curcumin for 48 hrs and stained with markers and DNA stains for apoptosis (Hoechst, Caspase-3, PARP), necrosis (Propidium Iodide) and autophagy (LC3B and Beclin-1). Cells were examined via fluorescence microscopy, Western Blot- and FACS analyses. MTT results showed no significant decrease in viability in the MCF-12A cell line after curcumin treatment. However, a significant decrease in viability was observed in MCF-7 cells after treatment with 200 μM curcumin (p < 0.05). Treated MCF-7 cells also show clear LC3B expression. FACS results show a significant difference in Hoechst mean fluorescence intensity in MCF-7 cells after curcumin treatment (p < 0.05). This study provides evidence that MCF-7 cells respond to a 200 μM dose of curcumin treatment through metabolic change and induction of the autophagic pathway. The model system used in this study provides groundwork for further cell culture based studies regarding breast cancer and curcumin.

Thesis (MSc (Physiological Sciences)--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Although muscle tissue demonstrates a remarkable capacity for regeneration following injury, this process is slow and often accompanied by the formation of scar tissue and a subsequent decrease in contractile capacity following regeneration. Treatment options are few and mostly supportive in nature. This regeneration process involves muscle stem cells (satellite cells) which ultimately give rise to the regenerated muscle. The contentious field of low level laser therapy (LLLT) has made remarkable claims in facilitating wound healing in soft tissue injuries of various types. Yet, the mechanism(s) invoked in these beneficial effects are poorly understood. We have investigated the effects of LLLT using a 638 nm laser on satellite cells in culture and in-vivo. Using an array of techniques we have measured, amongst other things, metabolic responses to laser irradiation, signaling pathways activated/altered and antioxidant status. In response to laser irradiation satellite cells in culture showed an increase in MTT values (a measure of metabolic activity) and a decrease in antioxidant status (measured using the ORAC assay). In addition laser irradiation also altered the expression and phosphorylation state of several signaling pathways, including Akt and STAT-3. Following on from this the effects of laser irradiation on satellite cells in-vivo was assessed in a rat model of contusion injury. No significant differences in satellite cell number was found following laser irradiation, changes were seen in tissue antioxidant status and blood antioxidant status (measured using the ORAC assay). In the course of this study several standard techniques were used to investigate the effects of laser irradiation on satellite cells both in-vitro and invivo. It has become apparent that several of these techniques have problems associated with them that possibly make them inappropriate for vi further use in studies involving laser irradiation. However the results indicate that laser therapy is induces satellite cell behavior and further study is warranted in this field. / AFRIKAANSE OPSOMMING: Alhoewel spierweefsel merkwaardige regenerasie kapasiteit vertoon ten opsigte van besering, is hierdie proses stadig en word soms vergesel met die vorming van letselweefsel asook ‘n gevolglike afname in kontaktiele kapasiteit na afloop van regenerasie. Behandelingsmoontlikhede is skaars en meesal ondersteunend van aard. Hierdie proses sluit spierstamselle (satelietselle), wat uiteindelik die ontstaan van die regenerasie van spier tot gevolg het, in. Die kontroversiële veld van lae vlak laserterapie (Engels: Low level laser therapy (LLLT)) het merkwaardige aansprake in die fasilitering met verskeie sagteweefsel wondgenesing. Nietemin, die meganisme(s) wat voordelige effekte induseer, word nog nie goed begryp nie. Ons het die effek van LLLT, deur gebruik te maak van ‘n 638 nm laser op kultuur in vitro satelietselle sowel in-vivo, ondersoek. Deur gebruik te maak van verskeie tegnieke is onder meer die metaboliese, sowel die seinstransduksie weë en antioksidantstatus na laserbestraling, gemeet. In reaksie op die laserbestraling het satelietselle (in kultuur) ‘n toename in MTT waardes getoon (‘n maatstaf van die metaboliese aktiwiteit) en ‘n afname in die antioksidantstatus (gemeet deur van die ORAC toets). Addisioneel het laserbestraling ook uitdrukking en fosforilering van verskeie proteïene betrokke in seintransduksieweë beïnvloed, insluitend Akt, STAT-3). Na afloop van hierdie effekte op satelietselle na laserbestraling, is daar gebruik gemaak van ‘n kneusbeseringsrotmodel om hierdie effekte in vivo te ondersoek. Geen betekenisvolle verskille in die aantal satelietselle na laserbestraling is opgemerk nie, maar veranderings is wel opgemerk in weefsel- en bloed-antioksidantstatus (gemeet deur van die ORAC toets gebruik te maak). Gedurende die verloop van die studie is van verskeie standaardtegnieke gebruik gemaak om die effekte van laserbestraling op beide satelietselle in vitro en in vivo te ondersoek. iv Dit het duidelik na vore gekom dat daar wel gepaardgaande probleme met van hierdie tegnieke voorgekom het, en dat van hierdie tegnieke nie gepas is vir ondersoek in laserbestralingsstudies nie. Nietemin, die resultate toon wel dat laserbehandeling. satelietselgedrag induseer wat verdere studie in hierdie veld noodsaak

Thesis (MScPhysio (Interdisciplinary Health Sciences. Physiotherapy))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The pelvic floor muscles (PFM) contribute to urinary continence and overactive PFM seem to be associated with pelvic pain syndrome (PPS). The literature indicates that ethnic differences regarding symptoms of urinary incontinence may exist. Research is needed to establish relationships between PFM function and symptoms reported by women of different ethnic groups. Objectives: To compare the PFM strength and endurance in black, white and coloured women. To investigate relationships between PFM strength, vaginal resting pressures, risk factors and symptoms associated with PFM dysfunction and PPS. Method: A cross-sectional study assessed the PFM strength and vaginal resting pressures of 122 nulliparous black (n=44), white (n=44) and coloured (n=34) university students. A self-developed questionnaire determined inclusion, demographic variables, factors affecting/factors associated with PFM strength and symptoms related to PPS. Maximum voluntary contraction pressure (cmH2O) and vaginal resting pressure (cmH2O) were measured with the Peritron TM 9300 (Cardio Design, Australia) used with the Camtech AS vaginal balloon sensor (Sandvika, Norway). Two sets of 3 maximum voluntary contractions of the PFM were recorded. Results: The mean age of the group was 22 ± 3.54 years and mean BMI of 23± 4.16 kg/m2. Black women (25 cmH2O ± 13.5) had significantly stronger PFM than white (p=0.02) or coloured (p<0.01) women, but no significant difference (p=0.78) in PFM strength existed between white (18.4 cmH2O ± 9.8) and coloured (15.6 cmH2O ± 8) women. In black women, PFM strength decreased significantly (p=0.02) between the sets, whereas no significant difference between sets was noted in the other ethnic groups. Increased PFM strength was associated with SUI (p=0.03) and amenorrhoea (p=0.01) and decreased PFM strength was associated with decreased frequency of bowel motion (p=0.01). In this sample, increased vaginal resting pressure was associated with menorrhagia (p=0.04). Conclusion: Black nulliparous women had stronger PFM than white and coloured women. There was no difference in PFM strength between white and coloured women. Endurance, as measured in this study, indicates that black women have decreased endurance of the PFM compared to white and coloured women. These findings inform the current research on ethnic differences in the prevalence of urinary incontinence. Preliminary data suggest that there was no relationship between vaginal resting pressures and symptoms of PPS and risk factors for PFM dysfunction, except for menorrhagia. / AFRIKAANSE OPSOMMING: Die bekkenvloer spiere (BVS) dra by tot urinêre kontinensie en ooraktiewe BVS kan moontlik geassosieer wees met pelviese pyn sindroom (PPS). Uit die literatuur blyk dit of daar etniese verskille bestaan in die simptome van urinere inkontinensie gerapporteer deur vroue. Navorsing is nodig om die verwantskap tussen BVS funksie en simptome wat deur pasiënte van verskillende etniese groepe gerapporteer word vas te stel. Doel: Om „n vergelyking te tref tussen BVS sterkte in swart, wit en kleurling vroue. Om vas te stel of daar assosiasies bestaan tussen BVS sterkte, rustende vaginale druklesings en risiko faktore en simptome geassosieer met bekkenvloer disfunksie en PPS. Metodologie: „n Dwarssnit studie het die BVS sterkte en rustende vaginale drukke van 122 nullipareuse swart (n=44), wit (n=44) en kleurling (n=34) universiteit studente geëvalueer. Insluiting, uitsluiting, demografiese veranderlikes, faktore wat kan affekteer/faktore geassosieer met BVS sterkte en simptome geassosier met PPS is deur „n self ontwikkelde vraelys geëvalueer. Maksimale willekeurige spiersametrekking drukke (cmH2O) en rustende vaginale drukke (cmH2O) was gemeet met „n Peritron™9300 perineometer (Cardio Design, Australië) wat saam „n vaginale ballon sensor (Camtech AS, Sandvika, Noorweë) gebruik is. Twee stelle van 3 maksimale willekeurige sametrekkings van die BVS was gemeet. Resultate: Die groep se gemiddelde ouderdom was 22±3.54 jaar en die gemiddelde liggaamsgewig indeks was 23±4.16kg/m2. Swart vroue (25 cmH2O ±13.5) het beduidend sterker BVS gehad as wit (p=0.02) en kleurling (p<0.01) vroue, maar daar was geen beduidende verskil (p=0.78) in BVS sterkte tussen wit (18.4 cmH2O ± 9.8) en kleurling (15.6 cmH2O ± 8) vroue nie. Die BVS sterkte in swart vroue het beduidend (p=0.02) verminder tussen die stelle, maar geen beduidende verskille was waargeneem in die ander etniese groepe tussen stelle. Verhoogde BVS sterkte was geassosieer met druklek (p=0.03), amenorrhoea (p=0.01) en verminderde BVS sterkte was geassosieer met verminderde frekwensie van opelyf (p=0.01). Verhoogde rustende vaginale drukke was geassosieer met menoragie in hierdie steekproef. Gevolgtrekking: Swart nullipareuse vroue het sterker BVS gehad as wit en kleurling vroue, Daar was geen verskil in BVS sterkte tussen wit en kleurling vroue nie. Uithouvermoë soos in hierdie studie getoets toon dat swart vroue verminderde uithouvermoë het i.v.m. wit en kleurling vroue. Hierdie bevindings dra by tot die huidige navorsing oor etniese verskille in die prevalensie van urinêre inkontinensie. Daar was geen verwantskap tussen vaginale rustende drukke en simptome van PPS en risiko faktore vir die ontwikkeling van bekkenvloer disfunksie, behalwe vir menoragie.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, activating a series of cellular events. Satellite cells (SC), the key role players in muscle regeneration, are activated to proliferate and develop into mature myoblasts, which could fuse to form new myotubes or to repair damaged fibres. Evidence suggests that anti-oxidants, such as those found in grape seed extract (GSE), enhance repair, but their effect on SCs is still unclear. This study aimed to harvest and culture primary rat myoblasts to investigate the effect of chronic in vivo GSE supplementation on SCs following a standardised crush injury. Using a modified pre-plate technique, myoblasts were harvested from rat muscle and then compared with the immortal C2C12 cell line for proliferation and differentiation competence. Several media options were compared: i) DMEM with or without L-glutamine, ii) Ham‘s F10 or iii) DMEM with L-glutamine and Ham‘s F10 combined. Primary myoblasts proliferated and differentiated at a much slower rate than C2C12 cells. The combined media was selected for further use. To investigate the effects of GSE on the recovery, rats were supplemented daily with GSE or placebo 14 days prior to a standardised mass-drop crush injury to the gastrocnemius. SCs were isolated and cultured from uninjured (NI, baseline) and from injured rats 4 hours (4h), 3 days (3d) or 14 days (14d) post-injury. Expression of myogenic proteins Pax7, M-cadherin, MyoD, CD56, desmin and CD34 was determined by flow cytometry. Myoblasts were sorted according to their CD56 and CD34 expression and three sub-sets were collected and re-cultured, namely CD56+/CD34-, CD56-/CD34+ and CD56+/CD34+. After 24 hours, sorted cells were stained for desmin expression. Pax7, M-cadherin and MyoD were present in 100% of isolated cells from all groups confirming their myogenic SC identity. For all groups, desmin was expressed only in ~80% of SCs. Lower adhesion competency in GSE supplemented groups resulted in lower yield obtained for culturing. Expression of CD56 increased significantly 3d post-injury in the placebo group. In contrast, with GSE, CD56 already increased 4h post-injury and decreased again 3d post-injury. Although CD34 expression did not differ dramatically, expression pattern resembled that of CD56. Immunocytochemistry revealed a range in morphology and desmin expression of sorted myoblasts. More myoblasts with high desmin expression were observed in the two CD56+ sub-sets (irrespective of CD34 expression), indicating that CD56 is still expressed in more mature myoblasts. Flow cytometry revealed a population of myoblasts expressing particularly high levels of desmin, primarily in the non-injured baseline GSE group. We hypothesise that this result is an indication of preparedness of myoblasts to respond earlier to injury, enabling quicker repair. This cell population with high desmin content is restored in skeletal muscle after repair (14d), only when supplemented with GSE. In conclusion, GSE attenuated adhesion competence of primary myoblasts in culture, but resulted in earlier maturation of SCs, possibly due to baseline preparedness of myoblasts in uninjured muscle for a quick response. Both reduced adhesion competence and early progression of myoblasts could enhance wound healing in skeletal muscle. / AFRIKAANSE OPSOMMING: Kneuswonde veroorsaak aansienlike skade aan skeletspier, wat ‘n reeks sellulêre prosesse in werking stel. Satellietselle, die hoofrolspelers tydens spierregenerasie, vermenigvuldig en ontwikkel tot volwasse mioblaste, wat saamsmelt om nuwe spiervesels te vorm. Antioksidante, soos die wat in druiwepit-ekstrak voorkom, bespoedig herstel, maar hul uitwerking op satellietselle is steeds onduidelik. Die doel van hierdie studie was om mioblaste uit rotspiere te isoleer en te kweek om die effek van langdurige in vivo aanvulling van druiwepit-ekstrak op satellietselle na ‘n kneusbesering te bepaal. 'n Aangepaste protokol is gebruik om primêre mioblaste te isoleer, wat daarna met C2C12 selle, ten opsigte van hul vermenigvuldigings- en differensiasievermoë vergelyk is. Verskeie groeimedia is gebruik: i) DMEM met of sonder L-glutamien, ii) Ham F10 en iii) ‘n kombinasie van DMEM, L-glutamien en Ham F10. Primêre mioblaste het stadiger vermenigvuldig en gedifferensieer as C2C12 selle. Die gekombineerde medium is vir verdere gebruik gekies. Om die uitwerking van druiwepit-ekstrak op spierherstel te ondersoek, is rotte vir 14 dae onderwerp aan daaglikse aanvullings van druiwepit-ekstrak of placebo voor ‘n gestandardiseerde kneusbesering aan die gastrocnemius. Satellietselle is geïsoleer vanuit onbeseerde spier (basiskontrole) en vanuit beseerde spier 4 ure (4h), 3 dae (3d) en 14 dae (14d) na die besering. Die uitdrukking van spierverwante proteïene Pax7, M-cadherin, MyoD, CD56, desmin en CD34 is vasgestel met 'n vloeisitometer. Mioblaste is daarna gesorteer op grond van hul CD56- en CD34-uitdrukking. Drie sub-groepe is versamel en verder gekweek, nl. CD56+/CD34-, CD56-/CD34+ en CD56+/CD34+. Na 24 uur is gesorteerde selle gekleur om desmin-uitdrukking te bepaal. Pax7, M-cadherin en MyoD is deur 100% satellietselle in alle groepe uitgedruk, wat hul spierverwante identiteit bevestig, alhoewel slegs 80% selle in alle groepe desmin uitgedruk. Druiwepit-ekstrak het die vermoë van selle om aan plate te heg onderdruk, wat gelei het tot ‘n laer opbrengs van mioblaste. Drie dae na die besering in die placebo groep het die CD56-uitdrukking beduidend toegeneem. In teenstelling hiermee het CD56-uitdrukking in die druiwepit-ekstrak groep 4 ure na die besering beduidend toegeneem en weer afgeneem na 3 dae. Hoewel daar nie sulke dramatiese verskille was tussen groepe ten opsigte van CD34-uitdrukking nie, was daar ‘n soortgelyke tendens as vir CD56-uitdrukking. Immunositochemie het ‘n verskeidenheid van morfologieë en variërende desminvlakke in gesorteerde mioblaste blootgestel. In die twee CD56+ groepe is meer mioblaste wat hoë desmin vlakke uitdruk gevind, wat aandui dat CD56 uitgedruk word deur meer volwasse mioblaste, ongeag van CD34-uitdrukking. Tydens vloeisitometrie is ‘n populasie selle wat hoë desminvlakke uitdruk, hoofsaaklik in die onbeseerde en 14d druiwepit-ekstrak groepe gevind. Dit is ‘n aanduiding dat sommige mioblaste voorbereid is om na 'n besering vinniger te reageer. Na die herstelproses word hierdie groep selle hernu in die teenwoordigheid van druiwepit-ekstrak-aanvulling. Die resultate het gevolglik daartoe gelei dat druiwepit-ekstrak die hegtingsvemoë van mioblaste verlaag, maar dat die aanvulling in vivo tot vroeër ontwikkeling van mioblaste lei, waarskynlik deur satellietselle voor te berei vir 'n vinnige respons na ‘n besering. Beide die onderdrukking van aanhegting aan kultuurplate en die vroeë ontwikkeling van mioblaste, kan die herstel van die skeletspier verbeter. / NRF and the Harry Crossley bursary for funding