Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Context: Cardiovascular diseases are the leading cause of death globally. Myocardial infarction is responsible for the highest number of deaths due to cardiovascular disease. Objective: We have previously shown that acute benfotiamine administration at the onset of reperfusion is associated with decreased infarct size and preserved contractile function in response to ischemia-reperfusion. We aimed to evaluate the involvement of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pro-survival signaling pathways in mediating these cardioprotective effects. Materials and Methods: Part One - Hearts were rapidly excised from Wistar rats and mounted on a Langendorff perfusion apparatus. After stabilization, hearts were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion. The control group received no treatment. Experimental groups were treated with 100 μM benfotiamine ± 0.1 μM Tyrphostin AG490 or Wortmannin (inhibitors of JAK2 and PI3K, respectively), dissolved in dimethyl sulfoxide. The vehicle control group received an equivalent dose of dimethyl sulfoxide. All treatments were administered for 20 minutes at the onset of reperfusion. Functional parameters were measured throughout, to test the effects of benfotiamine ± pro-survival pathway inhibitors on functional recovery. In addition, hearts were stained with Evans blue and triphenyltetrazolium chloride to assess the effects of benfotiamine ± pro-survival pathway inhibitors on infarct size. Part Two - Hearts that were perfused ± 30 minutes of global ischemia and ± 20 minutes of benfotiamine administration, were used to assess PI3K/Akt and JAK/STAT signaling in response to ischemia-reperfusion and benfotiamine treatment. As with previous experiments, benfotiamine was administered at a concentration of 100 μM, at the onset of reperfusion. Tissues were assessed by Western blot analysis. Results: 20 minutes of acute benfotiamine administration at the onset of reperfusion led to a decrease in infarct size (35.6 ± 2.4% vs. 55.7 ± 5.0% [p<0.05]). Inhibition of PI3K/Akt signaling by addition of Wortmannin abrogated this infarct-limiting effect (51.5 ± 1.3% vs. 35.6 ± 2.4% [p<0.05]). However, inhibition of JAK/STAT signaling had no effect. There were no significant differences in left ventricular developed pressure, coronary flow rate or heart rate during the experiments. In addition, 20 minutes of acute benfotiamine administration at the onset of reperfusion lead to an increase in phospho-FOXO/FOXO in the cytosolic fraction, but no significant change in phospho-STAT3/STAT3 in the nucleus. Conclusions: Our results suggest that acute benfotiamine administration at the onset of reperfusion may act to reduce infarct size via activation of PI3K/Akt pro-survival signaling. / AFRIKAANSE OPSOMMING: Konteks: Kardiovaskulêre siekte is die hoofoorsaak van sterftes wêreldwyd. Miokardiale infarksie is verantwoordelik vir die grootste aantal sterftes weens kardiovaskulêre siekte. Doel: Ons het voorheen getoon dat akute benfotiamientoediening met die aanvang van reperfusie geassosieer is met „n verkleining in die infarkgrootte, en dit het verder ook die kontraktiele funksie in reaksie op ischemie-reperfusie behou. Ons doel was om die betrokkenheid van die fosfatidielinositol 3-kinase/Akt (PI3K/Akt) en Janus kinase/seintransduseerde en aktiveerder van transkripsie (JAK/STAT) pro-oorlewings seinweg in die mediasie van hierdie kardiobeskermende effekte te evalueer. Materiale en Metodes: Deel een - Harte is vinnig vanuit Wistarrotte verwyder en op die Langendorff-perfusieapparaat gemonteer. Na stabilisering is die harte blootsgestel aan 30 minute regionale ischemie en 120 minute reperfusie. Die kontrole groep het geen behandeling ontvang nie. Eksperimentele groepe is met 100 μM benfotiamien ± 0.1 μM Tirfostien AG490 of Wortmannin (inhibeerders van JAK2 en PI3K, onderskeidelik) behandel, opgelos in dimetielsulfoksied. Die draer-kontrole groep het „n ekwivalente dosis van dimetielsulfoksied ontvang. Alle behandelings is toegedien vir 20 minute aan die begin van die reperfusie. Funksionele parameters is deurgaans gemeet om te toets vir die effekte van benfotiamien ± pro-oorlewingsweg inhibeerders op funksionele herstel. Verder is die harte met Evans-blou en trifenieltetrazoliumchloried gekleur om die effek van benfotiamien ± pro-oorlewingsweg inhibeerders op die infarkgrootte te bepaal. Deel twee - Harte is vir ± 30 minute perfuseer met globale ischemie en ± 20 minute met benfotiamientoediening. Dit was gebruik om PI3K/Akt en JAK/STAT seine as gevolg van ischemie-reperfusie en benfotiamienbehandeling te ondersoek. Soos met die vorige eksperimente, is benfotiamien toegedien by ‟n konsentrasie van 100 μM met die aanvang van reperfusie. Weefsel is ondersoek deur middel van Western blot analise. Resultate: 20 minute van akute benfotiamientoediening, met die aanvang van reperfusie, het tot „n verkleining in die infarkgrootte (35.6 ± 2.4% vs. 55.7 ± 5.0% [p<0.05]) gelei. Inhibering van die PI3K/Akt seinweg deur toediening van Wortmannin het die infark-beperkende effek opgehef (51.5 ± 1.3% vs. 35.6 ± 2.4% [p<0.05]). Inhibering van JAK/STAT seine het egter geen effek getoon nie. Daar was geen beduidende verskille in linkerventrikulêr-ontwikkelde druk, koronêre-vloeitempo of harttempo tydens die eksperimente nie. Verder, 20 minute van akute benfotiamientoediening met die aanvang van reperfusie het „n toename in fosfo-FOXO/FOXO in die sitosoliese-fraksie veroorsaak, maar geen beduidende verandering in fosfo-STAT3/STAT3 is in die nukleus waargeneem nie. Gevolgtrekkings: Ons resultate suggereer dat akute benfotiamientoediening met die aanvang van reperfusie moontlik die infarkgrootte via aktivering van die PI3K/Akt pro-oorlewingsein kan verklein.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: Neuroinflammation is central to the aetiology of HIV-associated neurocognitive disorders (HAND) that are prevalent in late stage AIDS. ARV treatments are rolled out relatively late in the context of neuroinflammatory changes, so that their usefulness in directly preventing HAND is probably limited. It is common practice for HIV+ individuals in developing countries to make use of traditional/complimentary medicines. One such medicine is Sutherlandia frutescens - commonly consumed as a water infusion. We have also identified a new candidate complimentary medicine for use in this context - grape seed-derived proanthocyanidolic oligomers (PCO) have significant anti-inflammatory action in the peripheral compartment in the context of e.g. skeletal muscle injury, but have not been investigated in the context of either neuroinflammation or HIV/AIDS. Here the efficacy of these two substances as an anti-inflammatory modality in this context was investigated in an in vitro co-culture model of the blood-brain barrier (BBB). Methods: Single cultures of human astrocytes, HUVECs and primary human monocytes, as well as co-cultures (BBB), were stimulated with HIV-1 subtype B & C Tat protein and/or HL2/3 cell secretory proteins after pre-treatment with S. frutescens or PCO extracts. Effects of this pre-treatment on pro-inflammatory mediator expression and monocyte migration across the BBB were assessed. Results: In accordance with others, B Tat was more pro-inflammatory than C Tat, validating our model. S. frutescens decreased IL-1β secretion significantly (P<0.0001), but exacerbated both monocyte chemoattractant protein-1 (P<0001) – a major role player in HIV-associated neuroinflammation – and CD14+ monocyte infiltration across the BBB (P<0.01). PCO pre-treatment resulted in a significantly dampened IL-1β (P<0.0001) response to stimulation with HIV-associated proteins. In contrast to S. frutescens, PCO modulated monocyte chemoattractant protein-1 (P<0001) response and decreased capacity for CD14+ monocytes to migrate across the simulated BBB (P<0.0001). Additionally, PCO pre-treatment decreased both GFAP (P<0.001) and HSP-27 (P<0.001) expression in the astrocytes of the BBB. Conclusions: Current data illustrates that the combined use of HL2/3 cells and the simulated BBB presents an accurate, disease relevant in vitro model with which to study neuroinflammation in the context of HIV/AIDS. In addition, our results caution against the use of S. frutescens as anti-inflammatory modality at any stage post-HIV infection. Novel data presented here illustrate that PCO is able to blunt the MCP-1 and IL-1β response to HIV-1 proteins in single cultures of human astrocytes and HUVECs, as well as in an in vitro simulation of the BBB. In addition, PCO was able to limit monocyte transmigration across the simulated BBB in response to HIV-1 proteins generated by HL2/3 cells. This suggests that grape seed-derived PCO could be considered as complimentary anti-neuroinflammatory drug in the context of HIV/AIDS. / AFRIKAANSE OPSOMMING: Agtergrond: Neuroinflammasie staan sentraal in die ontwikkeling van MIV-verwante toestande wat gekenmerk word deur neurokognitiewe afteruitgang, veral in die later stadia van die siekte. Aangesien anti-virale middels relatief laat toegedien word in die konteks van neuroinflammasie, is hul rol in die voorkoming van neuroinflammatoriese veranderinge heel moontlik weglaatbaar. MIV+ individue, veral in ontwikkelende lande, gebruik algemeen natuurlike medisinale preparate. Sutherlandia frutescens is een so „n middel wat as „n tee ingeneem word. Verder het ons ook „n nuwe kandidaat komplimentêre medisyne identifiseer – druiwepitekstrak wat polifenole bevat (PCO) het aansienlike anti-inflammatoriese eienskappe in die periferie, bv. in die konteks van skeletspierskade, maar die middel is nog nie voorheen in die konteks van neuroinflammasie of MIV/VIGS ondersoek nie. Hier word die anti-inflammatoriese effektiwiteit van beide middels in hierdie konteks ondersoek deur gebruik te maak van „n in vitro simulasie van die bloedbreinskans (BBS). Metodes: Kulture van menslike astrosiete, menslike naelstring endoteelselle (HUVECs) en primêre menslike monosiete, sowel as gesamentlike kulture (BBS) is met MIV-1 subtipe B en C Tat proteïen en/of HL2/3 selprodukte gestimuleer na voorafbehandeling met S. frutescens of PCO ekstrakte. Effekte op pro-inflammatoriese mediator uitdrukking sowel as monosiet migrasie oor die BBS is ondersoek. Resultate: In ooreenstemming met die literatuur was B Tat meer inflammatories as C Tat, wat die akkuraatheid en gepastheid van ons model bevestig. . S. frutescens het afskeiding van IL-1β betekenisvol verminder (P<0.0001), maar het afskeiding van beide monosiet chemoaantrekkingsproteïen-1 – „n groot rolspeler in MIV-verwante neuroinflammasie – en CD14+ monosiet migrasie oor die BBS vererger (P<0.0001 en P<0.01 onderskeidelik). PCO behandeling het „n betekenisvolle demping van die IL-1β reaksie (P<0.0001) op stimulasie met MIV-geassosieerde proteïene tot gevolg gehad. Anders as S. frutescens het PCO die MCP-1 reaksie, asook CD14+ monosiet migrasie betekenisvol inhibeer. Verder het PCO ook beide GFAP en HSP-27 uitdrukking in astrosiete van die BBS verminder (beide P<0.001). Gevolgtrekkings: Huidige data wys dat die gekombineerde gebruik van HL2/3 selle en die gesimuleerde BBS „n akkurate en fisiologies relevante in vitro model daarstel, waarmee neuroinflammasie in die konteks van MIV/VIGS bestudeer kan word. Ons resultate waarsku verder teen die gebruik van S. frutescens as anti-inflammatoriese middel in selfs die vroeë stadium na MIV infeksie. Oorspronklike data wat hier aangebied word illustreer dat PCO die pro-inflammatoriese reaksie op MIV-proteïene in kulture van astrosiete en HUVECs, asook die in vitro simulasie van die BBS, effektief demp. Verder het PCO die vermoë getoon om monosiet migrasie oor die BBS, in reaksie op MIV-1 proteïene wat hul oorsprong uit HL2/3 selle het, te beperk. Hierdie bevindings beteken dat PCO dus eerder as S. frutescens oorweeg moet word as komplimentêre anti-inflammatoriese medisyne in die konteks van MIV/VIGS.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition. / AFRIKKANSE OPSOMMING: Inleiding: Sirkadiese ritmes vorm ‘n integrale fisiologiese sisteem wat die sinkronisasie van alle metaboliese prosesse asook lig/donker siklusse se effektiwiteit optimaliseer. Onderbreking van hierdie sirkadiese ritmes word geïmpliseer in die ontstaan en bevordering van verskillende kankertipes, insluitend borskanker. Verskeie raakpunte bestaan tussen die sirkadiese proteïen, Per2, en die DNA skade-respons. Abnormale Per2 uitdrukking veroorsaak afstroom effekte op beide die selsiklus en apoptotiese teikens wat moontlik aanduidend van ‘n tumor-onderdrukkende rol vir Per2 kan wees. Daar bestaan ‘n groot nood vir meer effektiewe adjuvante terapieë om kankersel vatbaarheid vir chemoterapie te verhoog as gevolg van dosis-beperkende newe-effekte wat geassosieer word met huidige chemoterapeutiese strategieë, insluitende dié van doxorubicin. Ons hipotese is dus dat die manipulering van die sirkadiese Per2 proteïen tesame met doxorubicin ‘n meer effektiewe chemoterapeutiese strategie vir die behandeling van borskanker sal wees. Die doelwitte van hierdie projek was dus om: (i) Die rol van Per2 in normale borsepiteelselle sowel as in ER+ en ER- borsepiteel kankerselle te karakteriseer; (ii) die rol van Per2 in doxorubicin-geïnduseerde seldood te bepaal; (iii) te bepaal of Per2 ‘n rol in autofagie speel en laastens (iv) te bepaal of die farmakologiese inhibisie van Per2 met metformin chemo-weerstandbiedende MDA-MB-231 kankerselle kan sensitiseer vir doxorubicin-geïnduseerde seldood. Metodes: ‘n In vitro model vir borskanker is gebruik wat normale MCF-12A borsepiteelselle, estrogeen reseptor positiewe (ER+) MCF-7 en estrogeen reseptor negatiewe (ER-) MDA-MB-231 bors adenokarsenoomselle insluit. Sirkadiese ritmisiteit van Per2 proteïen uitdrukking is deur middel van die westelike kladtegniek bepaal en die sellulêre ligging van Per2 deur middel van fluoresensie mikroskopie. siPer2 is voorberei deur middel van endoribonuklease-siRNA en die effektiwiteit daarvan is deur middel van westelike kladtegniek getoon. Die rol van Per2 in doxorubicin-geinduseerde seldood en autofagie is bepaal deur MDA-MB-231 borskankerselle onder die volgende omstandighede te toets: (1) Kontrole, (2) 2.5 μM doxorubicin of 10 nM bafilomycin A1 (3) 30 nM esiPer2 en (4) 30 nM esiPer2 in kombinasie met 2.5 μM doxorubicin of 10 nM bafilomycin A1. Na die behandeling, is sellewensvatbaarheid bepaal deur gebruik te maak van ‘n MTT toets; westelike kladtegniek is gebruik om vir merkers van apoptose soos p-MDM2 (Ser166), p-p53 (Ser15), gekliefde caspase-3 en -PARP asook vir merkers van autofagie (AMPKα, mTOR en LC3) te toets. Die selsiklus, G2/M oorgang en seldood (Hoechst 33342 en propidium iodide kleuring) is deur middel van vloeisitometrie bepaal. Per2 is ook farmakologies geïnhibeer deur MDA-MB-231 selle met 40 mM metformin asook in kombinasie met 2.5 μM doxorubicin te behandel. Daarna is sellewensvatbaarheid (MTT) sowel as die selsiklus (vloeisitometrie) en apoptose (westelike kladtegniek vir Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) gemeet. Resultate en bespreking: ‘n Sirkadiese patroon vir Per2 proteïen uitdrukking is in die normale MCF-12A selle asook in die MDA-MB-231 kankerselle waargeneem met proteïenvlakke wat hul piek by ±700% and ±500% onderskeidelik in vergelyking met basislyn bereik het. Geen ritmiese patroon van Per2 proteïen uitdrukking is egter in die MCF-7 kankerselle waargeneem nie. Immunokleuring om Per2 ligging te bepaal het getoon dat Per2 in the sitoplasma sowel as in die nukleus in beide MCF-12A en MDA-MB-231 selle voorgekom het. Konsentrasie kurwes het aangetoon dat daar ‘n insiggewende vermindering in sellewensvatbaarheid voorgekom het na die behandeling van die selle met 2.5 μM doxorubicin vir 24 uur. Per2 proteïen uitdrukking is insiggewend verlaag met beide esiPer2 en metformin behandeling van die selle. esiPer2 aleen of in kombinasie met doxorubicin behandeling het selsiklus staking tot gevolg gehad asook ‘n beduidende toename in apoptose veroorsaak wat dus aangedui het dat esiPer2 effektief was om MDA-MB-231 kankerselle te sensitiseer vir die anti-karsinogeniese doxorubicin behandeling. Modulering van Per2 proteïen uitdrukking was effektief met metformin behandeling, alhoewel die afname onafhanklik van AMPKα fosforilasie plaasgevind het. ‘n Insiggewende toename in apoptose is waargeneem na metformin behandeling in kombinasie met doxorubicin. Geen veranderinge in die selsiklus is egter onder hierdie omstandighede waargeneem nie. Per2 blyk betrokke te wees in die regulering van autofagie aangesien ‘n insiggewende verhoging in autofagie omsetting waargeneem is na esiPer2 behandeling. Die toename in autofagie omsetting is geassosieer met ‘n insiggewende toename in seldood in MDA-MB-231 kankerselle wat verder verhoog is wanneer autofagie met bafilomycin A1 (autofagie inhibitor) in kombinasie met esiPer2 behandel is. Gevolgtrekkings: Per2 proteïen uitdrukking het ‘n 24 uur sirkadiese ritme in die MCF-12A normale selle, en tot ‘n mindere mate in die MDA-MB-231 selle getoon. Die MCF-7 selle het egter geen ritmiese patroon van Per2 proteïen uitdrukking getoon nie. Per2 kom hoofsaaklik in die sitoplasma voor en het slegs in die nukleus voorgekom wanneer die sitoplasmiese fluoresensie intensiteit laer was. esiPer2 was dus effektief om die chemo-weerstandbiedende MDA-MB-231 borskankerselle te sensitiseer vir doxorubicin-geïnduseerde seldood. / National Research Foundation

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Tumour necrosis factor alpha (TNFα) is a pleiotropic cytokine and has a wide variety of dose dependent cellular effects ranging from cell growth and differentiation, to inducing apoptosis. It has long been implicated in muscle and non-muscle inflammatory disorders, such as muscle wasting in chronic disease states, and rheumatoid arthritis. However, a physiological role for TNFα in muscle regeneration has been proposed as elevated levels of the cytokine are present when muscle regeneration processes are initiated: TNFα is secreted by infiltrating inflammatory cells, and by injured muscle fibres. Adult skeletal muscle contains a population of resident stem cell-like cells called satellite cells, which become activated, proliferate and differentiate following muscle injury to bring about repair of damaged muscle. Much research on the effects of TNFα on satellite cell differentiation has been conducted in recent years. It is however difficult to get a complete characterisation of the cytokine’s action as all models used slightly differ. We aimed therefore at providing comprehensive assessment of the effects of increasing doses of chronically supplemented TNFα on differentiating C2C12 cells. Cells were allowed to differentiate with or without TNFα supplementation for 7 days. Differentiation was induced at day 0. The effect on differentiation was assessed at days 1, 3, 5, and 7 by western blot analysis, and supplementary immunohistochemical analysis at days 1, 4, and 7 of markers of differentiation - muscle regulatory factors: MyoD and myogenin, markers of the cell cycle p21, PCNA, and the integral signalling molecule, p38MAPK. TNFα supplementation at day 1 tended to positively regulate early markers of differentiation. With continued supplementation however, markers of differentiation decreased dose dependently in treated cultures as the initial effect appeared to be reversed: A trend towards a dose dependent decrease in MyoD, myogenin and p21 protein existed in treated cultures at days 3, 5, and 7. These findings were significant at day 5 (p21, p<0.05), and day 7 (myogenin, p<0.05). A significant dose dependent decrease in p38 phosphorylation was evident at day 3 (p<0.05), while phospho-p38 was dose dependently increased at day 7 (p<0.05). Taken together, these data show that TNFα supplementation for 24 hours following the induction of differentiation in vitro, tends to increase levels of early markers of differentiation, and with continued TNFα supplementation decrease markers of differentiation in a dose dependent fashion. This study provides a comprehensive characterisation of the dose and time dependent effects of TNFα on satellite cell differentiaton in vitro. The model system used in the current study, allows us to make conclusions on more chronic disease states. / AFRIKAANSE OPSOMMING: Tumor nekrose faktor alfa (TNFα) is ‘n pleiotropiese sitokien wat ‘n wye verskeidenheid, dosis afhanklike, sellulêre effekte te weeg bring. Hierdie sellulêre effekte sluit sel groei en differensiasie tot sel dood in. TNFα is by beide spier en niespier inflammatoriese stoornisse soos spier tering in kroniese siektetoestande, en rumatiese artritis betrek. ‘n Fisiologiese rol vir TNFα is egter voorgestel aangesien verhoogde vlakke van die sitokien tydens inisiasie van spier herstel meganismes teenwoordig is: TNFα word deur infiltrerende inflammatoriese selle, asook deur beseerde spier vesels afgeskei. Volwasse skeletspier bevat ‘n populasie stamselagtige selle, sogenoemde satelliet selle. Laasgenoemde word geaktiveer, prolifereer en differensieër volgende spierbesering, om sodoende herstel van beskadigde spier te weeg te bring. Baie navorsing op die effekte van TNFα op satelliet sel differensiasie is onlangs uitgevoer. Dit is egter aansienlik moeilik om volgens hierdie navorsing‘n algehele beeld van TNFα se aksies te vorm aangesien alle modelle wat gebruik word verskil. Ons doel was daarom om ‘n omvangryke assessering van toenemende konsentrasies kronies gesupplementeerde TNFα op differensieërende C2C12 selle op ‘n enkele model uit te voer. Selle was vir 7 dae met of sonder TNFα supplementasie gedifferentieër. Differensiasie was by Dag 0 geïnduseer. TNFα se effek op differensiasie is op dae 1, 3, 5, en 7 deur middel van western blot analise geassesseer. Aanvullende immunohistochemiese bepalings op dae 1, 4, en 7 is verder deurgevoer. Merkers vir differensiasie het die spier regulatoriese faktore MyoD en miogenien, sel siklus merkers p21 en PCNA, asook die integrale sein transduksie molekule p38MAPK ingesluit. TNFα supplementasie by dag 1 het geneig om vroeë merkers van differensiasie positief te reguleer. Met voortdurende supplementasie is die vroeë positiewe effekte (op ‘n dosis afhanklike manier) egter omgekeer: ‘n neiging teenoor (‘n dosis afhanklike) vermindering in MyoD, miogenien en p21 proteïen het in behandelde kulture op dae 3, 5, en 7 bestaan. Hierdie bevindinge was beduidend by dag 5 (p21, p<0.05), en dag 7 (miogenien, p<0.05). A beduidende dosis afhanklike afname in p38 fosforilasie was duidelik by dag 3 (p<0.05), terwyl fosfo-p38 by dag 7 verhoog het met verhoogde konsentrasie TNFα (p<0.05). Bogenoemde saamgevat, dui aan dat TNFα supplementasie 24h volgende die induksie van differensiasie in vitro, verhoogde vlakke van vroeë differnsiasie merkers te weeg bring. Met voortdurende TNFα supplementasie, word differensiasie merkers egter met toenemende dosis verminder. Hierdie studie voorsien ‘n omvattende karakterisering van die dosis- en tyd afhanklike effekte van TNFα op satelliet sel differesiasie in vitro. Die model sisteem in hierdie studie gebruik, maak afleidings oor meer kroniese siektetoestande moontlik.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management. / AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.

This study was undertaken to redress the scant knowledge regarding the chemistry and mode of action of pregnancy-related traditional medicines, or isihlambezo (Zulu), which are used by 60 to 80% of women in South Africa. The three selected plants are among the six most frequently cited species from the approximately 90 used by traditional healers. The purpose of the study was to identify components which could cause uterine contractions, those with nutritional value for the foetus and mother, and those with any toxic effects. Plant root extracts were purified via silica gel column chromatography and bioassays were carried out on the fractions, using isolated rat uterine tissue. Purified compounds were identified via spectral techniques, and some were characterised by comparison to authentic standards using HPLC, and others by matching their GC-MS spectra to library standards. Thirty-eight compounds were identified in total, the majority of these being novel to the species concerned. Those isolated from Combretum kraussii were 1 sitosterol, 2 combretastatin, 3 3',4-tri-O-methylellagic acid, 4 combretastatin B-1, 5 combretastatin A-1, 6 3,3'-di-O-ellagic acid lactone, 7a ellagic acid lactone, 7b ellagic acid, 8 and 9 a mixture of combretastatin B-1 and A-1 glucosides, 10 and 11 partly characterised glucosides of ellagic acid. Those isolated from Gunnera perpensa were 12 3',4-tri-methylellagic acid, 13 ellagic acid lactone, 14 1,1'-biphenyl-4,4'-diacetic acid, 15 p-hydroxybenzaldehyde, 16 Z-methyl lespedezate, 17 and 18 partly characterized higher glucosides of Z-methyllespedezate. Those isolated rom Rhoicissus tridentata were 19 (-)-epigallocatechin, 20 (+)-gallocatechin, 21 procyanidin B3, 22 procyanidin B4, 23 (+)-catechin hydrate, 24 (+)-mollisacacidin, 25 (+)-epicatechin, 26 fisetinidol-(4a-8) catechin, 27 (-)-fisetinidol, 28 fisetinidol-(4b-8)catechin, 29 gallic acid, 30 epicatechin-3-0-gallate, 31 partly characterized hydrogel of glucose, 32 sitosterol, 33 sitosterolin, 34 y-sitosterol, 35 oleanolic acid, 36 lupen-3-one, 37 20-epi-y-taraxastananol and 38 triacontanol. The compounds with the greatest in vitro uteroactivity were predominantly proanthocyanidins or phenolic glucosides. It is proposed that effects of phenolic glucosides could be due to the interaction of the sugar moiety as well as the phenolic moiety with the receptor site in muscle tissue. The corresponding phenolic aglycones isolated were only moderately uterotonic, or unreactive by comparison. Non-polar compounds such as sitosterol and sitosterolin showed minimal enhancement of the uterine response at low concentrations, and inhibition at higher concentrations. Aqueous root extracts of the plants were all found to be non-toxic according to cell-viability tests using monkey vero cells and human fibroblasts. Extracts are therefore considered safe for human consumption, although it is recommended that Rhoicissus tridentata be used with caution because it showed the lowest cell viability of the three species, and uterine hyperstimulation has been attributed to this species, as well as CNS depression and respiratory arrest. Ions which could be nutritionally beneficial in pregnancy, calcium, iron, and phospate, were present in low in aqueous extracts. Levels of calcium and potassium ions were considered to be too low to directly stimulate uterine muscle. Proanthocyanidins, combretastatins, ellagic acid derivatives and phytosterols, with health-promoting properties, were also identified. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

Thesis (MScMedSc) -- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Osteopenia is a well documented complication of anorexia nervosa (AN). The pathogenesis of this bone loss is presently poorly defined in the literature. Pathogenetic mechanisms that have been implicated include certain nutritional factors, exercise abuse, hypogonadism, hypercortisolism and/or vitamin 0 deficiency. We studied, 59 Caucasian eating disorder patients aged 15-45yr. The eating disorder was classified by a single, qualified psychiatrist according to OSM IV R criteria as either anorexia nervosa (AN: n =25), bulimia nervosa (BN: n = 17) or eating disorder not otherwise specified (EONOS: n = 17). All patients were subjected to a detailed dietary and general history. We assessed the prevalence and severity (OEXA), the nature (osteocalcin, deoxypyridinoline) and site (vertebral versus hip) of osteopenla in these patients. he role of nutritional factors (energy intake, weight, height, BMI, plasma albumin, lipids), physical activity, hypercortisolemia (plasma and urinary free cortisol), vitamin 0 deficiency (plasma 250HD) and hypogonadism (amenorrhoea, E2, LH, FSH) in the pathogenesis of bone loss were also evaluated. Mild osteopenia (BMO decreased by more than 1SO below age-matched controls) was documented in 46% of the total study population, with more marked osteopenia (Z-Score < -2 SO) present in 15%. Both vertebral and hip osteopenia were documented. In the study population those patients with AN (Lumbar BMO (q/cm") = 0.869 ± 0.121) were most likely to develop osteoporosis, although a significant percentage of patients with BN (Lumbar BMO (q/crn") = 0.975 ± 0.16) and EONOS (Lumbar BMO (g/cm2) = 0.936 ± 0.10) were also osteopenic (29% and 35% respectively). Twenty four percent (24%) of the total patient population had a history of fragility fractures. These fractures were reported more commonly amongst patients with AN and EONOS (28% and29.4%). Fracture prevalence was however similar in patients with normal and low bone mass. Conventional risk factors were similar in patients with normal and low bone mass, except for a significantly longer duration of amenorrhoea (p = 0.009), a lower BMI (p = 0.0001) and greater alcohol consumption (p = 0.05) in the osteopenic patients. Nutritional parameters (S-albumin, protein, Ca, and P04 intakes), physical activity, as well as 25(OH) vitamin D levels were similar in AN and BN subjects, as well as in patients with a low versus normal BMD. Plasma and urine cortisol levels were also similar in these subgroups. With the exception of two patients with borderline osteopenia, significant bone loss was only documented in those patients with a past or current history of amenorrhoea. In the total patient population the duration of amenorrhoea was significantly (p<0.009) longer in patients with osteopenia versus those with a normal bone mass. A significant negative correlation between BMD (Z-Score) and duration of amenorrhoea was also documented in the total patient population (r = -0.4, P = 0.001) as well as in all three eating disorder groups (AN r - -0.4, P = 0.03; BN r = - 0.6, P = 0.008; EDNOS r = -0.6, P = 0.005). In the total patient population, those patients with amenorrhoea, had lower BMD and BMI values and lower estrogen levels compared to those with a normal menstrual cycle. We conclude that osteopenia commonly attends AN, as well as BN and EDNOS. Nutritional (with the exception of alcohol consumption) and mechanical factors as well as hypercortisolemia did not appear to contribute significantly to bone loss in this study population. Hypogonadism appeared to be the main cause of the bone loss observed in these patients. / AFRIKAANSE OPSOMMING: Osteopenie is In welbekende komplikasie van anorexia nervosa (AN). Die patogenese van hierdie beenverlies is swak in die huidige literatuur omskryf en nutrisiele faktore, 'n vita mien 0 gebrek, oormatige oefening, hiperkortisolemie en hipogonadisme word onder andere qeimpliseer. Vir die doel van die studie is In totaal van 59 Kaukasier eetsteurnis pasiente patients volledig ondersoek. Die tipe eetsteurnis is deur In enkel gekwalifiseerde psigiater volgens die DSM IV R kriteria geklassifiseer as anorexia nervosa (AN: n =25) of bulimia nervosa (BN: n = 17) of eetsteurnis nie anders gespesifiseer (EDNOS: n = 17). Elke pasient is ook aan In gedetailleerde dieet en algemene risikofaktor vraelys vir osteoporose onderwerp. Die voorkoms en graad (DEXA), die aard (osteokalsien, deoksipiridinolien) asook die tipe (werwelkolom/heup) osteopenie is ondersoek. Die rol van nutrisiele faktore (totale kalorie-inmame, gewig ,Iente LMI, plasma albumien, lipiede) en vitamien 0 gebrek, oefening, hiperkortisolemie (plasma en urinere kortisol) en hipogonadisme (amenoree, plasma E2, LH,FSH) in die patogenese van die beenverlies is ook evalueer. Matige osteopenie (BMD meer as 1 SO onder die van ouderdomskontrole) is in 46% van die totale pasientpopulasie gedokumenteer, met erge osteopenie (Z-Telling < -2) in 15%. Aantasting van beide werwelkolom en heup is aangetoon. In hierdie studiepopulasie kom osteopenie meer algemeen voor in die AN (Lumbaal BMD (g/cm2) = 0.869 ± 0.121) groep (64%) in vergelyking met BN (Lumbaal BMD (g/cm2) = 0.975 ± 0.16) (29%) en (EDNOS) (Lumbaal BMD (g/cm2) = 0.936 ± 0.10) (32%). Vier-en-twintig persent van die totale pasientpopulasie het In geskiedenis van frakture gehad. Frakture het meer algemeen in AN en EDNOS pasiente voorgekom (28% en 29%). Pasiente met 'n lae beenmassa was gekenmerk deur In betekenisvolle laer LMI (p = 0.0001), hoer alkolholverbruik (p = 0.05) en langer duurte van amenoree(p = 0.009). Nutrisiele parameters (s-albumien, protetene, Ca, P04 inname) oefening, asook 25(OH) vitamien 0 vlakke was soortgelyk in AN en BN pasiente. Hierdie parameters het ook nie verskil tussen pasiente met osteopenie en die met In normale BMD nie. Plasma en urinere vry kortisolvlakke was ook soortgelyk in hierdie twee groepe. Betekenisvolle beenverlies (met die uitsondering van twee pasiente met grenslyn osteopenie) het slegs voorgekom in pasiete met 'n huidige of In vorige geskiedenis van amenoree. In die totale pasientpopulasie was die duurte van amenoree (p< 0.009) betekenisvollanger in die pasiente met osteopenie. In Betekenisvolle negatiewe korrelasie tussen BMD (Z-Telling) en die duurte van amenoree in die toale pasient populasie (r = -0.4; P = 0.001) asook in al drie eetsteurnis groepe (AN: r = -0.4; P = 0.03; BN: r = -0.06; P = 0.008; EDNOS: r = - 0.6, P = 0.005) is aangetoon. In die groep as 'n geheel, het die amenoree pasiente In laer LMI, E2vlakke en BMD gehad in vergelyking met die pasiente met normale menses. Opsommend dus, kom osteopenie algemeen in pasiente met AN, asook BN en EDNOS voor. In Betekenisvolle bydrae van nutrisiele (met die uitsondering van alkoholinname) en meganiese faktore asook hiperkortisolemie tot been verlies, kon nie in hierdie tudie populasie gedemonstreer word nie. Hipogonadisme is as die hoofoorsaak van osteopenie in die pasiente qefdentifiseer.

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.