Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: OBJECTIVES: The primary objective was to establish and compare the injury incidence in forwards and backline players during the 2011 FNB Varsity Cup season. The secondary objective was to establish and compare the injury prevalence in forwards and backline players during the 2011 FNB Varsity Cup season. Lastly, to establish and compare the different training loads, types of injuries and injury rates amongst the various rugby teams during the 2011 FNB Varsity Cup. STUDY DESIGN: A retrospective, descriptive study was done to assess injury prevalence and incidence during the 2011 FNB Varsity Cup rugby (premier division) competition. PARTICIPANTS: Male rugby playing students, from eight teams. The total number of observed rugby players from the seven teams consisted of ±23 – 30 players, all between the ages of 18 and 25 years (23 ± 1.2 years). All players had to qualify according to the rules of the Varsity Cup 23,45. METHODS: The data collection procedure and injury definitions were aligned with the respective consensus statement for rugby injuries12. The injury surveillance included all injuries that were reported on the standardized IRB injury form (Addendum D), by each rugby team’s medical support staff. There were eight rugby teams partaking in the 2011 FNB Varsity cup, premier division tournament. The FNB Varsity cup took place at eight University venues in South Africa. The FNB Varsity Cup round robin began in February 2011 where games were played every Monday evenings over a seven to nine week period, on a home and away basis. The play-offs of the top four teams followed for two more weeks. Injury surveillance statistics were calculated and compared with training loads and the number of hours of exposure. Injury rates are expressed as the number of injuries sustained per 1000 hours a player is at risk. Descriptive statistics were used to report the prevalence and incidence of all injuries during the tournament. A significance level of p<0.05 was accepted. RESULTS: Seven out of the eight teams participating in the 2011 FNB Varsity Cup were able to submit injury and training statistics. Of these seven teams, there were 178 (6.1 injuries per 1000 hours) injuries in total reported throughout the season. 61 pre-season injuries were found (2.1 per 1000 hours) compared to 117 (4.0 per 1000 hours) in-season injuries reported. There were 125 match injuries (89 per 1000 hours) and 52 training injuries (1.58 per 1000 hours) which was statistically significant (p = 0.039). The total number of new injuries were 120 (4.1 per 1000 hours) with only 52 (2.0 per 1000 hours) recurrent. The lower limb was affected by 97.5% of the total injuries. Over-all the most injured sites showed a common trend, namely the ankle and foot with 15.9% and the head 15.3% of the total injuries. The shoulder (11.4%), hamstring (10.2%), knee (10.2%) and quadriceps (9.7%) were all similarly affected. The injury sites varied between forwards and backline players (forwards: 63.4% backline: 36.6%). The forwards most injured anatomical site showed a trend with the shoulder and ankle (0.5 per 1000 hours) being affected the most. The hamstring (0.4 per 1000 hours), ankle, head (0.3 per 1000 hours) and knee (0.2 per 1000 hours) were the most injured site amongst the backline players. The tackle was responsible for the cause of the majority of the injuries (total: 19.1 injuries per 1000 hours) amongst the forwards and backline players (forwards: 20.5 per 1000 hours backs: 13.6 per 1000 hours). The most common types of injuries were sprains (18.2 per 1000 hours) and strains (24.5 per 1000 hours) found amongst the forwards and backline players. The forwards had higher contusion and concussion (0.3 per 1000 hours) trend rates compared to the backline players. The backline had overall higher tendinopathy (0.2 per 1000 hours) trend rates. Amongst the forwards, the locks (15.2%) and props (12.9%) had the highest number of injuries and amongst the backline players were the wings (8.4%) and centres (9%) were the most injured players. The majority of the injuries occurred during the last part of the first half (26.7%) and the last part of the second half (30.7%). CONCLUSION: The prevalence and incidence of match injuries was significantly higher than during training (p = 0.039). Similarly to other injury surveillance studies, the tackle was the most dangerous phase of play. The Forwards who are more engaged in a greater number of physical collisions in a game resulted in more injuries compared to backline players 71. The backline players, due to their style of play had more running and accelerating injuries 5,24. Fatigue and other confounding factors such as a lack of physical conditioning, travel and academics could be a determinant to decreasing the threshold for injury’s occurring during the last part of each half of the game, during matches 22. Furthermore, the site, type and mechanism of injuries vary across individual playing positions as well as from team to team 24,26. This suggests that different training styles for the various positions should be recommended as an addition to an injury prevention protocol at this level of rugby. / AFRIKAANSE OPSOMMING: DOELWITTE: Die primêre doel van hierdie studie was om die beserings in voor- en agterspelers gedurende die 2011 FNB Universiteitsbeker seisoen vas te stel en te vergelyk. Die sekondêre doel was om die beserings in voorspelers en agterspelers gedurende die 2011 FNB Universiteitsbeker seisoen vas te stel en te vergelyk. Laastens, om die verskillende ladingsoefeninge, tipe beserings en hoeveelheid beserings onder die verskillende rugbyspelers gedurende die 2011 FNB Universiteitsbeker vas te stel en vergelyk. STUDIE ONTWERP: 'n Retrospektiewe beskrywende studie is gedoen waarin die geneigdheid tot, en die voorkoms van beserings gedurende die 2011 FNB Universiteitsrugby toernooi bepaal is. DEELNEMERS: Manlike studente rugbyspelers, vanaf agt spanne. Die totale aantal rugbyspelers wat geanaliseer was, was vanaf slegs sewe spanne, bestaande uit ± 23 - 30 spelers in elke span. Hierdie spelers was almal tussen die ouderdomme van 23 ± 1.2 jaar. Al die spelers moes 'n geregistreerde student by een van die agt deelnemende universiteite gewees het gedurende die toernooi regoor Suid-Afrika in 2011. METODE: Die data insamelingsproses en beseringsdefinisies is in lyn met die onderskeie konsensus ooreenkomste vir rugby beserings 12. Die besering opname sluit alle beserings in wat deur elke rugbyspan se mediese personeel op die gestandardiseerde IRB beseringsvorm aangedui is. Daar was agt deelnemende rugbyspanne in die 2011 FNB Universiteitsbeker-toernooi. Die FNB Universiteitsbeker het plaasgevind by agt Universiteit kampusse regoor Suid-Afrika. Die FNB Universiteitsbeker rondomtalie het in Februarie 2011 begin waar wedstryde elke Maandagaand, oor ‘n tydperk van sewe tot nege weke, gespeel is. Die uitspeelwedstryde van die top vier spanne het daarop gevolg vir ‘n verdere twee weke. Beide, die voorkoms van beserings en die frekwensies daarvan is aangeteken en vergelyk volgens die oefenprogramme en die hoeveelheid blootstellingsure van die spelers. Beseringsfrekwensies is gerraporteer as die aantal beserings per 1000 uur waar ‘n speler blootgestel word aan die risiko. Beskrywende statistiek is gebruik om die geneighdheid tot, en die voorkoms van alle beserings aan te meld tydens hierdie toernooi. Betekenisvolheidsvlak was geneem op p <0.05. RESULTATE: Sewe van die agt deelnemenede spanne aan die 2011 FNB Varsity Cup was in staat om besering- en oefenstatistiek in te dien. Van hierdie sewe spanne, was daar 178 (6.1 beserings per 1000 ure) beserings in totaal aangemeld regdeur die seisoen. Een en sestig voor-seisoenale beserings is gerapporteerd (2,1 per 1000 ure) in vergelyking met die 117 (4,0 per 1000 ure) inseisoense beserings wat aangemeld is. Daar was 125 wedstrydbeserings (89 per 1000 ure) en 52 beserings tydens oefeninge (1,58 per uur 1000) wat statisties betekenisvol (p = 0,039) was. Die aantal nuwe beserings was baie hoër as die herhalende voorkoms van ‘n besering. Die totale aantal nuwe beserings was 120 (4,1 per 1000 ure) met slegs 52 (2,0 per 1000 ure) herhalendes. Dit was die onderste ledemaat wat in die meerderheid beseringsgevalle (97.5%) geraak is. Oor die hele spektrum was die enkel en die voet die mees beseerde area (15,9%), met die kop (15,3%), skouer (11,4%), dyspier (10,2%), knie (10,2%) en kwadriseps (9,7%) wat soortgelyk geraak is. Areas vir individuelebesering het gewissel tussen voor-en agterspelers. Die voorspelers se mees beseerde anatomiese area was die skouer en enkel (0,5 per 1000 ure). Die dyspier (0,4), enkel-, kop (0,3) en knie (0,2) was die mees beseerde area onder die agterspelers. Die takel was verantwoordelik vir die meerderheid van die beserings (48.1%) onder die voor- en agterspelers. Die mees algemene vorme van beserings was verstuitings en spierverrekking onder die voor- en agterspelers. Die voorspelers het ‘n hoër voorkoms van kontusie en harsingskudding (0,3) in vergelyking met die agterspelers. Die agterlyn dui ‘n algehele hoër tendinopatie (0,2) aan. Onder die voorspelers het die slotte (15,2%) en die stutte (12,9%) die hoogste aantal beserings aangeteken en onder die agterspelers was die vleuels (8,4%) en die senters (9%) die mees beseerde spelers. Die meerderheid van die beserings het plaasgevind gedurende die laaste deel van die eerste helfte (26,7%) en die laaste deel van die tweede helfte (30,7%). GEVOLGTREKKING: Die prevalensie en insidensie van wedstrydbeserings was aansienlik hoër as tydens oefentye. Soortgelyk aan ander beseringsopname studies, was die takel die mees gevaarlike fase van die spel. Die voorspelers wat meer betrokke is in die fisiese kant van die spel het meer beserings in vergelyking met die agterspelers aangeteken 71. Die agterspelers, as gevolg van hul styl van die spel het meer hardloop en versnel beserings aangeteken5,24. Moegheid en 'n gebrek aan fisiese kondisionering kan 'n faktor wees in die meerderheid van hierdie beserings wat gedurende die laaste deel van elke wedstrydshelfte aangeteken word. Verder, die area, tipe en meganisme van beserings wissel oor individuele spelerposisies 24,26. Dit dui daarop aan dat verskillende oefenstyle vir die verskillende posisies aanbeveel moet word as 'n strategie vir besering voorkoming.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The cardiac isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid (FA) uptake; thus increased ACCβ activity decreases fatty acid utilization thereby potentially leading to intracellular myocardial lipid accumulation and insulin resistance (IR). Previous studies show that greater flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of IR. In light of this, we hypothesize that increased HBP flux induces ACCβ gene expression thereby contributing to the onset of IR. Our initial work focused on ACCβ gene promoter regulation and suggest that the HBP modulates upstream stimulatory factor 2 (USF2) thereby inducing ACCβ gene expression. Here, we further investigated HBP-mediated regulation of ACCβ gene expression by transiently transfecting cardiac-derived H9c2 cells with an expression vector encoding the rate-limiting HBP enzyme (GFAT) ± the full length ACCβ and 4 truncated promoter-luciferase constructs, respectively. GFAT overexpression increased ACCβ gene promoter activity for the full length and 3 larger deletion constructs (p<0.001 vs. controls). However, GFAT-mediated and USF2-mediated ACCβ promoter induction was blunted when co-transfected with the -38/+65 deletion construct suggesting that USF2 binds to the proximal promoter region (near start codon). Further investigation proves that USF2 binds to ACCβ promoter and activates it, but that USF2 is not O-GlcNAc modified even though there is a strong correlation between increased O-GlcNac levels and USF2 activation of ACCβ. This would suggest that there is another O-GlcNac modified factor involved in this regulatory pathway. Our study demonstrates that increased HBP flux induces ACCβ gene promoter activity via HBP modulation of USF2. We propose that ACCβ induction reduces fatty acid oxidation, thereby leading to intracellular lipid accumulation (FA uptake>>FA oxidation) and the onset of cardiac IR. / AFRIKAANSE OPSOMMING: Die kardiale isoform van asetiel-CoA karboksilase (ACCβ) produseer maloniel-CoA, ‘n kragtige inhibeerder van mitochondriale vetsuur (VS) opname, en om hierdie rede sal verhoogde ACCβ aktiwiteit, vetsuur gebruik verlaag en potensieël aanleiding gee tot intrasellulêre miokardiale lipiedophoping en insulienweerstand (IW). Vorige studies toon dat groter fluks deur die heksosamienbiosintetiese weg (HBW) bydra tot die ontwikkeling van IW. In die lig hiervan hipotetiseer ons dat verhoogde HBW fluks, ACCβ geenuitdrukking induseer, en sodoende tot die onstaan van IW bydra. Ons aanvanglike werk het op ACCβ geenpromotorregulering gefokus, en voorgestel dat die HBW die opstroom stimuleringsfaktor 2 (USF2) moduleer en dus ACCβ geen uitdrukking induseer. Hier het ons verder die HBW-gemedieërde regulering van ACCβ-geenuitdrukking deur kortstondige tranfeksie van kardiaalverkrygde H9c2 selle met ‘n uitdrukkingsvektor wat kodeer vir die tempo-bepalende HBW ensiem (GFAT) ± die volle lengte ACCβ, en vier afgestompte promotor-lusiferase konstrukte onderskeidelik, te ondersoek. GFAT ooruidrukking het ACCβ geenpromotor aktiwiteit vir die volle lengte, en drie groter uitwissingskonstrukte verhoog (p<0.001 vs. kontrole). Hoewel GFAT- en USF2-gemedieërde ACCβ promotorinduksie tydens ko-transfeksie van die -38/+65 uitwissingskonstruk versag was, is dit voorgestel dat USF2 aan die proksimale promotor area (naby die beginkodon) bind. Verdere ondersoek bewys ook dat USF2 aan die ACCβ promotor bind en dit aktiveer, maar dat USF2 nie O-GlcNAc gemodifiseer word nie ten spyte van ‘n sterk korrelasie tussen verhoogde O-GlcNac vlakke en USF2 aktivering van ACCβ. Dit kan dus voogestel word dat daar ‘n alternatiewe O-GlcNac gemodifiseerde faktor betrokke is in hierdie reguleringsweg. Ons studie demonstreer dat verhoogde HBW fluks ACCβ geenpromotor aktiwiteit via HBW modulering van USF2 veroorsaak. Ons stel voor dat ACCβ induksie vetsuuroksidasie verlaag en so tot intrasellulêre lipiedophoping (VS opname >> VS oksidasie) en die onstaan van kardiale IW lei.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction Cisplatin has been widely used to treat solid tumours and much success has come from the use of this drug in the treatment of head and neck, ovarian, testicular, cervical and small-cell lung cancers. However, the success of cisplatin treatment is limited due to its dose-limiting toxicity and its resulting side-effects, such as nephro- and ototoxicity. The devastating side-effects induced by cisplatin treatment provided the platform for this study whereby the aim was to lower the concentration of cisplatin while maintaining its cancer-specific cytotoxic action. Equally concerning is, cisplatin resistance which is becoming increasingly common, and this radically limits the clinical efficacy and utility of the drug. Adjuvant therapy has thus become necessary in an attempt to possibly curb or lessen the extent of cisplatin resistance. Due to the large body of evidence implicating the importance of autophagy in cancer, the prospect of targeting this mechanism has generally been accepted. Various chemotherapy agents induce autophagy in cancer cells; however the effect of cisplatin on autophagic induction has not been very well explored. We thus hypothesise that the manipulation of the autophagic pathway will sensitise cancer cells to a low concentration of cisplatin treatment. Furthermore, due to the functional interaction between Bcl-2 and Beclin-1 and its role in the regulation of autophagy, ratio analysis of Beclin-1 to Bcl-2 as means of detecting the role of autophagy within the cell under homeostatic and treatment/stress conditions has been conducted. Additionally, Bcl-2 has a prominent role in the malignant cell and it’s over-expression has been found to confer resistance in a variety of cancerous cell lines. We therefore hypothesise that the silencing of Bcl-2 prior to cisplatin treatment will sensitise cervical cancer cells to apoptosis and increase the Beclin-1/Bcl-2 ratio in favour of apoptosis. Materials and Methods Three human cervical cell lines were used: a non-cancerous ectocervical epithelial cell line (Ect1/E6E7) and two cancerous cervical cell lines (HeLa and CaSki). In order to determine a concentration of cisplatin that was non-toxic to the non-cancerous Ect1/E6E7 cell line, a dose-response was performed. With the use of an autophagy inhibitor (bafilomycin A1) and an autophagy inducer (rapamycin), autophagic flux capacities were assessed in each cell line through the Western blotting technique. In order to assess whether the chosen concentration of cisplatin induced autophagy, flow cytometry with the use of a Lysotracker™ dye was utilised, as well as analysis of autophagy protein levels (LC-3 II, Beclin-1 and p62). Autophagy modulation was achieved through two methods: pharmacological modulation with use of two recognised agents, namely bafilomycin A1 and rapamycin, and biological manipulation with the use of ATG5 and mTOR mRNA silencing. The effects of different treatment regimes on cell death was assessed with the use of PARP and caspase-3 cleavage through Western blotting, caspase-3/-7 activity (Caspase-Glo®), PI inclusion, LDH release and MTT reductive capacity. Additionally the effects of these treatment regimes on cell-cycle progression were also analysed. Beclin-1 and Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin in HeLa and CaSki cells. To assess the reliance of the cervical cancer cells on Bcl-2 after cisplatin treatment, Bcl-2 knock-down was achieved through RNA interference, where after the Beclin-1/Bcl-2 ratio was assessed as well as apoptosis with the use of cleaved PARP analysis (Western blotting) and Caspase-Glo©. For the ex vivo analysis, biopsies were collected from patients undergoing routine colposcopy screenings and hysterectomies at Tygerberg Hospital, Tygerberg, Western Cape. A total of 10 normal, 29 low-grade squamous intraepithelial lesions (LSIL), 33 high-grade squamous intraepithelial lesions (HSIL) and 13 carcinoma biopsies were collected for analysis, where after the expression profiles of two autophagy markers (mTOR and LC-3 II), as well as one anti-apoptotic marker (Bcl-2) were assessed. Protein levels were analysed through Western blot and confirmed through immunohistochemistry. Results Dose-response curves revealed that 15 μM of cisplatin did not induce cell death in the normal cervical epithelial cell line (Ect1/E6E7) and was therefore utilised through-out the remainder of the study. It was additionally determined that the CaSki cells were more resistant to cisplatin treatment when compared to the HeLa and Ect1/E6E7 cells. Autophagic flux analysis revealed that, although all three cell lines were cervix derived, their autophagic flux capacities differed. It was observed that the chosen concentration of cisplatin was able to induce autophagy in all three cell lines, with the HeLa cells demonstrating a particularly pronounced response. Autophagy modulation in conjunction with cisplatin treatment revealed the following: Autophagy inhibition with bafilomycin A1 lead to significant increases in caspase-3 and PARP cleavage and LDH release in both cervical cancer cell lines. The inhibition of autophagy through silencing of ATG5 induced caspase-3 cleavage and agrees with results obtained from pharmacological inhibition of autophagy with bafilomycin A1. In addition to autophagic induction, a low concentration of cisplatin induced the up-regulation of Bcl-2, which when silenced significantly improved cisplatin-induced apoptosis in both cervical cancer cell lines. Analysis of the expression profiles of mTOR and LC-3 in normal, pre-malignant (LSIL and HSIL) and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma of the cervix. Additionally, Bcl-2 expression is significantly increased in cervical carcinoma tissue, which agrees with results from other studies. Conclusion Autophagic flux capacities between the three cell lines investigated, derived from the same organ, differ significantly. This should be taken into consideration when autophagic modulation is being used as an adjuvant treatment. With regard to chemotherapy treatment in cervical cells, a low-concentration of cisplatin significantly induces autophagy in malignant and non-malignant cervix-derived cell lines where it serves a pro-survival mechanism. Inhibition of autophagy with bafilomycin A1 and ATG5 siRNA confirmed this survival effect in both cancerous cell lines where apoptosis was significantly increased. Interestingly, rapamycin pre-treatment together with cisplatin did not induce significant levels of apoptosis in HeLa cells where autophagy induction may have provided additional protection from the cytotoxic effects of cisplatin. Therefore the inhibition of autophagy through pharmacological and biological inhibition improves the cytotoxicity of a low concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer. Bcl-2 up-regulation in response to cisplatin treatment also serves as a protective mechanism by which cervical cancer cells survive. The extent of apoptotic cell death observed after biological inhibition of Bcl-2 reiterates the fact that this response may be exploited in order to favour the use of lower concentrations of cisplatin. Analysis of clinical specimens emphasised the value of the in vitro work: Cervical cancer biopsies had increased expression of both LC-3 II and Bcl-2, indicating autophagy induction and apoptosis inhibition, respectively. Thus two novel methods of improving cisplatin cytotoxicity have been demonstrated in the following study. Treatment regimens may administer more frequently and prolonged due to the minimal side-effects that accompanies low-dose cisplatin treatment. / AFRIKAANSE OPSOMMING: Inleiding Sisplatien word algemeen gebruik vir die behandeling van soliede gewasse. Baie sukses is reeds deur die gebruik van díe middel behaal in die behandeling van kop en nek, ovariale, terstikulêre, servikale en klein-sel kankers. Die sukses van Sisplatien-behandeling word wel ingeperk deur die dosis-beperkende toksisiteit en die gevolglike newe-effekte soos nefrotoksisiteit. Hierdie verwoestende newe-effekte wat deur sisplatien behandelings geïnduseer word, het as die platform vir hierdie studie gedien. Die doel was om die sisplatien konsentrasies te verlaag, maar terselfdertyd die kankerspesifieke sitotoksisiteit te behou. Nog ʼn punt van kommer is dat sisplatien-weerstandigheid aan die toeneem is, wat die kliniese effektiwiteit en gebruik van hierdie middel geweldig beperk. Byvoegmiddels het dus noodsaaklik geraak in die poging om die sisplatien-weerstandigheid te verhoed. As gevolg van verskeie bewyse wat die belangrikheid van outofagie in kanker impliseer, is die vooruitsig om hierdie meganisme te teiken, algemeen aanvaar. Verskeie chemoterapeutiese middels induseer outofagie in kanker selle, hoewel die effek van Sisplatien op outofagiese induksie nog nie goed ondersoek is nie. Ons hipotese is dus dat die manipulasie van die outofagiese pad die kankerselle sensitiseer tot ʼn lae konsentrasie van sisplatien. Verder, as gevolg van die funksionele interaksie tussen Bcl-2 en Beclin-1, en hul rol in die regulering van outofagie, is verhouding-analises van Beclin-1 tot Bcl-2 uitgevoer met die doel om die rol van outofagie in die sel onder homeostatiese en behandeling/stres kondisies te bepaal. Verder is Bcl-2 bekend daarvoor om ʼn prominente rol te speel in kwaadaardige selle, en die ooruitdrukking daarvan is gevind om weerstandigheid aan te help in ʼn verskeidenheid van kankeragtige sellyne. Ons hipotetiseer dus dat geenonderdrukking van Bcl-2 voor die behandeling met sisplatien die servikale kanker selle sal sensitiseer tot apoptose en ʼn verhoging in die verhouding van Beclin-1/Bcl-2 veroorsaak, wat in die guns van apoptose is. Materiale en Metodes Drie menslike servikale sellyne was gebruik: ʼn nie-kankeragtige servikale epiteel sellyn (Ect/E6E7) en twee kankeragtige servikale sellyne (HeLa en CaSki). Om ʼn konsentrasie van sisplatien te bepaal wat nie-toksies tot die nie-kankeragtige Ect1/E6E7 sellyn is, was ʼn dosisrespons uitgevoer. Met die gebruik van ʼn outofagiese inhibeerder (bafilomycin A1) en ʼn outofagiese induseerder (rapamycin), is die outofagiese-fluks kapasiteite van elke sellyn deur die Western Blotting tegniek geassesseer. Om te bepaal of die gekose konsentrasie van sisplatien outofagie induseer, is vloeisitometrie met ʼn Lysotracker™ kleurstof gebruik, sowel as analises op outofagie proteïenvlakke (LC-3 II, Beclin-1 en p62). Outofagie modulering is behaal deur twee metodes: farmakologiese modulering met twee erkende middels, naamlik bafilomycin A1 en rapamycin, en biologiese manipulasie met die gebruik van ATG5 en mTOR geenonderdrukking. Die effekte van die verskillende behandeling skedules op seldood was geassesseer deur gebruik te maak van PARP en kaspase-3 splitsing deur Western Blotting, kaspase-3/-7 aktiwiteit deur Caspase-Glo ®, PI-insluiting, LDH vrystelling en MTT reduserende kapasiteit. Verder is die effekte van hierdie behandeling skedules op selsiklus progressie ook geanaliseer. Beclin-1 en Bcl-2 uitdrukking was ook bepaal deur Western Blotting en immunohistochemie voor en na behandeling met sisplatien in HeLa en CaSki selle. Om die afhanklikheid van die servikale kankerselle op Bcl-2 na sisplatien behandelings te toets, is Bcl-2 onderdruk deur RNA-inmenging, waarna Beclin-1/Bcl-2 verhouding geassesseer is, sowel as opoptose deur die gebruik van gesplitste PARP analises (Western Blotting) en Caspase-Glo©. Vir die ex vivo analises is biopsies vanaf pasiënte wat roetine kolposkopie en histerektomies ondergaan, verkry (Tygerberg Hospitaal, Tygerberg, Westelike Provinsie). ʼn Totaal van 10 normale, 29 lae-graad plaveisel intraepiteel letsels (LSIL), 33 hoe-graad plaveisel intraepiteel letsels (HSIL) en 13 karsinoom biopsies is verkry vir analises. Die uitdrukkingsprofiel van twee outofagiese merkers (mTOR en LC-3 II), asook een merker vir apoptose (Bcl-2), was geassesseer. Proteïen vlakke was ook deur Western Blotting geanaliseer en deur immunohistochemie bevestig. Resultate Dosisrespons kurwes het getoon dat 15 μM sisplatien nie seldood in die normale sellyn (Ect1/E6E7) geïnduseer het nie, en was daarom gebruik deur die res van hierdie studie. Verder is daar ook gevind dat CaSki selle meer weerstandig tot sisplatien behandelings is wanneer vergelyk word met die HeLa en Ect1/E6E7 selle. Outofagiese-fluks analises het getoon dat, alhoewel al drie sellyne vanaf die serviks afkomstig is, daar verskille is in hul outofagiese-fluks kapasiteit. Daar is ook waargeneem dat die gekose konsentrasie van sisplatien in staat was om outofagie te induseer in al drie sellyne, met HeLa selle wat die mees merkbare respons getoon het. Modulering van outofagie in samewerking met sisplatien behandelings het die volgende onthul: inhibisie van outofagie deur bafilomycin A1 het gelei tot ʼn beduidende verhoging in kaspase-3, PARP splitsing en LDH vrylating in beide servikale kankersellyne. Geenonderdrukking van ATG5 induseer kaspase-3 splitsing en stem ooreen met resultate wat verkry is deur farmakologiese inhibisie van outofagie met bafilomycin A1. Bykomend tot outofagiese indusering, het ʼn lae konsentrasie sisplatien die opregulering van Bcl-2 geïnduseer. Wanneer Bcl-2 geenonderdrukking in hierdie scenario toegepas was, het dit ʼn beduidende verbetering in sisplatien-geïnduseerde apoptose in beide servikale kankersellyne getoon. Analises van die uitdrukkingsprofiel van mTOR en LC-3 in normale, pre-maligne (LSIL en HSIL) en kankeragtige servikale weefsel, het getoon dat outofagie beduidend opgereguleer is in HSILs en servikale karsinome. Verder is Bcl-2 uitdrukking ook gevind om beduidend verhoog te wees in servikale karsinoomweefsel, wat ooreenstem met resultate verkry in ander studies. Gevolgtrekking Outofagiese-fluks kapasiteite tussen die drie sellyne, afkomstig van dieselfde orgaan, toon beduidende verskille. Hierdie bevinding moet in ag geneem word wanneer outofagiese-modulering as ʼn bevorderingsbehandeling gebruik word. Met betrekking tot chemoterapie behandeling in servikale selle; ʼn lae konsentrasie van sisplatien veroorsaak ʼn beduidende indusering van outofagie in kwaadaardige en nie-kwaadaardige serviks-afkomstige sellyne, en dien as ʼn oorlewingsmeganisme. Inhibisie van outofagie met bafilomycin A1 en ATG5 siRNA het hierdie beskermings effek bevestig, aangesien apoptose beduidend verhoog was in beide kankersellyne. Interessant genoeg het rapamycin pre-behandeling tesame met sisplatien nie beduidende vlakke van apoptose in HeLa selle geïnduseer nie. Outofagie induksie mag dalk addisionele beskerming teen die sitotoksiese effekte van sisplatien gebied het. Daarom het die inhibisie van outofagie deur farmakologiese en biologiese inhibering die sitotoksisiteit van ʼn lae konsentrasie sisplatien bevorder, wat ʼn belowende bevinding is vir die toekomstige behandeling van servikale kanker. Bcl-2 opregulering as gevolg van sisplatien behandelings dien ook as beskermings meganisme waarby servikale kankerselle oorleef. Die mate van apoptotiese seldood wat waargeneem word na biologiese inhibering van Bcl-2, wys weer op die feit dat hierdie respons uitgebuit kan word vir die gebruik van laer konsentrasies van sisplatien. Analises van die kliniese monsters het ook die waarde van die in vitro werk versterk: Servikale kanker biopsies het verhoogde uitdrukking van beide LC-3 II en Bcl-2 getoon, wat aandui dat outofagie geïnduseer en apoptose geïnhibeer word. Daar is dus twee nuwe metodes vir die verbetering van sisplatien-toksisiteit in hierdie studie gedemonstreer. Behandeling regimes kan meer gereeld en vir langer tydperke toegepas word, aangesien die newe-effekte van lae-dosis sisplatien behandelings minimaal is. / MRC for funding

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background - Although acute hyperglycemic episodes are linked to lower glucose uptake, underlying mechanisms driving this process remain unclear. We hypothesized that acute hyperglycemia triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP) and protein kinase C (PKC) activation. These mechanisms attenuate cellular function, and may indeed decrease insulin-mediated cardiac glucose uptake. The role of the pentose phosphate pathway (PPP) under high glucose/diabetic conditions is a subject of contention. Activation of the PPP enzyme transketolase (TK) (by benfotiamine/BFT or thiamine) reduces flux via the other four NOGPs, and is associated with beneficial outcomes. Our aim was therefore to evaluate the effects of acute hyperglycemia on insulin-mediated glucose uptake in a cardiac-derived cell line. Specifically, we aimed to elucidate the role of ROS and NOGP induction under these conditions. Methodology - H9c2 cardiomyoblasts were exposed to 25 mM glucose for 24 hr vs. 5.5 mM glucose controls ± modulating agents during last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250 μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100 μM DPI), c) NOGP inhibitors – 100 μM aminoguanidine (AGE), 5 μM chelerythrine (PKC); 40 μM DON (HBP); and 10 μM zopolrestat (PP). We also employed BFT (50 and 100 μM) in vitro, while the effects of in vivo thiamine administration were assessed in hearts of an obese/diabetic rat model of pre-diabetes and diabetes, the OLETF strain. We evaluated insulin sensitivity by glucose uptake assay (flow cytometry), GLUT4 translocation (transfection of HA-GLUT4-GFP construct) and protein kinase B (Akt) activity assay. ROS levels (mitochondrial, intracellular) were measured by flow cytometry analysis of specific fluorescent probes. Markers of each NOGP were also assessed. Results - Acute hyperglycemia elevated ROS, activated NOGPs and blunted glucose uptake. However, TK activity (marker of PPP) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed BFT (known to shunt flux away from NOGPs and into the PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under acute hyperglycemic conditions. In vivo thiamine administration reduced markers of the other NOGP, while it attenuated (mainly in the pre-diabetic phase) the metabolic dysfunction observed in the OLETF rats. Conclusions - This study demonstrates that acute hyperglycemia elicits a series of maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance. / AFRIKAANSE OPSOMMING: Agtergrond – Alhoewel akute hiperglisemie voorvalle gekoppel is aan verlaagde glukose opname, is die onderliggende meganismes wat die proses dryf steeds onduidelik. Ons hipotetiseer dat akute hiperglisemie aanleiding gee tot die produksie van reaktiewe suurstofspesies (RSS) en toename in nie-oksidatiewe glukose weg (NOGW) aktivering, i.e. stimulering van gevorderde glukasie eindprodukte (GGE), poliolweg (PW), heksosamien biosintetiese weg (HBW) en proteïenkinase C (PKC) aktivering. Hierdie meganismes verminder sellulêre funksie, en mag inderdaad insulien-bemiddelde kardiêre glukose opname verlaag. Die rol van die pentosefosfaatweg (PFW) onder hoë glukose/diabetiese kondisies is ‘n onderwerp van stryd. Aktivering van die PFW ensiem transketolase (TK) (deur benfotiamien/BFT of tiamien) verminder fluks deur die ander vier NOGWë, en is geassosieer met voordelige uitkomste. Ons doel was dus om die effekte van akute hiperglisemie op insulien-bemiddelde glukose opname te evalueer in ‘n kardiaal-afkomstige sellyn. Meer bepaald het ons gepoog om die rol van RSS en NOGW induksie onder hierdie kondisies te verstaan. Metode – H9c2 kardiomioblaste is aan 25 mM glukose vir 24 h blootgestel vs. 5.5 mM glukose kontroles ± moduleeragente tydens die laaste uur van glukose blootstelling: a) anti-oksidant #1 vir mitochondriese RSS (250 μM 4-OHCA), b) anti-oksidant #2 vir NADPH oksidase-gegenereerde RSS (100 μM DPI), c) NOGW inhibeerders – 100 μM aminoguanidien (GGE), 5 μM cheleritrien (PKC); 40 μM DON (HBW); en 10 μM zopolrestaat (PW). Ons het ook BFT (50 en 100 μM) in vitro aangewend, terwyl die effek van in vivo tiamien aanwending geassesseer is in die harte van ‘n vetsugtige/diabetiese rotmodel van pre-diabetes en diabetes, die OLETF lyn. Ons het insuliensensitiwiteit deur ‘n glukose opname toets (vloeisitometrie), GLUT4 translokasie (transfeksie van HA-GLUT4-GFP konstruk) en proteïenkinase B (Akt) aktiwiteitstoets, geëvalueer. RSS vlakke (mitochondries, intrasellulêr) is gemeet deur vloeisitometriese analise van spesifieke fluoresserende peilers. Merkers van elke NOGW is ook geassesseer. Resultate - Akute hiperglisemie het RSS verhoog, NOGWë geaktiveer en glukose opname versag. TK aktiwiteit (merker van PFW) het egter nie verander nie. Onderskeidelike 4-OHCA en DPI behandeling het RSS produksie versag, NOGW aktivering verminder en glukose opname genormaliseer. NOGW onderdrukking studies het PKCβII geïdentifiseer as ‘n sleutel deelnemer in verlaging van insulien-bemiddelde glukose opname. Die aanwending van BFT (bekend vir die wegvoer van fluks vanaf NOGWë na die PFW), het RSS skepping en NOGW aktivering verlaag, en glukose opname herwin onder akute hiperglisemiese kondisies. In vivo tiamien toediening het merkers van die ander NOGW verlaag, terwyl dit die metaboliese disfunksie waargeneem in die OLETF rotte (hoofsaaklik in die pre-diabetiese fase) verminder het. Gevolgtrekking – Hierdie studie demonstreer dat akute hiperglisemie ‘n reeks van wanaangepaste voorvalle ontlok wat gesamentlik funksioneer om glukose opname te verlaag, en dat anti-oksidant behandeling en/of vermindering van NOGW aktivering (PKC, poliolweg), die aanvang van insulien weerstand mag beperk.

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.