Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Protein kinases and phosphatases play important roles in the phosphorylation state of intracellular proteins under both physiologic and pathophysiologic conditions. Compared to the large number of studies investigating the significance of kinases, in particular the mitogen-activated protein kinases (MAPKs) in myocardial ischaemia/reperfusion and ischaemic preconditioning, relatively few studies have been done on the protein phosphatases in this scenario. Although several role players in the signal transduction cascade of ischaemia/reperfusion and ischaemic preconditioning have been identified thus far, the exact mechanism of cardioprotection still remains unclear. Previous studies from our laboratory have shown that the stress kinase, p38 MAPK, has a dual role in preconditioning: it acts as trigger of the process, while attenuation of its activation during sustained ischaemia and reperfusion is required for cardioprotection. Since the activation of p38 MAPK is dependent on both the upstream kinases for phosphorylation and phosphatases for dephosphorylation, we hypothesized that the balance between the activation state of the MAPKs and the induction of phosphatases may play a major role in determining the fate of cardiomyocytes exposed to ischaemic stress. The objectives of this study were: (i) to assess the activity of the myocardial protein phosphatases (PSPs and PP1) during sustained ischaemia and during reperfusion of non-preconditioned and ischaemic preconditioned hearts; (ii) to evaluate the significance of these phosphatases in ischaemia/reperfusion as well as in ischaemic preconditioning using available appropriate inhibitors; (iii) to give particular attention to the role of the phosphatase, mitogen-activated protein kinase phosphatase-1 (MKP-1), in ischaemia/reperfusion. MKP-1 is upregulated by stress conditions and selectively inactivates p38 MAPK by dephosphorylation of the regulatory Thr and Tyr residues. The glucocorticoid, dexamethasone which increases MKP-1 expression, was used as agonist to upregulate MKP-1 experimentally. The isolated perfused working rat heart was used as experimental model. After stabilization, hearts were subjected to either a one-cycle or multi-cycle ischaemic preconditioning protocol, followed by sustained global or regional ischaemia and reperfusion. Non-preconditioned hearts were subjected to ischaemia/reperfusion only. For Western blot analysis of MAPKs, PKB/Akt and MKP-1, hearts were freeze-clamped at different times during the perfusion protocol. Endpoints were infarct size, functional recovery and phosphorylation of the MAPKs (ERK and p38 MAPK) and PKB/Akt during reperfusion. Expression of MKP-1 was monitored. The results obtained showed that activation of PSPs and PP1 does not occur during sustained global ischaemia or reperfusion of non-preconditioned and preconditioned hearts. The role of the phosphatases was subsequently further investigated using two inhibitors namely cantharidin (5 μM, a concentration which inhibits both PP1 and PP2A) and okadaic acid (7.5 nM, a concentration which inhibits PP2A selectively). Administration of cantharidin or okadaic acid during the preconditioning phase, completely abolished preconditioning induced cardioprotection as indicated by mechanical failure during reperfusion and increased infarct size, associated with increased phosphorylation of p38 MAPK and PKB/Akt and dephosphorylation of ERK42/44. These results suggest a role for PP2A in the trigger phase of preconditioning. Administration of cantharidin or okadaic acid during early reperfusion of preconditioned hearts improved functional recovery. This was associated with increased phosphorylation of ERK42/44 and PKB, but not p38 MAPK. Dexamethasone, administered intraperitoneally to rats for 10 days (3mg/kg/day) or directly added to the perfusate (1 μM) resulted in significant cardioprotection of hearts subjected to 20 min sustained global ischaemia, followed by 30 min reperfusion. This is associated with a marked upregulation of MKP-1 and dephosphorylation of p38 MAPK during reperfusion. These studies suggest that the phosphatases are definitely involved in the phenomenon of ischaemia/reperfusion and ischaemic preconditioning. However, it also become clear that extensive further research is required to fully elucidate which phosphatases are involved and the mechanisms thereof. Due to the large size of the protein phosphatase family, this may prove to be a formidable task and far beyond the scope of this thesis. The results also suggested that pharmacological targetting of phosphatases involved in phosphorylation of the reperfusion injury salvage kinase (RISK) pathway (e.g. ERK42/44 and PKB/Akt) or dephosphorylation of pro-apoptotic kinases, such as p38 MAPK, may have significant clinical potential. / AFRIKAANSE OPSOMMING: Proteïenkinases en fosfatases speel 'n belangrike rol in die fosforileringstatus van intrasellulêre proteïene in beide fisiologiese en patofisiologiese toestande. In teenstelling met die groot aantal studies gedoen ten einde die rol van die kinases, veral die mitogeen-geaktiveerde proteïenkinases (MAPKs), in iskemie/herperfusie en iskemiese prekondisionering te ondersoek, is relatief min bekend aangaande die rol van die fosfatases in hierdie scenario. Hoewel verskeie rolspelers in die seintransduksieprosesse van iskemie/herperfusie en iskemiese prekondisionering reeds geïdentifiseer is, is die presiese meganisme van miokardiale beskerming steeds onbekend. Vroeëre studies vanuit ons laboratorium het getoon dat die streskinase, p38 MAPK, 'n tweeledige rol in prekondisionering speel: dit is 'n sneller ("trigger") van die proses, terwyl verlaagde aktivering tydens volgehoue iskemie en herperfusie, noodsaaklik vir beskerming is. Ons hipotese is dus dat die balans tussen die aktiveringstatus van die MAPKs en induksie van fosfatases die oorlewing van kardiomiosiete blootgestel aan iskemiese stres, bepaal. Die doelwitte van hierdie studie was: (1) bepaling van die aktiwiteit van miokardiale proteïen fosfatases (PSPs en PP1) tydens volgehoue iskemie en herperfusie van nie-geprekondisioneerde en iskemies-geprekondisioneerde harte; (ii) evaluering van die belang van fosfatases in iskemie/herperfusie beskadiging sowel as in iskemiese prekondisionering deur van geskikte inhibitore gebruik te maak; (iii) ondersoek na die rol van die fosfatase, mitogeen-geaktiveerde proteïen kinase fosfatase-1 (MPK-1) in iskemie/herperfusie beskadiging. Dit is bekend dat MKP-1 deur strestoestande opgereguleer word en p38 MAPK selektief deur defosforilasie van die regulatoriese Thr en Tyr residue inaktiveer word. Die glukokortikoïed, deksametasoon, wat MKP-1 uitdrukking stimuleer, is as agonis gebruik ten einde MKP-1 eksperimenteel op te reguleer. Die geïsoleerde, geperfuseerde werkende rothart is as eksperimentele model gebruik. Na stabilisasie, is die harte aan 'n enkel- of veelvuldige siklus iskemiese prekondisioneringsprotokol onderwerp, gevolg deur volgehoue globale of streeksiskemie. Nie-geprekondisioneerde harte is slegs aan iskemie/herperfusie onderwerp. Harte is op verskillende tye tydens die perfusieprotokol gevriesklamp vir Western blot analise van die MAPKs, PKB/Akt en MKP-1. Infarktgrootte en funksionele herstel tydens herperfusie is as indikators van iskemiese beskadiging gebruik. Fosforilasie van MAPKs en PKB/Akt sowel as uitdrukking van MKP-1 tydens vroeë herperfusie is gemonitor. Die resultate toon dat aktivering van PSP en PP1 tydens volgehoue iskemie en herperfusie nie plaasvind nie. Die rol van die fosfatases is verder ondersoek deur van twee inhibitore gebruik te maak, naamlik cantharidin (5 μM inhibeer beide PP1 en PP2A) en okadaic suur (7.5 nM inhibeer PP2A selektief). Toediening van of cantharidin of okadaic suur tydens die prekondisioneringsprotokol, hef prekondisionering-geïnduseerde beskerming totaal op, soos aangetoon deur hartversaking tydens herperfusie en 'n toename in infarktgrootte, tesame met 'n toename in die fosforilering van p38 MAPK en PKB/Akt en defosforilering van ERK42/44. Hierdie waarnemings dui op 'n rol vir PP2A as sneller in prekondisionering. Toediening van hierdie inhibitore tydens vroeë herperfusie het ook die miokardium beskerm, soos aangetoon deur 'n verbeterde meganiese herstel van geprekondisioneerde harte, tesame met ‘n verhoogde fosforilering van ERK42/44 en PKB (maar nie p38 MAPK nie). Deksametasoon, intraperitoneaal toegedien, vir 10 dae (3mg/kg/dag) of direk by die perfusaat gevoeg (1μM), het tot 'n hoogs beduidende beskerming teen iskemiese beskadiging gelei van harte blootgestel aan 20 min globale iskemie en 30 min herperfusie. Hierdie toename in funksionele herstel en afname in infarktgrootte het met 'n toename in MKP-1 uitdrukking en defosforilasie van p38 MAPK gepaard gegaan. Bogenoemde resultate dui op 'n definitiewe betrokkenheid van fosfatases in iskemie/herperfusie en iskemiese prekondisionering. Dit is egter ook duidelik dat intensiewe verdere navorsing benodig word om die presiese rol van die fosfatases te bepaal. Vanweë die grootte van die fosfatase familie, val dit egter buite die beskek van hierdie studie. Ten slotte, die resultate toon dat farmakologiese manipulasie van fosfatases betrokke by die fosforileringstatus van anti-apoptotiese kinases soos ERK42/44 en PKB/Akt en defosforilasie van pro-apoptotiese kinases, soos p38 MAPK, besondere kliniese toepassings mag hê.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2007. / For spermatozoa to gain access to the oocyte for fertilization, lytic enzymes need to be released during the acrosome reaction. These enzymes, which are stored and transported within an organelle termed the acrosome, make it possible for spermatozoa to collectively penetrate the layers of cells and glycoproteins that surround and protect an oocyte. Acrosomes may thus be viewed as essential for fertilization and their shape, size and volume were examined morphometrically by utilizing automated morphometric analysis equipment. In addition to the acrosome being necessary for normal unassisted fertilization, spermatozoa also need the ability to migrate to the oocyte. Following zona pellucida binding, sperm tail thrust movement initiates zona penetration into the space created by the digestive action of the acrosomal enzymes. Therefore the motion characteristics of spermatozoa were also quantified in terms of kinematic properties. In the treatment of male sub fertility, assisted reproductive techniques are applied. In the application of such techniques, a motile sub-population of spermatozoa was obtained by employing a procedure (swim-up selection) that selects cells on the basis of their kinematic ability. This study presents an analysis of the morphometric and kinematic qualities of spermatozoa populations that are subjected to swim-up selection and investigates the relationship of these morphometrical and kinematic qualities. Computer-assisted semen analysis, swim-up selection and automated sperm morphology analysis tests were all used to evaluate spermatozoa populations. Results indicated that, irrespective of acrosome size, higher kinematic parameter measurements were observed post-swim-up. A significant inverse relationship between the population’s average acrosome size and a number of kinematic parameters was observed. Our results indicated that for a post-swim-up population of spermatozoa an increase in the average acrosome size was significantly related to a decrease in the kinematic parameters VAP, VCL and the VSL within the same population.

Thesis (MSCMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2005. / There is an urgent need for a safe, effective, affordable human immunodeficiency virus type 1 (HIV-1) vaccine that induces both cellular and humoral immunity. A popular strategy for vaccine design is the use of plasmid DNA encoding HIV-1 genes for priming vaccinations followed by either viral vector or recombinant protein boosting. DNA-based vaccines are attractive because they are safe, easily administered and can induce both cellular and humoral immune responses. In order for DNA vaccination to induce a potent immune response it is necessary for plasmid-encoded genes to be targeted to dendritic cells (DCs) as these are the key antigen presenting cells in natural HIV infection. The immunogenicity of all potential vaccine candidates needs to be assessed in animal models prior to entry into human trials. Nonhuman primates are the best alternative to humans for assessment of vaccine immunogenicity and protective efficacy. In order to clearly understand how DNA vaccines interact with DCs, suitable in vitro DC culture systems for nonhuman primates need to be developed. This study investigated the culture and characterisation of chacma baboon DCs in vitro, and was the first to assess the effect of various transfection methods on baboon DC maturation and function. The study also evaluated the efficacy of a candidate HIV-1 subtype C DNA vaccine at the level of baboon DC transfection, gene transcription and antigen presentation. Generation of immature DCs (iDCs) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) was accompanied by a loss in the monocyte marker CD14. Expression of the markers CD80 and CD83 was observed on a minority of iDCs, whereas CD86 was expressed on almost all iDCs. Following maturation, all these markers were expressed on an increased number of cells, a pattern of marker expression and upregulation that is similar to that observed in both human and macaque DCs. Transfection of baboon DCs by passive pulsing, lipofection and electroporation was evaluated and compared in several ways. Transfection efficiency, cytotoxicity, the effect of the transfection on DC maturation and subsequent presentation of plasmidencoded antigen to memory T lymphocytes was examined. Baboon DCs lipofected with pDNA efficiently took up HIV-1 subtype C plasmid DNA, transcribed plasmid-encoded genes into mRNA, translated the mRNA into protein, processed the protein and presented peptide antigens to antigen-specific memory T cells. The other methods of transfection were less effective than lipofection due to either decreased transfection efficiency or increased cell cytotoxicity. However, neither lipofection nor passive pulsing in any way negatively impacted on DC marker, CD83, or costimulatory molecule, CD80 and CD86, upregulation. Both methods were found to be as effective as a standard cytokine maturation cocktail in inducing DC maturation. Transfected DCs were also found to be more potent inducers of allogeneic T cell stimulation than their untransfected counterparts, which would appear to indicate enhanced major histocompatibility complex (MHC) expression concurrent with DC maturation marker expression. Lipofection with candidate HIV-1 subtype C vaccine plasmid DNA constructs led to antigen-specific expansion of autologous memory T cells, a finding which indicates the effective expression of plasmid-encoded HIV genes in baboon DCs. This study highlights the functional activity of in vitro generated baboon DCs and provides the groundwork for future studies addressing targeting of plasmid DNA to DCs and enhancement of expression of plasmid-encoded antigens in DCs. A more detailed evaluation of baboon DC interaction with simian immunodeficiency viruses/chimeric simian human immunodeficiency viruses (SIVs/SHIVs) may also reveal how the course of infection in this primate differs from that seen in the macaque or chimpanzee and also how it relates to HIV-1 infection in humans.

Thesis (MSCMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / In this study, we presented data on the role of free radicals in human spermatozoa, particularly in the context of centrifugation and the potential development of defective sperm function. In order to achieve this, methods were developed to directly measure intracellular free radicals in human sperm and the effects of exogenously applied free radicals on sperm function were established. The role of brief and prolonged centrifugation and the associated generation of free radicals was also investigated.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2006. / Acquired immunodeficiency syndrome (AIDS) was first reported in 5 homosexual men in Unite States of America in 1981 as a series of opportunistic infections which occasionally occurred in adults. Subsequently, it has been achieved that human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS and this aetiological agent has spread all over the world. The virus primarily attacks CD4+ T cells and gradually leads to progressive depletion of CD4 T lymphocytes from peripheral blood and lymphoid organs. Since CD4+ T cells are vital immune cells in induction and regulation of both cell-mediated and humoral immune responses, depletion of these cells ultimately results in a profound immunodeficiency characterized by susceptibility to variety of opportunistic infection. Apoptosis have been commonly proposed as the mechanism of CD4 depletion because elevated levels of apoptosis were observed in HIV-1 infected individuals (Ameisen et al., 1991; Groux et al., 1992 & Oyaizu et al., 1993). Nevertheless, there was evidence showing that HIV-1 infected cells died not from apoptosis (Bolton et al., 2002) and another study reported that inhibition of apoptosis resulted in high viral production (Antoni et al., 1995). These controversial views indicated that the mechanism of CD4 depletion and the immuno-pathogenesis of apoptosis should be considered. As a pilot sub-study, eight HIV-1 infected subjects were enrolled to determine the methods in measuring apoptosis. Three different cell separations: (1) whole blood cells, (2) buffy coat cells and (3) isolated PBMCs were prepared to determine whether different cell preparations result in different measurements of apoptosis. In addition, FITC-labelled Annexin V, an early marker of apoptosis, and flow-cytometer based scatter methods based on characteristics of apoptotic cells were used to investigate the difference in analytical methods in determining the levels of apoptosis. Firstly, it was found that whole blood samples yielded more precise measurements in measuring apoptosis, followed by Buffy coat and then PBMC samples. Secondly, this sub-study also indicated that the scatter method as well as fluorenscent labelled Annexin V could be useful markers for apoptosis. Secondly, different surface markers of apoptosis were used to investigate apoptosis in HIV-1 infected adults. Fifty-eight HIV-1 infected adults were involved in this sub-study. They were classified into three categories based on CDC CD4 category classification (CDC, 1993). According to the data, the level of apoptotic CD4+ T cells measured by the scatter method was high in CD4 category 1, decreased in category 2 and finally increased again in category 3. This tendency was in parallel with CD95 (Fas) expression on CD4+ T cells. The curve formed a “V” shape according to the three CD4 categories. Together with the gradually increased plasma viral load, these data reflect an activated immune response at early stage of infection and under controlled viraemia. This possibly represents the immune response trying to eliminate infected cells as a means of survival. The high level of apoptosis in category 3 could indicate a disordered immune system accounting for the rapid loss of CD4+ T cells and progression to AIDS. A novel finding of this study was the presence of two CD4+ populations in 10 HIV-1 infected subjects, which were CD4dim and CD4bright. These 10 subjects had relatively high CD4 count and low viral replication. Statistical analysis showed they had significantly higher levels of apoptosis in CD4 and CD8 T lymphocytes, measured by the scatter method, than those subjects presenting single CD4 population. In addition, when comparing the two CD4 subpopulations, it was found that CD4dim cells had significant higher level of apoptosis and CD95 expression than the CD4bright cells. Finally, the virological and immunological effects of antiretroviral therapy (ART) were investigated in two cohorts of HIV-1 infected children. Fourteen HIV-1 infected children were involved in investigation of 12-month long-term effect, while another five children were involved in a short-term 1-month follow-up study. In addition, a different assay of detecting apoptosis: terminal deoxynucleotidyltransferase deoxyuridine triphosphates nick end labeling (TUNEL) was conducted to measure the level of apoptotic PBMCs. According to the findings from 12-month and 1-month sub-studies, it appeared that ART could be effective in suppression of viral replication at an early stage. However, the immunological effect, such as CD4 reconstitution, could only be seen as a long-term effect, since immune recovery would take a long time. In addition, different regimens containing protease inhibitors (PIs) might be more effective in inhibiting apoptosis than non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell membrane. It also promotes glucose utilisation in type 2 diabetic patients via increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13- kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection exists between these two pathways upstream of GLUT4 translocation. Understanding this connection is important in the development of treatment strategies for type 2 diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK) activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4- carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'- aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue. Aim: To investigate the effect of two pharmacological AMPK-activating compounds, ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt as well as possible feedback on insulin-stimulated glucose uptake and GLUT4 translocation. Materials and Methods: Adult ventricular cardiomyocytes were isolated from male Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13- kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation. PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain results were confinned or refuted by repeating experiments using the isolated rat heart model. Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but in fact inhibited basal glucose uptake, although GLUT4 translocation from cytosol to membrane occurred. Both compounds also attenuated insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin, AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and ZMP increased glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. However both compounds increased GLUT4 translocation, clearly demonstrating that translocation and activation of GLUT4 are separate processes. 8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP indicating that there was no involvement of the purinergic receptors. Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. Conversely, both compounds inhibited both basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation. Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto unknown effects of AICAR and ZMP on PKB phosphorylation. / AFRIKAANSE OPSOMMING: Agtergrond: Oefening verhoog skeletspier glukose opname via AMP-geaktiveerde protein kinase (AMPK) aktivering en GLUT4 translokering vanaf die sitosol na die selmembraan. Dit verbeter ook glukose verbruik in tipe 2 diabetes pasiënte via verhoogde insulien sensitiwiteit. Insulien stimuleer GLUT4 translokering deur P13- kinase en protein kinase B (PKB/Akt) te aktiveer. Dit word dus gepostuleer dat daar 'n verbinding tussen hierdie twee paaie, wat beide betrokke is by GLUT4 translokering, bestaan. Dit is belangrik om hierdie verbinding te verstaan aangesien dit in behandelingstrategieë van tipe 2 diabetes geteiken kan word. Die oefening geïnduseerde verhoging in AMPK aktivering, kan deur 'n farmakologiese middel 5'- aminoimidasool-4-karboksamied ribonukleosied (AICAR), wat intrasellulêr omgesit word na 5'-aminoimidasool-4-karboksamied-ribonukleosiedfosfaat (ZMP), 'n AMP analoog, nageboots word. Doel: Om die effek van twee farmakologiese AMPK-aktiveringsmiddels, AICAR en ZMP, op die fosforilering van AMPK en PKB/Akt, sowel as moontlike effekte daarvan op insulien-gestimuleerde glukose opname en GLUT4 translokering, te ondersoek. Materiale en Metodes: Volwasse ventrikulêre kardiomiosiete is uit manlike Wistar rotharte geïsoleer d.m.v kollagenase perfusies en behandel met 1 mM AICAR of 1 mM ZMP in die teenwoordigheid of afwesigheid van 100 nM insulien of 100 nM wortmannin. Glukose opname is gemeet via intrasellulêre [3H]-2-deoksiglukose akkumulasie; PKB/Akt en AMPK fosforilering sowel as GLUT4 translokering is bepaal deur Western blot analises. Purinergiese reseptore is geblokkeer met 8-siklopentiel- 1,3-dipropielxanthien (8CPT) en die effek daarvan op AMPK fosforilering genoteer. Ten einde resultate wat in die geïsoleerde kardiomiosiet-model verkry is, te bevestig, is sekere eksperimente in die geïsoleerde rothart herhaal. Resultate: Beide AIGAR en ZMP stimuleer AMPK fosforilering. Die middels kan nie glukose opname verhoog nie, inteendeel, basale glukose opname is onderdruk alhoewel GLUT4 translokering vanaf die sitosol na die selmembraan wel plaasgevind het. Wortmannin kon insulien gemedieerde glukose opname en PKB/Akt fosforilering onderdruk. In die teenwoordigheid van wortmannin het beide AIGAR en ZMP PKB/Akt fosforilering verhoog. Alhoewel beide AIGAR en ZMP glukose opname in skeletspier verhoog, was dit nie die geval in kardiomiosiete nie. Beide middels het wel GLUT 4 translokering verhoog, wat duidelik demonstreer dat die translokering en aktivering van GLUT4, verskillende prosesse is. 8GPT het geen effek gehad op die fosforilering van AMPK deur AIGAR of ZMP nie, wat bewys dat daar geen betrokkenheid van die purinergiese reseptore was nie. Gevolgtrekking: Alhoewel AIGAR en ZMP glukose opname in skeletspier verhoog is dit nie die geval in kardiomiosiete nie. Beide middels inhibeer basale en insuliengestimuleerde glukose opname maar stimuleer GLUT4 translokeering. Inhibisie van PI3-kinase in die teenwoordigheid of afwesigheid van insulien, ontmasker voorheen onbekende effekte van AIGAR en ZMP op PKB/Akt fosforilering.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Please see fulltext for abstract / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

The 2 in VO2 is in subscript. / Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Please see fulltext for abstract / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Please see fulltext for abstract / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one third of the world’s population and kills 1.7 million people per year. The increasing prevalence of multi- and extensively drug resistant M. tuberculosis strains means that there is an urgent need to develop new anti-TB drugs. The genome of M. tuberculosis has five copies of the ESAT-6 gene clusters (ESX-1, -2, -3, -4 and -5), which are essential for the survival (ESX-3) and pathogenicity (ESX-1 and ESX-5) of the bacterium. The ESX clusters encode for proteins which form a novel secretion system which has been shown to secreted small T-cell antigens of the esx gene family, as well as other proteins such as the PE and PPE’s. The mycosins are a family of genes situated in the ESX clusters which encode for putative subtilisin-like serine proteases. These proteins are the most conserved proteins within the five clusters. Apart from their conserved protein sequence, mycosin-3 is also an essential protein specific to the mycobacteria, which makes it an attractive potential drug target. Identifying the substrate(s) of mycosin-3 could help to understand the function of this enzyme and discover novel inhibitors from which new drugs could be designed. We hypothesize that the secreted products of the ESX system could be potential substrates for the mycosins. Specifically, we hypothesize that PE5, PPE4, esxG and esxH (all found in ESX-3) might be the substrates for mycosin-3. Mycosin-3, PE5, PPE4, esxG and esxH were thus cloned, expressed and purified respectively. The four substrates were used for protease assays using mycosin-3 as the protease. The protease-substrate mixture were subsequently separated on 2-D SDS-PAGE gels to check whether there were any cleavage of the four substrates. Although all the target fusion proteins were cloned and expressed successfully, the protease assay results showed no cleavage for any of the four substrates. Possible explanations for the failure of cleavage were: (1) impure enzyme and substrate(s); (2) inappropriate buffer conditions; (3) the hypothesized substrates might not be the substrates of mycosin-3; and (4) incorrect folding or modification of the target fusion proteins might have taken place. Future research will aim to address these possible limitations in order to fully elucidate the function and substrate specificity of mycosin-3 and to use this information for the design of novel drugs against M. tuberculosis. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat tuberkulose (TB) veroorsaak, infekteer `n derde van die wêreld se bevolking en veroorsaak die dood van 1.7 miljoen mense per jaar. Die verhoogde voorkoms van multi- en ekstensiewe middelweerstandige stamme van M. tuberculosis beteken dat daar `n ernstige nodigheid is om nuwe anti-TB middels te ontwikkel. Die genoom van M. tuberculosis het vyf kopieë van die ESAT-6 geengroepe (ESX-1, -2, -3, -4 en -5), wat essensieel is vir die oorlewing (ESX-3) en patogenisiteit (ESX-1 and ESX-5) van die bakterium. Die ESX groepe enkodeer vir proteïene wat `n nuwe uitskeidingssisteem vorm wat bewys is om klein T-sel antigene van die esx geenfamilie, sowel as ander proteïene soos die PE en PPE proteïene uit te skei. Die mycosins is `n familie gene wat in die ESX geengroepe voorkom en wat waarskynlik enkodeer vir subtilisin-agtige serine proteases. Hierdie proteïene is die mees gekonserveerde proteïene in die vyf geengroepe. Mycosin-3 is `n essensiële protein wat spesifiek in die mikobakteriëe voorkom, sodat dit `n aantreklike teiken vir die ontwikkeling van middels is. Die identifisering van die substrate van mycosin-3 kan moontlik help om die funksie van die ensiem te verstaan en om nuwe inhibeerders vir die ensiem te ontdek, wat kan lei tot die onwikkeling van nuwe middels. Ons hipotese is dat die uitgeskeide proteïene van die ESX sisteem moontlik die substrate van die mycosin proteïene kan wees. Meer spesifiek, ons hipnotiseer dat die proteïene PE5, PPE4, esxG en esxH (wat almal in ESX-3 voorkom) die substrate vir mycosin-3 kan wees. Mycosin-3, PE5, PPE4, esxG en esxH is afsonderlik gekloneer, uitgedruk en gesuiwer. Die vier substrate is gebruik vir protease proewe met mycosin-3 as die protease. Die protease-substraat mengsel is hierna deur middel van 2-D SDS-PAGE geanaliseer om te kyk of daar enige kliewing van die vier substrate voorgekom het. Alhoewel al die teiken fusieproteïene suksesvol gekloneer, uitgedruk en gesuiwer is, het die protease proewe geen kliewing getoon vir enige van die vier potensiële substrate nie. Moontlike verklarings vir hierdie negatiewe resultaat is die volgende: (1) ensiem en substrate was moontlik onsuiwer; (2) bufferkondisies was moontlik nie korrek nie; (3) gehipotiseerde substrate mag moontlik nie substrate van mycosin-3 wees nie; en (4) nie-korrekte vouing of modifisering van die teiken proteïene kon moontlik plaasgevind het. Toekomstige navorsing sal daarop gemik wees om hierdie beperkinge aan te spreek om sodoende die funksie en substrate van mycosin-3 te kan ontdek en nuwe middels teen M. tuberculosis te ontwerp.