Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Background: Understanding the contribution of divergent individual response patterns remains a key objective in identifying mechanisms of inflammation and potential factors limiting the resolution of inflammation. The purpose of this research project was to investigate downstream effects of inflammation following exercise-induced muscle damage in human subjects. Methods: For three different studies, a total of 53 untrained healthy male participants were recruited and divided into a non-exercising control (n=13) and exercise-induced muscle damage groups (n=40). The study design for the three studies was the same (with few exceptions): Downhill running (DHR) (12 x 5min bouts, 10% decline, 15 km.h-1) with blood samples taken pre, post, after 2 and 4 hours post-exercise (2h, 4h) and on days 1, 2, 3, 4 and 7 (d1-d7). Serum was analysed for creatine kinase activity (CK), myoglobin (Mb), cortisol, cytokine (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokine (G-CSF, MIP-1β) and adhesion factor (sICAM-1, sP-selectin) concentrations. Tissue degradation was assessed by serum matrix metalloprotease (MMP-9) and myeloperoxidase (MPO) content. White blood cell differential count was determined and the surface expression of various cluster of differentiation factors (CD11b, CD163, CD68, CD88, CD34) as well as intracellular MPO were assessed in whole bood using flow cytometry. Nuclear localization of the inflammatory mediator NFĸB in isolated perhipheral blood mononuclear cells (PBMCs) was determined using immunofluorescence microscopy. Muscle biopsies (vastus lateralis) taken at baseline, 4h, d1 and d2 were analysed for fibre type, inflammatory and stress-induced pathways (STAT3, IĸBα, p38MAPK), myogenic factors (MyoD, myogenin), neutrophil activity (MPO) and satellite cell number (Pax7). Results: Participants in the DHR group were subdivided into those with a normal recovery (DHR1) and those who developed secondary damage (DHR2). CK peaked on d1 in both subgroups (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) and again on d4 only in the DHR2 group (1110 ± 184 u.L-1). A similar IL-6 and IL-10 response was evident immediately post DHR in all individuals. Additional IL-6 was released in the DHR2 subgroup peaking at 4h (10.3 ± 4.2 pg.mL-1) whereas IL-10 had returned to baseline. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) and CD163+ (3%) monocytes were significantly higher in the DHR2 subgroup. Neutrophil count at 2h (DHR1: 8.6 ± 0.8 x109 cells.L-1, DHR2: 11.4 ± 1.8 x109 cells.L-1) was significantly (p<0.02) correlated to CK activity on d4. PBMC NFĸB p65 nuclear localization was slightly less at 2h in the DHR2 compared to the DHR1 and control groups. Intramuscular STAT3 signalling and MPO were significantly higher in the DHR2 compared to the DHR1 subgroup at 4h and d2 respectively. The progenitor cell response was similar for all DHR individuals with an increase in Pax7+ SC observed at 4h (0.06 ± 0.01 Pax+ SCs/fibre) and d1 (0.07 ± 0.02 Pax+ SCs/fibre). Conclusion: Healthy young men can be divided into those with a adequate and those with a less efficient capacity to control the post damage inflammatory response. The early cytokine response, especially IL-6, seems to be a key role player in the cascade of events leading to late secondary skeletal muscle damage. / AFRIKAANSE OPSOMMING: Agtergrond: Die begrip van uiteenlopende individuele reaksie patrone, is belangrik in die identifisering van faktore asook meganismes betrokke in die ontwikkeling en resolusie van inflammasie. Die doel van hierdie navorsingsprojek was om die gevolge van oefening-geïnduseerde spierskade en inflammasie te ondersoek in menslike proefpersone. Metodiek: ‘n Totaal van 53 gesonde mans is tydens drie verskillende studies, gegroepeer in ’n kontrole (geen oefening) (n=13) en oefening geinduseerde spier skade (DHR) groep (n=40). Die uitleg van de studies was eenders (met min uitsonderings): Afdraende hardloop (12 x 5min hardloop sessies, 10% afdraende, 15km.h-1) met bloed monsters geneem voor, na, 2 ure, 4 ure (pre, post, 2h, 4h) en op dag 1, 2, 3, 4 en 7 (d1-7). Serum is ontleed vir die volgende: kreatien kinase aktiwiteit (CK), kortisol, sitokiene (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokien (G-CSF, MIP-1β) en adhesie molekuul (sICAM-1, sP-selectin) konsentrasies. Weefsel degradasie is vasgestel deur die teenwoordigheid van matriks metalo-protease-9 (MMP-9) en miëloperoksidase (MPO) in serum te meet. Differensiële witbloed sel (WBC) telling asook die teenwoordigheid van sekere differensiasie faktore (CD11b, CD163, CD68, CD88, CD34) op die sel oppervlak asook intrasellulêre MPO vlakke is bepaal deur gebruik te maak van vloeisitometrie. Die lokalisering van NFĸB in die selkerne van geïsoleerde bloed mononukleêre selle (PBMC) is bepaal deur fluoriserende mikroskopie. Spierbiopsies (vastus lateralis) geneem tydens rus (basislyn), na 4h, en op d1 en d2 is ontleed vir veseltipe, inflammatoriese en stresverwante faktore (STAT3, IĸBα, p38 MAPK), miogeniese faktore (myoD, myogenin), neutrofiel aktiwiteit (MPO) en aantal satelliet selle (Pax7). Resultate: Deelnemers in die DHR-groep is onderverdeel in twee groepe. Persone wat normaalweg herstel het is saam gegroepeer (DHR1) en diegene wat sekondêre skade ontwikkel het is saam gegroepeer (DHR2). CK aktiwiteit in serum het hoogtepunte bereik op d1 in beide subgroepe (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) en weer op d4 in die DHR2 groep (1110 ± 184 u.L-1). 'n Soortgelyke IL-6 en IL-10 reaksie is onmiddellik na oefening (in al die proefpersone) waargeneem. Addisionele IL-6 is vrygestel in die DHR2 subgroep en het ’n hoogtepunt bereik na 4h (10.3 ± 4.2 pg.mL-1), terwyl IL-10 reeds teruggekeer het na rustende waardes. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) en CD163+ (3%) monosiete was aansienlik hoër in die DHR2 subgroep. Neutrofieltelling na 2h (DHR1: 8.6 ± 0.8 x109cells.L-1, DHR2: 11.4 ± 1.8 x109cells.L-1) het verband (p <0,02) gehou met CK-aktiwiteit op d4. In vergelyking met die DHR1 en kontrole groep was die lokalisering van NFĸB p65 in PBMC selkerne na 2h effens minder in die DHR2 subgroep. STAT3- en MPO-vlakke in die spiere was aansienlik hoër in die DHR2 subgroep as in die DHR1 subgroep na 4h en op d2 onderskeidelik. Die spierherstel proses was eenders vir alle individue wat aan die oefening deelgeneem het; 'n toename in Pax7+ Satelietselle (SC) is waargeneem na 4h (0.06 ± 0.01 Pax+ SC/spiervesel) en op d1 (0.07 ± 0.02 Pax+ SC/spiervesel). Gevolgtrekking: Gesonde jong mans kan verdeel word in diegene met 'n bevoegde en diegene met 'n minder doeltreffende vermoë om oefenings-geïnduseerde spierskade en die inflammatoriese reaksie te beheer. Die sitokien-reaksie, veral IL-6, blyk om 'n belangrike rolspeler in die ontwikkeling van sekondêre skeletspierskade te wees.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher inhospital mortality. Hyperglycemia-induced oxidative stress results in DNA damage and subsequent activation of poly-ADP-ribose polymerase (PARP) as a restorative mechanism. However, PARP attenuates glyceraldehyde–3-phosphate dehydrogenase (GAPDH) activity, thereby diverting upstream glycolytic metabolites into damaging non-oxidative glucose pathways (NOGP). For example, hyperglycemia-induced stimulation of four NOGP, i.e. the polyol pathway, hexosamine biosynthetic pathway (HBP), advanced glycation end products (AGE), and PKC activation elicit cardiovascular complications. The current thesis examined the regulation of NOGP in the setting of ischemia and reperfusion under hyperglycemic conditions. Here we hypothesized that administration of two unique therapeutic interventions, i.e. oleanolic acid (OA; clove extract) and benfotiamine (BFT; vitamin B1 derivative), can blunt oxidative stress and NOGP-induced cardiac dysfunction under hyperglycemic conditions following ischemia and reperfusion. Our choice for these agents was based on the principle that OA possesses antioxidant properties; and BFT stimulates transketolase (pentose phosphate pathway [PPP] enzyme) thereby shunting flux away from the NOGP pathways. Additionally, hyperglycemia-induced oxidative stress can also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfolded proteins. There are conflicting data whether increased/decreased UPS is detrimental with hyperglycemia and/or in response to ischemia and reperfusion. In light of this, we also hypothesized that BFT and OA act as novel cardio-protective agents by diminishing myocardial UPS activity in response to ischemia and reperfusion under acute hyperglycemic conditions. Materials and Methods For the first part of the study, we employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 μM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20 min regional ischemia and 2 hr reperfusion 4) In vivo coronary ligations were performed on streptozotocin-diabetic rats ± 0.45 mg/kg OA administration within the first two minutes of reperfusion; and 5) Effects of long-term OA treatment (2 weeks) on heart function were assessed in streptozotocin (STZ)-diabetic rats. Here, STZ was dissolved in citrate buffer (p.H 6.3) and diabetes was induced by administering 60 mg/kg i.p Tissues were collected at the end of the global ischemia experiments and analyzed for oxidative stress, apoptosis, UPS activity and HBP activation. For the second part of the study we employed several experimental systems: 1) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 90 min, followed by 30 min global ischemia and 60 min reperfusion ± 25, 50 and 100 μM BFT treatment, respectively, added during the first 20 min of reperfusion; 2) Infarct size determination as in #3 above but with 30 min regional ischemia and 2 hr reperfusion ± 100 μM BFT treatment; and 3) In vivo coronary ligations performed on streptozotocin-diabetic rats ± 0.50 mg/kg BFT treatment within the first two min of reperfusion. In parallel experiments, NOGP inhibitors were added during the first 20 min of reperfusion. The following inhibitors were individually employed: AGE pathway (100 μM aminoguanidine); PKC (5 μM chelerythrine chloride); HBP (40 μM 6-diazo-5-oxo-L-norleucine); and polyol pathway (1 μM zopolrestat); Infarct size determination as in #2) with 30 min regional ischemia and 120 min reperfusion ± similar treatments. Results Our data show decreased cardiac contractile function in response to ischemia and reperfusion under hyperglycemic conditions. This was linked to increased PARP and attenuated GAPDH activities, together with higher activation of the NOGP. Moreover, we found elevated myocardial oxidative stress, UPS and cell death under these conditions. OA treatment resulted in cardio-protection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia and reperfusion under hyperglycemic conditions. In parallel, OA decreased oxidative stress, apoptosis, HBP flux and UPS activity following ischemia and reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. Our data also reveal that acute BFT treatment significantly decreased myocardial oxidative stress and apoptosis, and provided cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions. In parallel, BFT blunted hyperglycemia-induced activation of four NOGP in the rat heart. Acute administration of each of the NOGP inhibitors decreased PARP and enhanced GAPDH activities, while diminishing oxidative stress and myocardial apoptosis. Moreover, each of the NOGP inhibitors (individually) employed blunted activation of the other three pathways here examined. Hearts treated with NOGP inhibitors also displayed improved functional recovery and smaller infarct sizes following ischemia and reperfusion. Interestingly, NOGP inhibitors resulted in the same degree of change (for all above-mentioned parameters evaluated) when compared to each other. Conclusions This study shows that acute and chronic hyperglycemia trigger myocardial oxidative stress that eventually results in NOGP activation and contractile dysfunction following ischemia and reperfusion. Moreover, our findings establish - for the first time as far as we are aware - that there is a convergence of downstream NOGP effects in our model, i.e. increased myocardial oxidative stress, further NOGP pathway activation, apoptosis, and impaired contractile function. Thus a vicious metabolic cycle is established whereby hyperglycemia-induced NOGP further fuels its own activation by generating even more oxidative stress, thereby exacerbating damaging effects on the heart under these conditions. We also found that both OA and BFT treatment blunted high glucose-induced detrimental effects and provided robust cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions (acute and chronic). These findings suggest that the UPS may be a unique therapeutic target to treat ischemic heart disease in individuals that present with stress-induced, acute hyperglycemia. Moreover, BFT exhibited its cardio-protective effects by NOGP inhibition after ischemia and reperfusion under acute and chronic high glucose conditions. A similar effect was observed at baseline although the underlying mechanisms driving this process still need to be elucidated. In summary, the findings of this thesis are highly promising since it may eventually result in novel, cost-effective therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding Diabetes skep ‘n groot gesondheidsuitdaging. Omrede kardiovaskulêre komplikaseis algemeen onder diabetiese pasiënte is, sal dit oorkoepelend die las van hierdie siekte verder laat toeneem. Verder word stresgeïnduseerde hiperglukemie in nie-diabetiese pasiënte met akute miokardiale infarksie geassosieër met ‘n hoër binne-hospitaalmortaliteit. Hiperglukemies-geïnduseerde oksidatiewe stres veroorsaak DNA skade, en gevolglike aktivering van poli-ADF-ribose polimerase (PARP), as ‘n herstelmeganisme. Nietemin, PARP verminder gliseraldehied–3-fosfaatdehidrogenase (GAPDH) aktiwiteit om sodoende die opstroom glikolitiese metaboliete te herlei na skadelike nie-oksidatiewe glukose weë (NOGW). Byvoorbeeld, hiperglukemie-geïnduseerde stimulasie van vier NOGW, i.e. die poliolweg, heksosamienbiosintetiese weg, (HBW), gevorderde glukasie eindprodukte (GGE), en PKC aktivering, lei tot kardiovaskulêre komplikasies. Die huidige tesis ondersoek die regulering van NOGW in ‘n isgemiese-reperfussie onder hiperglukemiese toestande. Ons hipotetiseer dat die toediening van twee unieke terapeutises intervensies, i.e. oleanoliese suur (OS, naaltjie ekstrak), en benfotiamien (BFT, vitamien B1 derivaat) oksidatiewe stress kan versag, en NOGW geinduseerde kardiale disfunksie onder hiperglukemiese toestande na ischemie en reperfussie. Ons keuse vir hierdie middels is gebaseer op die beginsel dat OS antioksidanteienskappe bevat, en dat BFT transketolase (pentosefosfaat weg (PFW) ensiem) stimuleer en sodoende die fluks weg van die NOGW weg veroorsaak. Addisioneel kan hiperglukemiegeïnduseerde oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS) wat wangevoude protïene verwyder, aanleiding gee. Daar bestaan kontrasterende data oor ‘n verhoogde/verlaagde UPS, tesame met hiperglukemie en/of in reaksie tot isgemie-reperfussie. In die lig hiervan, hipotetiseer ons dat BFT en OS as ‘n nuwe kardiobeskermingsmiddel kan optree deur miokardiale oksidatiewe stres en UPS aktiwiteit in reaksie op isgemie-reperfussie tydens akute hiperglukemiese toestande kan verlaag. Materiale en Metodes Vir die eerste deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1) H9c2 kardiale mioblaste is aan 33 mM glukose vir 48 uur vs. kontrole (5 mM glukose) blootgestel; en gevolglik met twee OS dosisse (20 en 50 μM) vir 6 en 24 hr, onderskeidelik behandel; 2) geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie ± OS behandeling; 3) Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie 4) In vivo koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.45 mg/kg OS toedienning binne die eerste twee minute van reperfussie; en 5) effekte van langtermyn OS behandeling (2 weke) op hartfunskie is in hierdie streptozotosien-diabetiese rotte ondersoek. Hier is STZ opgelos in ‘n sitraatbuffer (pH 6.3), en diabetes is geinduseer deur 60mg/kg i.p. toe te dien. Weefsels is aan die einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose, UPS aktiwiteit en HBW aktivering, ontleed. Vir die tweede deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1) geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 90 min geperfuseer. Daarna is dit gevolg met 30 min globale isgemie en 60 min reperfussie ± 25, 50 en 100 μM BFT behandeling onderskeidelik, gevolg, bykomend, gedurende die eerste 20 min reperfussie; 2) Infarkgrootte is bepaal soos in #3 hierbo, maar met 30 minute regionale ischemie en 2 uur reperfussie ± 100 μM BFT behandeling; en 3) In vivo koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.50 mg/kg BFT behandeling binne die eerste twee minute van reperfussie. Met parallele eksperimente is NOGW inhibeerders bygevoeg binne die eerste 20 min van reperfussie. Die volgende inhibeerders is individueel ontplooi: GGE weg (100 μM aminoguanidien); PKC (5 μM chelleritrienchloried); HBW (40 μM 6-diazo-5-oxo-L-nor-leusien); en poliolweg (1 μM zopolrestaat); 2) Infarkgrootte is bepaal soos in #2) met die uitsondering van 30 min regionale isgemie en 120 min reperfussie ± identiese behandelings. Resultate Ons data toon aan dat kardiale kontraktiele funksie, in reaksie op isgemie-reperfussie onder hiperglukemiese toestande, verlaag. Dit is verwant aan verhoogde PARP en verminderde GAPDH aktiwiteit, tesame met ‘n hoër aktivering van die NOGW. Verder het ons bevind dat verhoogde miokardiale oksidatiewe stres, UPS en seldood onder die toestande voorkom. OS behandeling lei tot kardiale beskerming, i.e. vir ex vivo en in vivo rotharte wat aan isgemie-reperfussie onder hiperglukemiese toestande blootgestel is. Parallel hiermee het OS oksidatiewe stres, apoptose, HBW invloed, en UPS aktiwiteit na isgemie-reperfussie, verlaag. Langtermyn OS behandeling het ook hartfunksie in streptozotosien-diabetiese rotte verbeter. Ons data vertoon verder dat akute BFT behandeling, miokardiale oksidatiewe stres en apoptose, betekenisvol verlaag het in reaksie op isgemie-reperfussie onder hiperglukemiese toestande. Parallel hiermee het BFT hiperglukemiegeïnduseerde aktivering van vier NOGWë in die rothart, verminder. Akute toediening van die elk van die NOGW inhibeerders het PARP verlaag, en GAPDH aktiwiteite verhoog, terwyl oksidatiewe stres, en miokardiale apoptose verminder. Verder het elk van die NOGW inhibeerders wat (individueel) toegedien is, aktivering van die ander drie weë, hier ondersoek, verlaag. Die harte wat met NOGW inhibeerders behandel is het ook ‘n verbeterde herstel en kleiner infarkgrootte na isgemie-reperfussie getoon. Interessant is hoe die NOGW inhibeerders tot dieselfde graad verandering (vir al die bogemelde parameters wat geevalueer is) indien dit vergelyk word teen mekaar, gelei het. Gevolgtrekking Hierdie studie het bevind dat akute en chroniese hiperglukemie, miokardiale oksidatiewe stres ontlok, en dat dit geleidelik tot NOGW aktivering en kontraktiele wanfunksionering na isgemie-reperfussie lei. Verder het ons bevindinge vir die eerste keer, volgens ons wete, bewys dat daar ‘n ineenloping is van afstroom NOGW effekte in ons model, i.e. verhoogde miokardiale oksidatiewe stres, verdere NOGW weg aktivering, apoptose, en ingeperkte kontraktiele funksie. Dus, ‘n gebrekkige metaboliese siklus word verkry waarby hiperglukemies-geïnduseerde NOGW verder sy eie aktivering aanvuur deur meer oksidatiewe stres, en sodoende die skadelike effekte op die hart onder hierdie toestande verder versleg. Ons het verder bevind dat beide OS en BFT behandeling, hoë glukose-geïnduseerde skadelike effekte onderdruk, en kragtige kardiale-beskerming in reaksie op isgemie-reperfussie onder hiperglukemiese toestande (akuut en chronies), teweeg bring. Hierdie bevindinge dui moontlik daarop dat die UPS ‘n unieke terapeutiese teiken kan wees vir die behandeling van isgemiese hartsiekte in individue wat presenteer met stres-geïnduseerde, akute hiperglukemie. BFT het ook sy kardiale beskermende effekte getoon deur NOGW inhibering na isgemie-geïnduseerde reperfussie onder aktute en chroniese hoë glukose toestande. ‘n Soorgelyke effek is tydens die basislyn waargeneem, alhoewel die onderliggende meganisme wat hierdie proses dryf verder ondersoek moet word. Opsommend is ons bevindinge baie belowend omrede dit daartoe kan aanleiding gee tot ‘n nuwe, meer koste-effektiewe terapeutiese intervensie vir die behandeling van akute hiperglukemie (in niediabetiese pasiënte) en diabetiese pasiënte met geassosieërde kardiovaskulêre komplikasies. / Oppenheimer, Beit Trust and Harry Crossley

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term effects include onset of insulin resistance and cardiovascular diseases. Increased oxidative stress and dysregulation of the ubiquitin-proteasome system (UPS) are implicated in protease-inhibitor (PI)-mediated cardio-metabolic pathophysiology. We hypothesized that PI treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the UPS, thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for eight weeks vs. vehicle and sham controls. Subsequently, we evaluated metabolic parameters and heart function (ex vivo and in vivo methods) at baseline and following ischemia-reperfusion. PI-treated rats exhibited weight gain, increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. It also upregulated hepatic gene expression of acetyl-CoA carboxylase β and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. Further, PI-treated hearts displayed impaired UPS, increased superoxide dismutase (SOD) activity and unaltered superoxide levels, and elevated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) peptide levels. Perfusion data revealed contractile dysfunction at baseline and following ischemia-reperfusion, while post-ischemic hearts exhibited decreased ATPase specific activity vs. matched controls. Early changes initiated by PI treatment resemble the metabolic syndrome and reflect a pre-atherogenic profile. Moreover, the effects of PIs on cardiac contractile function may in part be triggered by impaired UPS activity together with strain on the mitochondrial energetic system. Our study alerts to cardio-metabolic side effects of PI treatment and raises the question of the most appropriate co-therapies for patients on chronic antiretroviral treatment. / AFRIKAANSE OPSOMMING: Alhoewel anti-retrovirale behandeling MIV-VIGS morbiditeit/mortaliteit verlaag, bestaan daar langtermyn effekte soos die aanvang van insulienweerstandigheid en kardiovaskulêre siektes. Verhoogde oksidatiewe stres en wanregulering van die ubikwitien-proteosoomsisteem (UPS) word geïmpliseer met protease-inhibeerder (PI) gemediëerde kardio-metaboliese patofisiologie. Ons hipotetiseer dat PI behandeling (Lopinavir/Ritonavir) miokardiale oksidatiewe stres verhoog, en gevolglik die UPS inhibeer waardeur dit kardiale funksie verander. Lopinavir/Ritonavir is in 1% etanol (draer) opgelos en in ‘n mini-osmotiese pomp ingespuit wat chirurgies in Wistar rottes ingeplant is vir agt weke vs. draer en valskontroles. Gevolglik het ons die metabolise parameters en hartfunksie (ex vivo en in vivo metodes) op basislyn en na afloop van ischemie-reperfusie ondersoek. PI-behandelde rotte het ‘n toename in massa getoon asook verhoogde serum LDL-cholesterol, hoër weefseltrigliseriede (hart, lewer), maar geen bewys van insulienweerstandigheid nie. Dit het ook hepatiese asetielko-ensiem A karboksilase β en 3-hidrokise-3-metielglutariel KoA reduktase geenuidrukking opwaarts gereguleer, wat sleutel reguleerders van vetsuuroksidasie en cholesterolsintese onderskeidelik is. Verder, het PI-behandelde harte ingeperkte UPS, verhoogde SOD aktiwiteit en onveranderde superoksiedvlakke vertoon, asook verhoogde peroksisoomproliferator-geaktiveerde reseptor-γ ko-aktiveerder 1-α (PGC-1α) peptiedvlakke. Perfusie data toon kontraktiele wanfunskionering gedurende basislyn en na afloop van ischemie-reperfussie, terwyl post-ischemiese harte verlaagde ATPase spesifieke aktiwiteit vs gepaarde kontrole vertoon. Vroeë veranderinge wat deur PI behandeling veroorsaak word, kom ooreen met die metabolise sindroom en reflekteer op ‘n pre-aterogeniese profiel. Bowendien kan die effekte van PIs op kardiale kontraktiele funksie deels veroorsaak word deur die ingeperkte UPS aktiwiteit tesame met die las op die mitochondriale energie sisteem. Ons studie waarsku teen kardio-metaboliese newe effekte met PI behandeling en rig die vraag; wat die mees gepaste ko-behandeling vir pasiënte op chroniese anti-retrovirale behandeling is.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Physiological cardiac hypertrophy is characterized by the heart’s ability to increase mass in a reversible fashion without leading to heart failure. In contrast, pathological cardiac hypertrophy leads to the onset of heart failure. For this study, we investigated a model of physiological hypobaric hypoxia-mediated right ventricular (RV) hypertrophy (RVH). Here our hypothesis was that the hypertrophic response and associated changes triggered in the RV in response to chronic hypobaric hypoxia (CHH) (increased RV mass, function and respiratory capacity) are reversible. To test our hypothesis we exposed male Wistar rats to 3 weeks of CHH and thereafter removed the hypoxic stimulus for 3 and 6 weeks, respectively. Adaptation to 3 weeks of CHH increased the RV to left ventricle (LV) plus interventricular septum ratio by increased (223.5 ± 7.03 vs. 397.4 ± 29.8, p<0.001 versus normoxic controls), indicative of RVH. Hematocrit levels, RV systolic pressure and RV developed pressure (RVDP) were increased in parallel. Mitochondrial respiratory capacity was not significantly altered when using both carbohydrate and fatty acid oxidative substrates. After the 3-week normoxia recovery period, the RV to LV ratio was increased but to a lesser extent compared to the 3-week hypoxic time-point, i.e. 244.7 ± 11.2 vs. 349.64 ± 3.8, p<0.001 versus normoxic controls. Moreover, hematocrit levels were completely normalized. However, the RV systolic pressure and the functional adaptations, i.e. increased RVDP induced by CHH exposure still persisted in the 3-week recovery (3HRe) group. Also, pyruvate utilization was increased versus matched controls (p<0.04 vs. matched controls). Interestingly, we found that at the 6-week recovery time point functional parameters were largely normalized. However, the RV to LV ratio was still increased by 269.3 ± 14.03 vs. 333.9 ± 11.7, p<0.0001 vs. matched controls. Furthermore, palmitoylcarnitine utilization was increased (p<0.03 vs. matched controls). In conclusion, we found that exposure to CHH resulted in various adaptive physiological changes, i.e. enhanced hematocrit levels, increased RV mass linked to greater RV contractility and respiratory function. It is important to note that all these changes only occurred in the RV and not in the LV. Furthermore, when a normoxic recovery period (3 and 6 weeks, respectively) were initiated, these physiological parameters largely normalized. Together, the findings of this thesis clearly show the establishment of a reversible model of RV physiological hypertrophy. Our future work will focus on disrupting signaling pathways underlying this process and to thereafter ascertain whether reversibility is abolished. Elucidation of such targets should provide a unique opportunity to develop novel therapeutic agents to treat patients and thereby reduce the burden of heart disease.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Aims and Background: According to the World Health Organization type 2 diabetes will constitute a major global burden of disease within the next few decades. In agreement, reports show that rapid urbanization and lifestyle changes in South Africa are major factors responsible for these projections. Therefore, any perturbations that alter the regulatory steps that control myocardial glucose uptake by the cardiac-enrich glucose transporter, GLUT4, will lead in the development of diabetic cardiomyopathy and cardiac hypertrophy. Although considerable efforts are been put into unraveling molecular mechanisms underlying this process, less is known regarding the spatio-temporal regulation of GLUT4. In light of this, our specific aim was to establish in vitro fluorescence microscopy- and flow cytometry-based models for visualization and assessment of myocardial GLUT4 translocation using H9c2 cardiac-derived myoblasts. After successful establishment of our in vitro-based model for myocardial GLUT4 translocation, our second aim was to determine the role of the hexosamine biosynthetic pathway (HBP) in this process. Here, we employed HBP modulators to alter flux and subsequently evaluate its effect on myocardial GLUT4 translocation. To further strengthen our hypothesis, we also investigated the role of the HBP in hearts of an in vivo type 2 diabetes mouse model. Hypothesis: We hypothesize that increased flux through the HBP impairs myocardial GLUT4 translocation by greater O-linked glycosylation of the insulin signaling pathway, ultimately leading to myocardial insulin resistance. Methods: Rat cardiac-derived H9c2 myoblasts were cultured until ~ 80-90 % confluent for 3 days and thereafter subcultured in Lab-Tek chamber slides (~ 15, 000 cells per well) for 24 hours. Cells were then serum starved for 3 hours by insulin administration of 100 nM for 0, 5 and 30 minutes, respectively. We employed a method to quantify the relative proportion of GLUT4 at the sarcolemma using immunofluorescence microscopy- and flow cytometry-based models for visualization and assessment of myocardial GLUT4 translocation. Using these methods we investigated the role HBP have during GLUT4 translocation. The HBP were then activated through the following: a) high glucose and glutamine concentrations; b) low glucose and glucosamine stimulation; and c) over-expression of the HBP rate- limiting enzyme, i.e. GFAT. Subsequently, cardiac-derived myoblasts were fixed and probed for ~ 24 hours with antibodies specific for intracellular- and membrane-bound GLUT4, anti-myc GLUT4 (9E10) and O-GlcNAc. To assess GLUT4 translocation and O-GlcNAcylation we employed the following secondary antibodies: FITC Green for intracellular-bound GLUT4; and b) Texas Red for membrane-bound GLUT4 (immunofluorescence microscopy) and Phycoerythrin for flow cytometry-based model. Cells were thereafter viewed by multi-dimension imaging using an inverted system microscope (Olympus IX81) and a BD FACS Aria cell sorter for flow cytometric analysis. We also assessed HBP in an in vivo context by probing heart tissue - from insulin resistant db/db mice - with a GFAT monoclonal antibody. Results: The db/db mouse represents an ideal model to confirm our hypothesis in an in vivo context. In agreement, our preliminary results show increased GFAT expression versus heterozygous db/+ controls. Our in vitro model show myocardial GLUT4 translocation at 5 minute peak response when H9c2 cardiac-derived myoblasts were stimulated with 100 nM insulin, and GLUT4 vesicles return to normal after longer insulin stimulatory times (10, 15 and 30 minutes. Myocardial Glut4 v translocation was impaired when cells were stimulated with 100 nM wortmannin. Our transfection based model (immunofluorescence microscopy- and flow cytometry-based models) confirms 5 minute peak response under real time conditions. High glucose concentration (25 mM glucose), glucosamine concentrations (2.5 mM, 5 mM, and 10 mM) and over-expression of GFAT led to an impairment of myocardial GLUT4 translocation. Employment of an HBP activator (50 μM PUGNAc) also caused impairment of myocardial GLUT4 translocation. Myocardial GLUT4 translocation was restored when cells were treated with an HBP inhibitor (40 μM DON). High glucose concentrations (25 mM glucose), glucosamine concentrations (2.5 mM, 5 mM, and 10 mM) and over-expression of GFAT resulted in an increase in O-GlcNAcylation. HBP activation (50 μM PUGNAc) showed an increase in O-GlcNAcylation, while administration of 40 μM DON reversed this effect. Discussion and conclusion: We successfully established an in vitro experimental system to assay myocardial GLUT4 translocation. Our data show that dysregulated flux through the HBP impairs myocardial GLUT4 translocation. It is likely that the HBP becomes dysregulated during the pre-diabetic/early diabetic state and that O-GlcNAcylation of members of the insulin signaling pathway occurs during this stage. This will lead to myocardial insulin resistance, and in the long term, will contribute to the onset of the diabetic cardiomyopathy. Investigations to find unique inhibitors of this maladaptive pathway should therefore result in the development of novel therapeutic agents that will lead to a reduction in the growing global burden of disease for type 2 diabetes and associated cardiovascular diseases.

Thesis (MPhil (Physiological Sciences))--University of Stellenbosch, 2006. / The responses of central systems to oxygen deprivation have been well characterised while adaptations in peripheral systems, such as skeletal muscles, have presented confounding variations. Several reasons for these discrepancies are purported, amongst them being the duration of exposure to hypoxia and variations in fibre composition. Moreover, in real-life high altitude situations there may be a combination of factors which have the ability to modify or alter the effect of hypoxia. This study investigates the effect of short duration hypoxia per se on substrate utilisation in different types of skeletal muscles.

Thesis (MPhil (Physiological Sciences))--University of Stellenbosch, 2006. / Background. Creatine monohydrate is widely used for its purported ergogenic and anabolic properties. The mechanism by which creatine supplementation enhances muscle growth is not understood. This study was undertaken to determine whether creatine monohydrate supplementation increases the conversion rate of testosterone to dihydrotestosterone. An increase in dihydrotestosterone could partly explain the beneficial effect of creatine monohydrate on muscle hypertrophy. Methods. Subcommittee C of the research committee of the University of Stellenbosch approved the study. Project number 2001/ C045. The study was designed as a double blind crossover with subjects (n = 20) in each leg of the study. Group 1 (n = 10) taking creatine monohydrate and group 2 (n = 10) glucose during the first leg of the study. In accordance with crossover study design the groups were reversed in the second leg of the study. Gelatin capsules were filled with 5g of either creatine monohydrate or 5g of glucose. Subjects taking creatine monohydrate also took 25g of glucose to improve absorption of creatine. Subjects took creatine monohydrate 25g plus 25g of glucose (ten capsules in all) or glucose ten capsules per day for seven days in the loading phase. In the maintenance phase they took 5g of creatine monohydrate plus 25g of glucose (six capsules in all) per day or six capsules of glucose, for 14 days. The groups were reversed after a six-week washout period and the dosages repeated as per crossover study design. Blood samples were taken on day zero of the study as baseline measurements, repeated on day 7, (after the loading phase), and again on day 21, (after the maintenance phase). These were again repeated in the second leg of the study as per crossover design. Serum was separated within one hour of collection and stored at minus 70 oC. Testosterone and dihydrotestosterone concentrations were determined using a radio-immunoassay kit by an accredited university laboratory. The percentage conversion of testosterone to dihydrotestosterone was calculated. The results were statistically analyzed: A paired t - tests at the beginning of each leg of the study and repeated measure analysis of variance, for the pooled data for each condition over the whole study. Results. The difference in blood levels of testosterone and dihydrotestosterone on both days 0, were not statistically significant. This made the pooling of the data possible. The difference in the percentage conversion of testosterone to dihydrotestosterone over the study period between the creatine monohydrate condition and the glucose condition, was however significant (p < 0.0001). In this small study highly significant statistical results were obtained. The answer to how creatine taken as a supplement exerts its effect may lie in the increased rate of conversion of testosterone to dihydrotestosterone. Conclusion. With the known greater androgenic effect of dihydrotestosterone as opposed to testosterone, the increase in testosterone conversion to dihydrotestosterone could explain how creatine supplementation exerts its anabolic effect in susceptible individuals. A larger study should be done to confirm these results and answer the questions arising from the findings.

Thesis (MSc) -- Stellenbosch University, 1995. / ENGLISH ABSTRACT: The aim of this study was to investigate the influence of the allergic reaction on the respiratory system. The influence of parasite infestation (with specific reference to the roundworm, Ascaris lumbricoides) on the normal functioning of the respiratory system was also investigated. Firstly a pilot study was done to determine the normal immunoglobulin E (IgE) levels of coloured newborns in the Western Cape. The cord blood IgE values of a group of coloured newborns were determined by using the RIA technique. Although a mean value was reached, it is important for more controlled studies, that would take into account a variety of factors, to be done before determining separate reference values for this population. The levels of one of the most important effector cells in the allergic reaction, namely eosinphils, were determined in a group of asthma patients by using the counting chamber method. In all the cases the eosinophil levels were elevated The influence of Ascaris lumbricoides on the respiratory system was investigated from two different points of view, namely the possible allergic reactions it could induce in the host and secondly by determing the influence of this parasite on one of the parametres of the respiratory system, namely the peak expiratory flow (PEF) values. In the first case the presence of specific Ascaris allergens in a group of asthma patients was determined by using the RAST technique. The incidence of these specific allergens was however not high. Secondly the PEF values of two groups (the one group was infested with the roundworm and the other not) were compared. Wet stool mounts were investigated under a light microscope to identy parasite ova. There was however no significant difference in PEF values between these two groups. This study underlines the complex interaction between parasites and the respiratory system. Further studies in this regard are needed in order to fully understand the nature of this interaction. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om ondersoek in te stel na die invloed wat die allergiese reaksie uitoefen op die respiratoriese sisteem. Daar is verder ook aandag gegee aan die uitwerking van parasiet infestasies (met spesifieke verwysing na die rondewurm, Ascaris lumbricoides) op die normale funksionering van die respiratoriese sisteem. 'n Loodsstudie is eerstens uitgevoer ten einde normale immunoglobulien E (IgE) vlakke vir Kleurling pasgeborenes te bepaal. lgE bepalings is gedoen op die nawelstring bloed van 'n groep Kleurling babas in die Wes-Kaap. Die RIA tegniek is vir hierdie doel gebruik. Hoewel 'n gemiddelde waarde verkry is, is dit egter noodsaaklik dat meer gekontrolleerde studies wat 'n wye verskeidenheid van faktore in ag neem, uitgevoer sal moet word alvorens aparte verwysingswaardes vir hierdie bevolkingsgroep saamgestel kan word. Die voorkoms van een van die belangrikste effektor selle in die allergiese reaksie, naamlik eosinofiele, is bepaal in die bloed van 'n groep asma pasiente. Die eosinofiele is getel deur gebruik te maak van die telkamer metode. In al die gevalle was die eosinofiel vlakke verhoog. Die invloed van Ascaris lumbricoides op die respiratoriese sisteem is vanuit twee oogpunte ondersoek, naamlik die uitlokking van 'n allergiese respons in ~ie gasheer en die invloed van hierdie parasiet op een van die parameters van longfunksie, naamlik "peak expiratory flow" (PEF) vlakke. In die eerste geval is die mate van teenwoordigheid van Ascaris allergene in 'n groep asma pasiente bepaal deur gebruik te maak van die RAST tegniek. Die insidensie van allergie teen hierdie allergene was egter nie hoog nie. Tweedens is die PEF waardes van twee groepe proefpersone (die een groep gei:nfesteer met Ascaris lumbricoides en die ander groep nie) met mekaar vergelyk. Daar was egter nie 'n beduidende verskil tussen die waardes van die twee groepe nie. Parasiet ova is gei:dentifiseer deur die maak van eenvoudige nat stoelgang smere en die ondersoek daarvan onder 'n ligmiroskoop. In hierdie studie word die komplekse aard van die interaksie tussen parasiete en die respiratoriese sisteem duidelik na vore gebring. Verdere studies is nodig ten einde die verband tussen hierdie twee faktore te bepaal. / SNO and Stellenbosch 2000 scholarship

Thesis (MSc) -- Stellenbosch University, 1990. / ENGLISH ABSTRACT: The main aim of this workpiece was to establish the physiological parameters against which a vitamin Bu deficiency could be measured. A comparison between the hematological values of healthy patients and those suffering from pernicious anemia due to vitamin Bu deficiency was done. A specific case of pernicious anemia was used in the comparison of abnormal values to the values of normal healthy patients. The comparison consisted of blood analyses with the help of specified instruments, photomicrographs of bone marrow and blood smears and statistical data. A Coulter Counter Model ZF was used for the hematological analyses of blood, a radio-isotope assay for serum vitamin B u was done and photomicrographs were taken with a NIKON Microflex camera with photomicrographic attachments. The importance of vitamin Bu has been shown in this workpiece. With the use of techniques and certain instruments, the effects of a shortage of vitamin Bu has been shown. Analyses of the blood from normal ,healthy patients was compared to that of patients suffering from pernicious anemia. It was demonstrated that pernicious anemia is characterized by a low erythrocyte count, hematocrit (Het), hemoglobin (Hb) and vitamin Bu levels together with a higher mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV). In severe cases of pernicious anemia these levels are extremely high or low as the case may be. Together with these values, the investigation of pernicious anemic blood and bone marrow smears revealed abnormally large erythrocyte precursors and fewer leucocytes than normal. Abnormally shaped cells, called macrocytes, were seen which was due to the disruption in deoxyribonucleic acid (DNA) synthesis caused by the vitamin Bu deficiency. This study produced a set of hematological reference values. The comparative study between healthy and pernicious anemic patients demonstrated a significant drop in serum vitamin B12 values during pernicious anemia. The hematological effect was illustrated by the Coulter Counter blood analysis results and the microscopic examination revealed the presence of megaloblastic erythrocytes, oval erythrocytes, pear-shaped poikilocytes and polymorphonuclear neutropbils with hypersegmented nuclei in blood smears I during vitamin B12 deficiency. This dianoses can be supported by the presence of megaloblasts and metamyelocytes in pernicious anemic bone marrow. / AFRIKAANSE OPSOMMING: Die hoof doel van hierdie werkstuk was om fisiologiese grense te bepaal waarteen 'n vitamien B12 tekort gemeet kan word. 'n Vergelyking tussen die hematologiese waardes van gesonde persone en die van pasiente met pernisieuse anemie wat ontstaan het as gevolg van 'n vitamien B12 tekort was uitgevoer. Die waardes verkry vanaf 'n spesifieke geval van pernisieuse anemie. was vergelyk met waardes vanaf normale gesonde persone. Hierdie vergelyking het bestaan uit bloed analises, fotomikrograwe van bloed en beenmurg smere en statistiese data. Die hematologiese bloed analises was met behulp van 'n Coulter Teller model ZF uitgevoer. 'n Radio-isotoop bepaling vir serum vitamien B12 was gedoen en fotomikrograwe was met 'n NIKON Microflex kamera geneem. Die belang van 'n vitamien B12 tekort was in hierdie werkstuk gedemonstreer. Die effek van hierdie tekort is deur die gebruik van sekere instrumente en tegnieke aangedui en die resultate hiervan is vergelyk tussen gesonde persone en pasiente met 'n vitamien B12 tekort. Hierdie studie het bevestig dat pernisieuse anemie gekenmerk word deur verlaagde eritrosiet, hematokrit (Het), hemoglobien (Hb) en vitamien B12 vlakke tesame met verhoogde gemene korpuskulere hemoglobien (GKH) en gemene korpuskulere volume (GKV) vlakke. Gedurende ernstige gevalle van pernisieuse anemie kan hierdie waardes uitermatig hoog of laag wees. Benewens hierdie waardes het die ondersoek van bloed en beenmurg gedurende vitamien B12 tekort, abnormale groot eritrosiet voorgangers en 'n verminderde hoeveelheid leukosiete getoon. Abnormale sel vorms was ook sigbaar a.g.v. die onderbreking in DNA sintese wat deur 'n vitamien B12 tekort veroorsaak word. Pernisieuse anemie word verkry wanneer daar 'n vitamien B12 en/of folaat tekort in die dieet is of wanneer hierdie vitamiene nie geabsorbeer kan word nie. Die teenwoordigheid van makrosiete, ovaal eritrosiete, peervormige poikilosiete en polimorfonuklere neutrofiele met hipergesegmenteerde keme in bloedsmere dui op 'n megaloblastiese anemie. Hierdie diagnose kan ondersteun word deur die aanwesigheid van megaloblaste en reuse metamielosiete in die beenmurg. Die bepaling van vitamien B12 en folaat vlakke in die bloed kan as addisionele bewysstukke vir 'n volledige diagnose dien. Gedurende hierdie studie is daar 'n stel hematologiese verwysingswaardes vasgestel. Die vergelykende studie tussen gesonde persone en pasiente met pernisieuse anemie het getoon dat daar 'n beduidende verlaging in serum vitamien B12 waardes gedurende pernisieuse anemie is. Die hematologiese effek was ook duidelik waameembaar in die Coulter teller se bloed analiese en mikroskopiese ondersoeke het die · teenwoordigheid van makrosiete, ovaal eritrosiete, peervormige poikilosiete en polimorfenuklere neutrofiele met hipersegmenteerde keme in bloedsmere aangedui. Hierdie diagnose kan ondersteun word deur die aanwesigheid van megaloblaste en reuse metamielosiete in die beenmurg. / This study was financially aided by a bursary from the CSIR.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: Cancer is an emerging health problem in South Africa, with breast cancer being one of the leading cancers affecting women globally. Therefore, there is a need to find novel targets to improve the therapeutic options for these patients. A recently proposed target is the mitogen-activated protein kinase phosphatase-1 (MKP-1). Studies have suggested that mitogen-activated protein kinase phosphatases are involved in the development of cancer and play an important role in the response of cancer cells to chemotherapy. Additionally, numerous studies have indicated that there is increased expression of MKP-1 in breast cancers where its over-expression is proposed to be a significant mediator in chemo-resistance. We propose that inhibition of MKP-1 will increase the cytotoxic effect of doxorubicin in breast cancer cells, thus making the cells more responsive to treatment leading to increased cell death through autophagy and apoptosis. Methods: In MDA-MB231 cells, MKP-1 was inhibited using sanguinarine or MKP-1 siRNA and this was compared to a known inducer of MKP-1, dexamethasone. MDA-MB231 cells were treated with doxorubicin alone or in combination with MKP-1 inhibitors or an inducer. Following treatment, cell death was determined by trypan blue and a caspase glo assay as well as with western blotting. Autophagy was determined by western blotting and flow cytometry. LC3 and p62 were used as markers of autophagy and caspase 3 and PARP as apoptosis markers. Likewise, the level of MKP-1 expression under conditions of MKP-1 induction, inhibition or silencing was evaluated by means of western blotting. C57BL6 tumour bearing mice was used to analyse apoptosis and autophagy in vivo under conditions of MKP-1 inhibition, using sanguinarine, together with doxorubicin treatment. Western blotting was used to determine levels of caspase 3, LC3, p62 and MKP-1 expression. Results and discussion: A concentration and time curve indicated that 5 μM doxorubicin reduced cell viability in the MDA-MB231 cells significantly after 24 hours of treatment. MKP-1 expression was significantly reduced with sanguinarine and MKP-1 siRNA. Furthermore, our results indicate a significant increase in apoptosis in MDA-MB231 cells when treated with doxorubicin, under conditions of MKP-1 inhibition or MKP-1 silencing. Also, an increase in autophagic activity was observed following treatment with doxorubicin in combination with sanguinarine. Whole excised tumours of C57BL6 mice also showed an increase in apoptosis and autophagy following treatment with sanguinarine in combination with doxorubicin. This indicates that the inhibition of MKP-1 with sanguinarine sensitized the MDA-MB231 cells and E0771 cell tumours to doxorubicin-induced-apoptosis through a mechanism involving autophagy. Conclusion: This is an encouraging finding that could hopefully be used in future studies to overcome doxorubicin-resistance in breast cancer cells overexpressing MKP-1. Targeting MKP-1 can have potential therapeutic benefits for breast cancer patients by making chemotherapy more effective. Sanguinarine thus has potential to be developed as a clinically relevant inhibitor of MKP-1 which could provide a novel avenue for therapeutic intervention in combination with chemotherapy in breast cancer patients. / AFRIKAANSE OPSOMMING: Inleiding: Kanker is 'n vinnig groeiende gesondheidsprobleem in Suid-Afrika, met borskanker as een van die vernaamste kankers wat vroue wêreldwyd raak. Daar is dus 'n behoefte aan nuwe terapeutiese opsies vir hierdie pasiënte en mitogeen-geaktiveerde proteïenkinase fosfatase-1 (MKP-1) is onlangs voorgestel as ‘n moontlike teiken. Verskeie studies toon dat mitogeen-geaktiveerde proteïenkinase fosfatases betrokke is by die ontwikkeling van kanker en ook belangrike rolspelers is in die reaksie van kanker op chemoterapie. Daarbenewens toon talle studies dat daar verhoogde MKP-1 uitdrukking in borskanker is, asook dat dit ‘n belangrike bemiddelaar is vir die weerstand wat borskanker teen chemoterapie bied. Ons het dus voorgestel dat die inhibisie van MKP-1 die sitotoksiese effek van doxorubicin op borskanker selle sal verhoog; sodoende sal die kanker selle beter reageer op behandeling en dit sal dus lei tot verhoogde seldood deur autofagie en apoptose. Metodes: MKP-1 is geïnhibeer met behulp van sanguinarine of MKP-1 siRNA in MDA-MB231 selle en dit is vergelyk met 'n bekende MKP-1 induseerder, dexamethasone. MDA-MB231 selle is met doxorubicin alleen behandel of in kombinasie met MKP-1 inhibeerders of ‘n induseerder. Seldood is bepaal deur middel van ‘n trypan blou en kaspase toetsingsmetode, asook met die westelike kladtegniek. Autofagie is bepaal deur westelike kladtegniek en vloeisitometrie. LC3 en p62 is gebruik as merkers van autofagie en kaspase 3 en PARP is as apoptose merkers gebruik. MKP-1 uitdrukking is geëvalueer deur middel van westelike kladtegniek. C57BL6 muise met kankeragtige gewasse is gebruik om apoptose en autofagie in vivo te ondersoek. MKP-1 is geïnhibeer met sanguinarine en die muise is behandel met ‘n kombinasie van sanguinarine en doxorubicin. Kaspase 3, LC3, p62 en MKP-1 uitdrukking is bepaal deur middel van die westelike kladtegniek. Resultate en bespreking: ‘n Konsentrasie en tyd kurwe het aangedui dat 5 μM doxorubicin die MDA-MB231 selle se lewensvatbaarheid aansienlik verminder het na 24 uur. MKP-1 uitdrukking is ook aansienlik verminder met sanguinarine en MKP-1 siRNA. Verder dui die resultate op 'n beduidende toename in apoptose in MDA-MB231 selle na behandeling met doxorubicin onder toestande van MKP-1 inhibisie. 'n Toename in autofagiese aktiwiteit is waargeneem na behandeling met doxorubicin en sanguinarine. Die kankeragtige gewasse van die C57BL6 muise toon ook 'n toename in apoptose en autofagie na behandeling met sanguinarine en doxorubicin. Hierdie resultate dui daarop dat die inhibisie van MKP-1 met sanguinarine die MDA-MB231 selle en E0771 sel gewasse gesensitiseer het tot doxorubicin-geïnduseerde apoptose deur middel van ‘n meganisme wat autofagie insluit. Gevolgtrekking: Hierdie bevinding kan hopelik in toekomstige studies gebruik word om doxorubicin weerstand te oorkom in borskanker selle waar MKP-1 verhoog is. Deur MKP-1 te teiken, kan dit lei tot potensiële terapeutiese voordele vir borskanker pasiënte en sodoende kan dit chemoterapie meer effektief maak. Sanguinarine het dus die potensiaal om ontwikkel te word as ‘n klinies relevante inhibeerder van MKP-1 wat sodoende kan dien as terapeutiese intervensie in kombinasie met chemoterapie vir borskanker pasiënte.