Thesis (M.A.)--University of North Carolina at Chapel Hill, 2004. / Includes bibliographical references (leaves 48-52). Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

Thesis (M.A.)--University of North Carolina at Chapel Hill, 2004. / Includes bibliographical references (leaves 48-52).

Thesis (MScMedSc)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Obesity is a major risk factor for ischaemic heart disease. Obesity-induced metabolic abnormalities have been associated with increased oxidative stress which may play an important role in the increased susceptibility to myocardial dysfunction and ischaemiareperfusion (I/R) injury seen in obesity. The pineal gland hormone, melatonin, has powerful antioxidant properties. Previous studies have shown that short-term or acute melatonin administration protects the normal healthy heart of lean animals against I/R damage. However, the effects of melatonin on the heart in obesity remain unknown. Moreover, the myocardial signalling mechanisms associated with the cardioprotective effects of melatonin have not been established. Using a rat model of diet induced obesity, we set out to: 1) investigate the effects of chronic melatonin administration on the development of diet-induced systemic alterations including biometric and metabolic parameters and oxidative stress, 2) determine whether chronic melatonin treatment protects the myocardium against ischaemia-reperfusion injury, and 3) determine whether melatonin treatment confers cardioprotection by altering the reperfusion injury salvage kinase (RISK) pathway signalling and the pro-apoptotic p38 MAPK, AMPK and GLUT-4 expression. Male rats weighing 200±20g were randomly allocated to four groups: 1) C, control rats receiving a standard commercial rat chow and drinking water without melatonin; 2) CM, control rats receiving melatonin (4mg/kg/day) in drinking water; 3) D, diet-induced obesity rats, receiving a high calorie diet and drinking water without melatonin; 4) DM, diet-induced obesity rats, receiving melatonin in drinking water. After 16 weeks of treatment and feeding, rats were weighed and blood and myocardial tissue collected to document biochemical and molecular biological changes. Hearts were perfused on the isolated working rat heart perfusion apparatus for the evaluation of myocardial function and infarct size. The Reperfusion Injury Salvage Kinases (RISK) pathway (PKB/Akt (Ser-473), ERK p42/ p44) and p38 MAPK (mitogenactivated protein kinase) were investigated in pre-and post-ischaemic hearts using Western blotting techniques. Post-ischaemic activation of AMPK (5’AMP-activated protein kinase) (Thr- 172) and GLUT-4 (glucose transporter) expression were also investigated. Serum and baseline myocardial glutathione (GSH) content were measured. In addition, serum lipid peroxidation products: thiobarbituric reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxide (LOOH), were also determined. The high-calorie diet caused increases in body weight, visceral adiposity, heart weight, serum insulin, leptin, blood triglycerides, and low HDL-cholesterol levels. Blood glucose levels were similar for both diet fed rats and controls. Myocardial glutathione, serum glutathione, total cholesterol, TBARS, LOOH, CD as well as total cholesterol (TC) levels were not affected by the high calorie diet. Chronic melatonin treatment reduced body weight gain, visceral adiposity, heart weight, blood triglycerides, serum insulin, HOMA index, serum leptin (DM vs D, p<0.01), and increased blood HDL-C in diet treated rats while there was no effect on these parameters in control rats, despite the reduction in body weight, heart weight and visceral adiposity. Melatonin treatment had no effect on myocardial or serum GSH and LOOH in either control or diet animals. It however reduced TBARS and CD in the diet and control groups, respectively. At baseline, chronic melatonin treatment caused a significant increase in phospho-PKB/total PKB ratio and a concomitant reduction in phospho-p38 MAPK/total p38 MAPK ratio of control hearts while there were no such effects on diet-induced-obesity hearts. Infarct size was significantly reduced by melatonin in both diet and control groups (DM: 16.6±2.0%; D: 38.4±2.6% (p < 0.001), and CM: 12.8±1.5%; C: 30.4±1.0%, p<0.001). After coronary artery occlusion and 30 minutes of reperfusion, melatonin increased percentage recovery of aortic output (DM: 28.5±6.5%; D: 6.2±6.2%, p<0.01), cardiac output (DM: 44.4±5.2%; D: 26.6±5.1%, p < 0.01) and total work (DM: 34.5±5.6%; D: 20.4±7.9%, p<0.05) of diet-induced obesity hearts, while having no effect on control hearts. During reperfusion, hearts from melatonin treated rats had increased activation of PKB/Akt (p<0.01), ERK42/44 (p<0.05), and reduced p38 MAPK activation (p<0.05). There was no difference in post-ischaemic activation of AMPK (Thr-172) and GLUT-4 expression in either control or diet fed rats. We successfully demonstrated that chronic melatonin treatment prevented the development of diet-induced metabolic abnormalities and improved ex vivo myocardial function. Melatonin protected the heart against ischaemia-reperfusion injury that was exacerbated in obesity. This was achieved by activation of the RISK pathway. The antioxidant properties of melatonin were involved in these cardioprotective effects. / AFRIKAANSE OPSOMMING: Vetsug of obesiteit is een van die hoof risikofaktore vir iskemiese hartsiekte. Obesiteitgeinduseerde metaboliese abnormaliteite gaan met verhoogde oksidatiewe stres gepaard wat op sy beurt ‘n belangrike rol mag speel in die miokardiale wanfunksie en verhoogde vatbaarheid vir iskemie-herperfusie (I/H) beskadiging, kenmerkend van vetsug. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige anti-oksidant. Vorige studies het getoon dat kort-termyn of akute toediening van melatonien die normale hart van gesonde diere teen I/H beskadiging deur middel van sy anti-oksidant aksies beskerm. Die effek van melatonien op die hart in obesiteit is egter nog onbekend. Hierbenewens is die miokardiale seintransduksie meganismes geassosieer met die beskermende effekte van die hormoon nog nie ontrafel nie. ‘n Model van dieet-geinduseerde obesiteit in rotte is gebruik om die volgende te bepaal: (i) die effek van kroniese melatonientoediening op die ontwikkeling van dieet-geinduseerde sistemiese veranderinge soos biometriese en metaboliese parameters en oksidatiewe stres (ii) die effek van kroniese melatonienbehandeling op die respons van die hart op I/H beskadiging en (iii) die rol van herperfusie beskadiging op die aktivering van PKB/Akt en ERK42/44 (die sg RISK seintransduksiepad), die pro-apoptotiese p38MAPK, AMPK sowel as die uitdrukking van GLUT-4. Manlike Wistar rotte (200±20g) is ewekansig in vier groepe verdeel: (i) C, kontrole rotte wat ‘n standaard rotdieet en drinkwater sonder melatonien ontvang (ii) CM, kontrole rotte wat melatonien (4mg/kg/dag) ontvang (iii) D, dieet-geϊnduseerde vet rotte wat ‘n hoë kalorie dieet en drinkwater sonder melatonien ontvang (iv) DM, dieet-geϊnduseerde vet rotte wat melatonien (4mg/kg/dag) in die drinkwater ontvang. Na 16 weke van behandeling, is die rotte geweeg, bloed en hartweefsel gekollekteer vir biochemiese en molekulêre biologie bepalings. Harte is geperfuseer volgens die werkhartmodel, blootgestel aan iskemie/herperfusie vir evaluering van funksionele herstel en infarktgrootte. Uitdrukking en aktivering van PKB/Akt (Ser-473), ERKp42/p44 en p38MAPK van pre-en postiskemiese hartweefsel is met behulp van Western blot bepaal. Postiskemiese aktivering van AMPK (5’AMP-aktiveerde proteϊen kinase) (Thr-172) en GLUT-4 (glukose transporter) is op soortgelyke wyse bepaal. Serum en basislyn hartweefsel glutatioon (GSH) inhoud asook tiobarbituursuur reaktiewe substans (TBARS), gekonjugeerde diene (CD) en lipiedhidroperoksied (LOOH) konsentrasies is bepaal. Resultate Die hoë kalorie diet het ‘n toename in liggaamsgewig, visserale vet, hartgewig, serum insulien, leptien, plasma trigliseried en lae HDL-cholesterol vlakke teweegebring. Bloed glukosevlakke was egter dieselfde in die vet en kontrole rotte. Miokardiale glutatioon, serum glutatioon, totale cholesterol, TBARS, LOOH, CD is nie deur die dieet beinvloed nie. Chroniese melatonien behandeling het die liggaamsgewig, visserale vet, hartgewig, plasma trigliseried, serum insulien en leptien, HOMA indeks verlaag (DM vs D, p<0.05) en die HDL-cholesterol verhoog in die dieetrotte, terwyl dit geen effek op hierdie parameters in kontrole rotte gehad het nie (uitgesonderd ‘n afname in liggaamsgewig, hartgewig en visserale vet). Melatonien behandeling het geen effek op hart of serum GSH en LOOH in kontrole en vet rotte gehad nie. Dit het egter die TBARS en CD in beide vet en kontrole rotte verlaag. Chroniese melatonien toediening het ‘n beduidende toename in basislyn fosfo PKB//totale PKB ratio en ‘n afname in fosfo p38MAPK/totale p38MAPK ratio teweegebring in harte van kontrole rotte, maar soortgelyke effekte is nie in die harte van die vet rotte waargeneem nie. Infarktgrootte is beduidend deur melatonienbehandeling verlaag in beide dieet en kontrole groepe (DM: 16.6± 5.2%, D: 38.4 ±2.6% (p<0.001); CM: 12.8± 1.5%; C 30.4±1.0 (p<0.001). Na koronere arterie afbinding en 30 min van herperfusie, het melatonien die persentasie herstel van aorta omset (DM: 28.5± 6.5%; D: 6.2± 6.2%, p<0.01), kardiale omset ( DM: 44.4± 5.2%D: 26.6±5.1%, p<0.01) en totale werk (DM: 34.5 5.6%; D 20.4± 7.9%, p<0.05) in die harte van dieetrotte verbeter, terwyl dit sonder effek was in kontrole harte. Tydens herperfusie het harte van melatonienbehandelde rotte verhoogde aktivering van PKB/Akt (p<0.01) en ERKp42/p44 (p<0.05) getoon, terwyl aktivering van p38MAPK verlaag is (p<0.05). Geen verskil in postiskemiese aktivering van AMPK en GLUT-4 uitdrukking is in beide kontrole en dieetrotte waargeneem nie. Ons het daarin geslaag om aan te toon dat chroniese melatonienbehandeling die ontwikkeling van dieet-geϊnduseerde metaboliese abnormaliteite beduidend kan voorkom en ex vivo miokardiale funksie verbeter. Melatonien het ook die hart teen iskemie/herperfusie beskadiging beskerm in beide kontrole en dieetrotte. Bogenoemde veranderinge het met aktivering van PKB/Akt en ERKp42/p44 gepaard gegaan. Die anti-oksidant effekte van melatonien was heelwaarskynlik hierby betrokke.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity. / AFRIKAANSE OPSOMMING: INLEIDING: Die meganisme verantwoordelik vir vetsug-verwante kardiomiopatieë is huidiglik nie bekend nie maar kardiale insulienweerstandigheid word geïmpliseer as een van die hoof bemiddelaars van vetsug-verwante hartsiektes. Verskeie studies het versteurings in die insulien-gemediëerde PI3K/PKB/Akt pad geassosieer met die bevordering van hierdie insulienweerstandigheid. Daarbenewens is dit getoon dat hierdie pad betrokke is in die regulering van miokardiale apoptose, wat op sy beurt geïmpliseer is in 'n aantal kardiovaskulêre siektes. Daar is tans min studies beskikbaar wat die vroeë en laat gevolge van obesiteit op die hart vergelyk. DOELWITTE: Om die vroeë en gevorderde stadiums van vetsug te vergelyk in terme van miokardiale (i) PI3K/PKB/Akt seintransduksie, (ii) apoptotiese seintransduksie en (iii) mitokondriale integriteit. Verder, het die studie ten doel om die kardiale mitokondriale (i) PI3K/PKB/Akt en (ii) apoptotiese seintransduksie en (iii) integriteit in die gevorderde stadiums van vetsug te bepaal. METODES: Manlike Wistar rotte is ewekansig toegewys aan óf 'n kontrole of dieet-geïnduseerde vetsug (DIO) groep. Kontroles is met 'n normale rotkos dieet en die DIO groep met 'n hoë kalorie dieet (normale rotkos aangevul met sukrose en kondensmelk) gevoed. Die dieet is vir 8 of 20 weke volgehou en daarna was die liggaamsgewig, intra-peritoneale vet massa, en vastende bloed glukose en insulien vlakke (insluitende intra-peritoneale glukose toleransie toets (IPGTT`s)) bepaal. Gevriesklampte harte van beide groepe is onderwerp aan sitosoliese WB-analise vir die PI3K p85 subeenheid, PKB / Akt, GSK-3α/β, Bad, Bax en Bcl-2. `n Fraksie van hierdie harte is ook onderwerp aan westerse klad analise (WK-analise) van die mitokondriale elektron vervoer ketting (EVK) komplekse (I-V). Daarna is bogenoemde proteïene ondersoek in mitokondrieë geïsoleer uit die 20 weke groep ná die toediening van insulien en die blootstelling van die harte aan iskemie. Die oksigraaf mitokondriale oksidatiewe fosforilering (OXPHOS) kapasiteit analise is dan op mitokondrieë van 20 weke DIO en kontrole groepe uitgevoer en daarna is 'n sitraatsintase (SS) aktiwiteitstoets gedoen. RESULTATE: Na die 8 en 20 weke dieet, het die intra-peritoneale vet massa, vastende plasma glukose en insulien vlakke in die DIOs aansienlik toegeneem, in vergelyking met hul kontroles. Sitosoliese WK-analise: Die tp85, pp85 en pPKB/Akt vlakke was beduidend hoër in die DIOs in vergelyking met die kontroles, na 8 weke van die dieet. Verder is die pBad en Bax vlakke beduidend verhoog in hierdie diere. Na 20 weke van die dieet, het die pp85, tPKB/Akt en pPKB/Akt vlakke beduidend afgeneem in die DIOs, in vergelyking met die kontroles. Die tBad was beduidend verhoog, terwyl die Bad verhouding van gefosforileerde oor die totale proteïen uitdrukking (P/T)-verhouding) beduidend verminder het in hierdie diere. SS aktiwiteitstoets: SS aktiwiteit is beduidend verminder in die DIOs, teenoor die kontroles, op 20 weke. Mitokondriale EVK WK-analise: Die subeenheid uitdrukking in komplekse I-III en V was nie beduidend verskillend na 8 weke nie. Na 20 weke egter, was die uitdrukking aansienlik laer in komplekse I en II. Interessant genoeg, is die uitdrukking aansienlik verhoog in komplekse III en V. Mitokondriale OXPHOS analise: Die ADP/O verhouding met (1) glutamaat of (2) palmitiel-L-karnitien as substraat, het beduidend afgeneem in die DIOs teen 20 weke. Mitokondriale WK-analise: Die pp85 subeenheid was beduidend verhoog in die kontrole en DIO groepe, blootgestel aan insulien en iskemie, in vergelyking met die onbehandelde kontroles. Die Bcl-2 vlakke was beduidend verminder in die insulien en isgemie DIOs, in vergelyking met onbehandelde DIOs. Die tBad uitdrukking het nie beduidend verskil tussen die insulien en onbehandelde kontroles nie, terwyl die tBad beduidend verhoog was in die isgemie kontroles versus onbehandelde kontroles. Alle beduidende verskille is geneem as p<0.05. GEVOLGTREKKING: Die resultate dui daarop dat die eerste fase van dieet-geïnduseerde obesiteit geassosieer is met kardiale beskerming want `n toename in PI3K/PKB/Akt seintransduksie en 'n afname in apoptotiese merkers is waargeneem. In teenstelling, in die gevorderde stadium van vetsug is daar 'n afname in aktiwiteit in die PI3K/PKB/Akt pad wat verband hou met verhoogde miokardiale apoptose en verminderde mitokondriale funksie en integriteit.

Sedentary lifestyles and energy-rich diets are driving an increasing prevalence of abdominal obesity, which is associated with cardiovascular risk. Reliable estimates of the worldwide prevalence of abdominal obesity are needed to quantify the associated health risk. The International Day for the Evaluation of Abdominal obesity (IDEA) study is a large, international epidemiological cross-sectional study designed to provide reliable data on the distribution of waist circumference according to region, gender, age, and socio-economic level in 177 345 primary care patients from 63 countries across five continents. Any non-pregnant patient aged 18–80 consulting one of the randomly selected primary care physicians on two pre-defined half days was eligible to participate in the study. The primary objective was to estimate the prevalence of abdominal obesity in primary care, in each participating country. Secondary objectives were to estimate the prevalence of hypertension, type 2 diabetes, dyslipidaemia, and smoking, and to evaluate their associations with abdominal obesity, according to age, gender, and socio-economic level and region. The IDEA study will provide the first global map of the prevalence of abdominal obesity and associated comorbidities in primary care practice.

Genome wide association studies (GWAS) have revolutionized our approach to mapping genetic determinants of complex human diseases. However, even with success from recent studies, we have typically been able to explain only a fraction of the trait heritability. GWAS are typically analysed by testing for the marginal effects of single variants. Consequently, it has been suggested that gene-gene interactions might contribute to the missing heritability of complex diseases. GWAS incorporating interaction effects have not been routinely applied because of statistical and computational challenges relating to the number of tests performed, genome-wide. To overcome this issue, I have developed novel methodology to allow rapid testing of pairwise interactions in GWAS of complex traits, implemented in the IntRapid software. Simulations demonstrated that the power of this approach was equivalent to computationally demanding exhaustive searches of the genome, but required only a fraction of the computing time. Application of IntRapid to GWAS of a range of complex human traits undertaken by the Wellcome Trust Case Control Consortium (WTCCC) identified several interaction effects at nominal significance, which warrant further investigation in independent studies. In an attempt to fine-map the identified interacting loci, I undertook imputation of the WTCCC genotype data up to the 1000 Genomes Project reference panel (Phase 1 integrated release, March 2012) in the neighbourhood of the lead SNPs. I modified the IntRapid software to take account of imputed genotypes, and identified stronger signals of interaction after imputation at the majority of loci, where the lead SNP often had moved by hundreds of kilobases. The X-chromosome is often overlooked in GWAS of complex human traits, primarily because of the difference in the distribution of genotypes in males and females. I have extended IntRapid to allow for interactions with the X chromosome by considering males and females separately, and combining effect estimates across the sexes in a fixed-effects meta-analysis. Application to genotype data from the WTCCC failed to identify any strong signals of association with the X-chromosome, despite known epidemiological differences between the sexes for the traits considered. The novel methods developed as part of this doctoral work enable a user friendly, computationally efficient and powerful way of implementing genome-wide gene-gene interaction studies. Further work would be required to allow for more complex interaction modelling and deal with the associated computational burden, particularly when using next-generation sequencing (NGS) data which includes a much larger set of SNPs. However, IntRapid is demonstrably efficient in exhaustively searching for pairwise interactions in GWAS of complex traits, potentially leading to novel insights into the genetic architecture and biology of human disease.