Omi/HtrA2 is a mitochondrial serine protease that is known to translocate to the cytoplasm upon induction of apoptosis and to activate caspase-dependent and caspase-independent cell death. The molecular mechanism of Omi/HtrA2's function is not clear but involves degradation of specific substrates. These substrates include cytoplasmic, mitochondrial, as well as nuclear proteins. We have isolated a new Omi/HtrA2 interactor, the THAP5 protein. THAP5 is a fifth member of a large family of transcription factors that are involved in cell proliferation, apoptosis, cell cycle control, chromosome segregation, chromatin modification and transcriptional regulation. THAP5 is an approximately 50kDa nuclear protein, with a restricted pattern of expression. Furthermore, there is no mouse or rat homolog for this protein. THAP5 mRNA is highly expressed in the human heart but some expression is also seen in the brain and skeletal muscle. The normal function of THAP5 in the heart or heart disease is unknown. THAP5 protein level is significantly reduced in the myocardial infarction (MI) area in the heart of patients with coronary artery disease (CAD). This part of the heart sustains most of the cellular damage and apoptosis. Our data clearly show that THAP5 is a specific substrate of the proapoptotic Omi/HtrA2 protease and is cleaved and removed during cell death. The molecular mechanism of THAP5's function is unclear. THAP5 can bind to a specific DNA sequence and repress transcription of a reporter gene. Our work suggests that THAP5 is a tissue specific transcriptional repressor that plays an important role in the normal function of the human heart as well as in the development of heart disease.

Thesis (Ph.D. (Health Sciences)) -- University of Limpopo, 2022 / Background: Cardio Vascular Diseases(CVD) are presently the main cause of high mortality rate around the world. In sub-Saharan Africa, CVDs contributes to about 13% of deaths, with 80% deaths being recorded from developing countries. Behavioural and metabolic risk factors contribute to high mortality rate related to development of CVD which self-management strategies and practices could effectively prevent. Objective: To explore and develop ―self-management strategies‖ to prevent the development of CVD among patients living with chronic conditions at Ga Molepo Area clinics in the Limpopo Province, South Africa. Methods: “A mixed method exploratory sequential design study was conducted in four primary health care settings in a rural setting.‖ A semi structured one-on-one interviews were conducted with 43 patients selected by non-probability homogenous purposive sampling for a qualitative strand. Qualitative findings that was obtained with thematic ―Tesch‘s open coding data analysis method guided the quantitative strand‖. A simple random sampling was used to sample 347 respondents for the quantitative strand.Descriptive statistics and chi-square were applied to analyze data from the quantitative strand. ``Results: The qualitative results revealed that there is different self management strategies used by patients living with hypertension and Diabetes Mellitus as risk factors of CVDs. The self-management strategies include engaging in physical activity, eating a healthy balanced diet, drinking a lot of water and adhering to treatment. The quantitative revealed majority of participants(74%) perform mild exercises with 26% performing moderate exercis.On the other hand 7% are having stress and depression and while 7% are smoking.`` Conclusions:The findings indicated that by adopting self-management strategies could be an effective way of preventing the development of CVD which complicates to hypertension and diabetic mellitus .Therefore are encouraged to adhere to strategies adhere to treat,healthy diet and perform mild exercises Recommendations Hence, the government needs to appoint CHW permanently to strengthen the guidelines on the prevention of CVD and also the risk factors that put the communities at risk of developing CVD. Communities need to adhere to preventative strategies ti order prevent CVD. / SPICES Project - (Scaling -up Packages of Interventions for CVD prevention in Europe and sub-Saharan)

The purpose of this study was to verify the efficiency of McDowell's leisure counseling model for potential use with cardiac rehabilitation and intervention programs in effecting leisure attitudes, work attitudes, work self-concept, leisure self-concept and leisure satisfaction. To test the effectiveness of McDowell's model, an experimental research design, utilizing a pretest-posttest control group design, was used. The independent variable was counseling and the dependent variables, stated as alternative research hypotheses, were a positive effect on leisure self-concept, work self-concept, leisure attitudes, work attitudes and leisure satisfaction. Over a three month period, 24 adult subjects from an Adult Fitness Intervention Program volunteered for leisure counseling and were randomly assigned to one of two groups after which treatment was also randomly assigned. A test for homogeneity of regression revealed the subjects to be representative of the population on all variables except Work Self-Concept. In the study, a client typically was married, middle-aged, white-collar male worker. To measure the dependent variables, five instruments designed to be used with McDowell's model were used. The data obtained from the leisure counseling sample was first subject to a test for homogeneity of regression after which hypotheses one, two, three and five, were subjected to an analysis of covariance. The findings of the analysis of data allowed for the rejection of the null hypotheses and the acceptance of the research hypotheses for hypotheses two and five. For hypotheses one and three, the researcher failed to reject the null hypothesis, and rejected the research hypothesis. An interpretation of these findings suggested that leisure counseling using McDowell's model can affect one's leisure self-concept and leisure satisfaction. From the study it was concluded that for the sample studied McDowell's leisure counseling model can be said to have positively increased the clients' leisure self-concept and leisure satisfaction on a short term basis. It was also concluded that leisure counseling as a component of cardiac rehabilitation/intervention programs using McDowell's model was said to be neither effective or ineffective for the sample studies. These results must account for the fact that the research methodology was not able to identify the possible reactive effect of the pretest. / Ed. D.

Previous research has shown that excessive cardiovascular reactivity may be important in the development of coronary heart disease. The present study examines the role of masculine cognitive appraisal of stress as a mediator of cardiovascular reactivity in men. The reactivity of men who differed on a measure of cognitive appraisal of masculine gender role stress (MGRS) was compared using the cold-pressor test under conditions of high and low masculine performance challenge. Under conditions of minimal challenge, it was predicted that high and low MGRS men would not differ on reactivity. Under high challenge, high MGRS men were expected to show greater reactivity than low MGRS men. Since coping responses are related to appraisal of stressful situations and impact on cardiovascular reactivity, subjects' coping responses were also assessed. Analysis of results for systolic blood pressure confirmed the major predictions. High MGRS men showed greater systolic blood pressure reactivity than low MGRS men under high challenge and equal or less reactivity under low masculine performance challenge. In general, the high and low MGRS groups did not differ in their use of coping strategies as a function of the high and low challenge condition. The implications of MGRS appraisal for men's health are discussed. / Master of Science

Twenty subjects were audio and video taped during their exercise leadership examination. The tapes were used to code the interactions that occurred between the exercise leaders and the participants in the simulated cardiac rehabilitation exercise session using an interaction analysis system developed specifically for this physical activity setting group. The system identified interactions that could occur during the warm-up, stimulus, and cool-down phases of the session. The tapes were coded using the Datamyte 801 Observational Recorder. The phases of the sessions were coded individually. A frequency count was made as the interaction categories occurred. The frequencies were converted into rates of interaction (f•min⁻¹) for comparison. The mean rates of interaction were low for the phases and overall (warm-up= .38/min; stimulus= .59/min; cool-down= .29/min; total= .46/min). The individual subject's rates of interaction were all less than 1 interaction per minute (minimum= .28/min; maximum= .72/min). Related t-tests across category facets between phases showed the instruction and explanation facets in the warm-up phase differed significantly from the same facets in the stimulus phase. The compliance facet differed significantly in the stimulus phase from the compliance facet in the other two phases. The monitor facet in the stimulus phase differed significantly from the monitor facet in the cool-down phase. There was no significant differences across facets between the warm-up and cool-down phases. Higher rates of interaction occurred more frequently in the stimulus phase. The coding showed the differences in the interactions of the exercise leaders in the different phases in the simulated exercise session. The low rates of interaction suggest that the exercise leaders may have been reactive to the specific examination situation in which these data were collected. / M.S.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2010 / This main aim of this study was to assess patients in a general cardiology practice in order to determine the systolic and diastolic profiles of these patients. The aim was also to determine what effect life style and risk factors may have on the echocardiographic variables measured during such an examination. The specific aim of this study was the importance of not only examining the systolic function but the necessity to also examine the diastolic profile of patients. Life-style plays an important role, with the main culprit being obesity. Obesity was the single most important factor that affected the diastolic profile of patients seen in this study. With obesity a combination of other risk factors related to obesity was observed. Most abnormalities found due to these risk factors were associated with diastolic changes in the left ventricle. Echocardiography is routinely used in daily practice, but the diagnostic value of this tool can be enhanced if proper analyses of the systolic as well as the diastolic profiles are determined. Many cardiologists only measure the systolic function of the heart as an indication of the well- being of the left ventricle, although in this study it was proven that systolic function did not alter with ageing or with changes in the risk profile. Hundred-and-twelve patients, divided into three age groups, were evaluated in this study. Both systolic and diastolic variables were measured and analysed for abnormalities. None of these patients had systolic function abnormalities, although they had detectable anatomic changes due to ageing, obesity and hypertension. Several abnormalities were found on the diastolic profile of these patients. Muscle thickness increased due to obesity and hypertension and even with ageing, but with no significant abnormalities in the systolic function of the heart. There was a slight increase in the circumferential shortening of the left ventricle and that both the septal and longitudinal functions decreased with ageing. It is noteworthy that even where the systolic function remained normal in ageing subjects, their diastolic profiles changed significantly. Assessment of left ventricular function required a meticulous and systematic approach. In this study forty- one percent of patients visiting this general practice had abnormalities of their diastolic function although their systolic function was normal. It was found that with ageing, especially in the older age group, important abnormalities occur in their diastolic profile. The most common changes were that the E- peak velocity decreased and that the Apeak velocity of the trans-mitral flow increased. It seemed that passive filling decreased with ageing but that active filling increased simultaneously, causing the cardiac output to remain constant in older subjects. This is important to know because diseases affecting the atrium may have a profound effect on the cardiac output of older patients, even if they have normal systolic function, (due to the decreased passive filling they need their active filling or atrial contraction to support a normal cardiac output). An important marker will be to look at the ratio of the E/A- velocities in older patients to determine the ratio of active against passive filling. Other than that, a relatively new tool in echocardiography called tissue Doppler was used to determine what happened to the muscle with ageing. Here it was demonstrated that the different layers of the left ventricle acted differently with ageing. Results showed that the longitudinal fibres weakened with ageing although the circumferential fibres remained unchanged or even strengthened with ageing. It was apparent in this study that the traditional use of only systolic function may not be adequate when evaluating relative asymptomatic patients presenting at a general cardiology practice. It is important to also evaluate the diastolic profiles of these patients in order to scientifically quantify their heart health, even in asymptomatic patients. It is important to routinely evaluate the diastolic profile of patients so that early detection of these diastolic variables can be detected and timely consideration for its treatment can be given by their cardiologist. It is also important to take note of the significance of the obesity problem and the effect it has on the heart’s health. In conclusion, this study emphasizes the importance of the echocardiographic evaluation of diastolic cardiac function in addition to routine systolic evaluation in asymptomatic patients. This will enable the clinician to detect abnormalities early and tailor therapy accordingly. Lifestyle related risk factors, especially obesity, also have significant effects on diastolic cardiac function.

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.

Thesis MSc (Physiological Sciences))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Ischaemic heart disease is a leading cause of death worldwide and is also largely contributing to deaths in Africa. Better treatment or even prevention of ischaemia/reperfusion injury in the heart, necessitates a better understanding of the molecular pathways and mechanisms of cell death. Three types of cell death can occur in the diseased myocardium. Type I, better known as apoptotic cell death, is characterised by cell shrinkage and chromatin condensation, type II, known as autophagic cell death, is characterised by intracellular accumulation of double membranes vacuoles and type III, necrotic cell death, is characterised by cellular swelling and loss of membrane integrity. Many signaling pathways are activated during ischaemia/reperfusion injury which include the mitogen activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK) and p38 MAPK. These kinases are dephosphorylated by appropriate phosphatases. MAPK phosphatase-1 (MKP-1), a dual specificity phosphatase, inactivates the MAPKs by dephosphorylating specific Thr/Tyr residues. Upregulation of MKP-1 during ischaemia/reperfusion injury has been shown to be cardioprotective, however no knowledge regarding a role of MKP-1 in autophagy exists. Therefore the aim of this study is to investigate the role of MKP-1 in autophagy, apoptosis and necrosis during simulated ischaemia/reperfusion injury in the heart.METHOD: H9C2 cells (rat cardiomyocytes) were cultured under standard conditions. Upon reaching 75-80% confluency, cells were treated for 30 min during normoxic conditions with dexamethasone, to induce MKP-1 expression, or sanguinarine, to inhibit MKP-1 induction. Thereafter, they were exposed to 3 hrs simulated ischaemia (induced by an ischaemic buffer and 5% CO2/1% O2) in the presence of the above mentioned treatments. Cells were then allowed to reperfuse for 30 min in the presence of dexamethasone or sanguinarine. Samples were analysed after simulated ischaemia and after reperfusion. Cell viability was measured by MTT assay. Propidium iodide and Hoechst staining were used to assess morphological markers of apoptosis and necrosis. LDH release during reperfusion was assessed as indicator of necrotic cell death. LysoTracker®Red was used to visualise the autophagic flux occurring during ischaemia/reperfusion in the cell. Flow cytometry was used to quantify cells stained with acridine orange as indicator for autophagy. Autophagic and apoptotic protein markers as well as MAPK and MKP-1 activity were analysed by Western Blotting. RESULTS: Our results indicate a clear relationship between MKP-1 induction, autophagy and cell survival during simulated ischaemia/reperfusion (SI/R). MKP-1 inhibition during SI/R resulted in decreased autophagy activity accompanied by significant apoptotic and necrotic cell death. Increased MKP-1 induction, on the other hand, during SI/R resulted in increased levels of autophagy activity and subsequent attenuation of apoptotic and necrotic cell death. p38 MAPK phosphorylation was significantly higher while MKP-1 was inhibited and significantly lower while MKP-1 was induced. This strongly indicates that upregulation of MKP-1, known to attenuate ischaemia/reperfusion injury, has an important role in cell survival during ischaemia/reperfusion injury in the heart, through its involvement in the regulation of autophagic activity as a stress response against apoptotic or necrotic cell death. / AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die grootste oorsake van sterftes wêreldwyd en dra ook beduidend by tot sterftes in Afrika. Om iskemiese hartsiektes te behandel of selfs te voorkom, is 'n goeie begrip van die molekulêre paaie wat betrokke is tydens iskemie/herperfusie, noodsaaklik. Drie tipes seldood kom tydens patologiese toestande in die hart voor. Tipe I, ook bekend as apoptotiese seldood, word gekenmerk deur selkrimping en kromatien kondensasie, tipe II, ook bekend as autofagiese seldood word gekenmerk deur intrasellulêre opeenhoping van dubbelmembraan vakuole en tipe III, bekend as nekrotiese seldood, word deur sellulêre swelling en verlies van membraan integriteit gekenmerk. Iskemie/herperfusie lei tot die aktivering van seintransduksiepaaie wat die MAPKs, soos p38, ERK en JNK insluit. Hierdie kinases word deur die gepaste fosfatases gedefosforileer. MKP-1, 'n dubbele spesifieke fosfatase, deaktiveer MAPKs deur hul Thr/Tyr eenhede te defosforileer. Alhoewel daar al voorheen getoon is dat verhoogte MKP-1 ‘n beskermende funksie in die hart tydens iskemie/herperfusie het, is daar nog geen bewyse vir ‘n rol van MKP-1 tydens autofagie nie. Die doel van hierdie studie is dus om die rol van MKP-1 in autofagie, apoptose en nekrose te ondersoek tydens gesimuleerde iskemie/herperfusie in die hart. METODE: H9C2 selle (rot ventrikulêre hartselle) is onder standaard toestande gekweek. Wanneer die selle 75-80% konfluensie bereik het, is dit behandel met dexamethasone of sanguinarine onder standaard toestande vir 30 min. Daarna is selle blootgestel aan 3 ure iskemie, in die teenwoordigheid van dexamethasone of sanguinarine. Selle is dan toegelaat om vir 30 min te herperfuseer, weer in die teenwoordigheid van dexamethasone of sanguinarine. Monsters is na iskemie en herperfusie geneem vir analise. Selvatbaarheid is gekwantifiseer deur ‘n MTT bepaling. Morfologiese merkers van seldood is bepaal met behulp van propidium iodide en Hoechst kleuringsmetodes. Laktaatdehidrogenase (LDH) vrystelling tydens herperfusie is as merker van nekrose gebruik. Autofagie is gevisualiseer deur gebruik te maak van LysoTracker®Red kleuring tydens iskemie en herperfusie. Akridienoranje is gebruik om suur kompartemente te kleur. Vloeisitometrie is as kwantifiseringstegniek vir autofagie gebruik. Western Blotting is gebruik om uitdrukking van merkerproteïene van autofagie en apoptose sowel as MAPK en MKP-1 aktiwiteit tydens iskemie/reperfisie te bepaal. RESULTATE: Ons resultate toon ‘n verband tussen MKP-1 induksie, autofagie en seloorlewing gedurende gesimuleerde iskemie/herperfusie (SI/R) aan. MKP- 1 inhibisie gedurende SI/R het tot ‘n afname in autofagie gelei tesame met ‘n beduidende toename in apoptotiese en nekrotiese seldood. Verhoogde MKP-1 induksie gedurende SI/R, daarteenoor, het autofagiese aktiwiteit verhoog, gepaardgaande met ‘n verlaging in apoptose en nekrose. p38 MAPK fosforilasie was beduidend hoër tydens MKP-1 inhibisie en laer met MKP-1 induksie. Hierdie resultate toon dat MKP-1 ‘n belangrike rol in seloorlewing speel tydens iskemie/herperfusiesskade in die hart, deur sy deelname in die regulering van autofagiese aktiwiteit as ‘n stres reaksie teen apoptotiese en nekrotiese seldood.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced.Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure. / AFRIKAANSE OPSOMMING: Hartversaking as gevolg van kardiomiopatie of kardiale geleidingsiekte is ‘n hoof-oorsaak van mortaliteit and morbiditeit in beide ontwikkelde en ontwikkelende lande. Alhoewel gedefinieer as verskillende kliniese entiteite is oorerflike vorms van kardiomiopatie en kardiale geleidingsstoornisse geïdentifiseer met oorvleuelende kliniese eienskappe en/of molukulêre oorsake. Die doelwit van hierdie studie was om die molukulêre oorsaak van progressiewe familiële hartblok tipe II (PFHBII), ‘n oorerflike kardiale geleidingsstoornis, wat in ‘n Suid-Afrikaanse Kaukasiër familie segregeer (Brink en Torrington, 1977), te identifiseer. Die beskikbaarheid van familie data, beteken dat koppelingsanalise gebruik kan word om die chromosomale posisie van die siekte-veroorsakende geen te identifiseer. Menslike Genoom Projek (MGP) databanke het addisionele hulpbronne beskikbaar gestel om die identifikasie van posisionele kandidaat gene te vergemaklik. Kliniese ondersoeke is uitgevoer op PFHBII-geaffekteerde familielede, en waar beskikbaar is kliniese rekords van persone, wat in ‘n vorige studie deur Brink en Torrington (1977) geassesseer was, herontleed. Retrospektiewe data-analise het die kliniese herdefinisie van PFHBII voorgestel. Daarna is koppelingsanalise gebruik om dilateerde kardiomiopatie (DKM), hipertrofiese kardiomiopatie (HKM) en kardiale geleidingssiekte-veroorsakende loki op chromosoom 1, 2, 3, 6, 7, 9, 11, 14, 15 en 19 te ondersoek vir hul moontlike bydrae tot die ontwikkeling van PFHBII. Toe die lokus gekarteer was, is bioinformatiese ondersoeke gebruik om posisionele kandidaat gene te identifiseer en prioritiseer vir mutasie analise. Die retrospektiewe en prospektiewe kliniese ondersoek het PFHBII herdefinieer as ‘n geleidingsstoornis en DKM-verbonde siekte, en terselfde tyd het dit gelei tot die opsporingvan nog familielede. Toevallig het kandidaat loki-analise die PFHBII lokus op chromosoom 1q32 gekarteer, na ‘n gebied wat met ‘n voorheen-beskyfde DKM-verbonde stoornis (CMD1D) oorvleuel, met die opwekking van ‘n makisimum paargewyse lod-getal van 3.13 by D1S3753 (theta [θ] = 0.0) en ‘n maksimum multipunt lod-getal van 3.7 tussen D1S3753 en D1S414. Genetiese fynkartering en haplotipe-analise het die PFHBII-veroorsakende lokus afwaards van die CMD1D lokus geplaas, in ‘n 3.9 centimorgan (cM) gebied op chromosoom 1q32.2-q32.3, telomeries van D1S70 en sentromeries van D1S505. Bioinformatiese analise het daarnatoe gelei dat sewe kandidaat gene vir mutasie analise geprioritiseerd is, naamlik, gene wat onderskeidelik ‘n kalium kanaal (KCNH1), ‘n ekstrasellulêre matriksproteïen (LAMB3), ‘n proteïen fosfatase (PPP2R5A), ‘n aansluiter proteïen wat met ‘n sitoskilet proteïen bind (T3JAM), ‘n asieltansferase (KIAA0205) en twee gene moontlik betrokke in energie homeostase (RAMP en VWS59) enkodeer. Die PFHBII-veroorsakende geen is nie geïdentifiseer nie, alhoewel enkele volgorde-wisselings geïdentifiseer is in vier van die sewe geanaliseerde kandidaat gene. Alhowel die molekulêre oorsaak van die siekte nie vasgestel is nie, het die huidige studie die onderliggende betrokkenheid van DKM in die pathogenese van PFHBII gedefinieer. Die nuwe kliniese klassifikasie van PFHBII is gepubiliseer (Fernandez et al., 2004) en sal lei tot die identifisering van nog geaffekteerde persone in Suid Afrika of in ander lande. Die identifikasie van ‘n nuwe siekte-verbonde lokus mag lei tot die toekomstige identifikasie van ‘n nuwe DKM-verbonde genetiese oorsaak wat, opsig self, dalk insig kan gee in die molekulêre patofisiologie van hartversaking.

published_or_final_version / Community Medicine / Master / Master of Philosophy