Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The prevalence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis has been increasing to alarming levels globally. This has been exacerbated by tuberculosis (TB) co-infection with HIV where the epidemic is endemic. South Africa as a developing country is hit hard by TB and efforts to develop TB drugs that are compatible with anti-retroviral medication and also effective against MDR/XDR, could help shorten the treatment duration of the current TB treatment regimens. This thesis presents the identification and characterisation of a novel furanone based compound (F1082) and its derivatives as leads for anti-TB drug development. Furanones are generally known for an array of biological activities ranging from antibacterial, antifungal and antitumor. F1082 has an aromatic benzene structure and was identified from screening synthetic compounds against M. tuberculosis. It is potent against M. tuberculosis at minimum inhibitory concentration (MIC) of 8 μg/ml. It is selective for mycobacteria since it did not inhibit the growth of Gram-positive and Gram-negative bacteria at concentrations five times the MIC for M. tuberculosis. F1082 is generally bacteriostatic around MIC concentrations in its effects against M. tuberculosis however; it may be bactericidal at higher concentrations. It is as effective against MDR, XDR and clinical isolates of M. tuberculosis at the same concentration as the M. tuberculosis H37Rv reference strain. This suggests that F1082 may have a different mechanism of action compared to current TB drugs. It has been shown to have no antagonistic effect with the first-line anti-TB drugs and it has been shown to synergize with rifampicin by reducing the MIC of rifampicin. A drawback of F1082 is that it is cytotoxic to human cell lines, but this is presently being addressed through the synthesis of analogues that have shown improved activity and less cytotoxicity. The synthesis of more than 40 analogues has led to identification of 4 compounds that have more than five times higher activity and more than 100 times less cytotoxicity against human cell-lines. Microarray analyses have identified possible metabolic pathway/s in M. tuberculosis that is/are affected by F1082. One subset of genes which showed the most prominent alteration encodes the siderophores, which are involved with iron homeostasis in the M. tuberculosis bacillus. Of these genes, 7 were of interest (mbtB, mbtC, mbtD, mbtE, mbtF, mbtH and bfrB) as they all fall in the same cluster and are involved in iron acquisition. Due to the involvement of iron we also show that F1082 generates oxidative stress that is metal (iron) dependent. From the results we conclude that F1082 is a promising antituberculosis lead compound with unique target properties and also specificity against mycobacteria. / AFRIKAANSE OPSOMMING: Die voorkoms van veelvuldige middelweerstandige M.tuberculosis (MDR) en uiters middelweerstandige M.tuberculosis (XDR) is besig om toe te neem teen ‘n kommerwekkende tempo wêreldwyd. Hierdie situasie word vererger met die ko-infektering van M.tuberculosis en HIV. Suid- Afrika, as ontwikkelende land, word sleg benadeel met tuberkulose siekte. Antituberkulose middels wat kan saamwerk met bestaande antiretrovirale middels en ook effektief is teen MDR en XDR stamme, kan alles meewerk om die behandelingstyd van tuberkulose te verkort. In hierdie tesis identifiseer en karakteriseer ons ‘n furanoon-gebaseerde verbinding (F1082) en derivate daarvan as voorloper-middels vir anti-tuberkulose middelontwikkeling. Furanone is algemeen bekend vir ‘n verskeidenheid van biologiese aktiwiteite insluitende antibakteriële-, antifungale- en antitumor aktiwiteite. F1082 bevat ‘n aromatiese benseenstruktuur en is oorspronklik geïdentifiseer gedurende die skandering van sintetiese middels teen M.tuberculosis. Dit het ‘n sterk werking teen M.tuberculosis met ‘n minimum inhibitoriese konsentrasie (MIC) van 8ug/ml. Dit is baie selektief vir mikobakterieë aangesien dit nie gram-positiewe of gram-negatiewe bakterieë teen 5 maal die MIC, soos vir M.tuberculosis, geïnhibeer het nie. F1082 is bevind om, by laer konsentrasies, bakteriostaties te wees in sy aktiwiteit teen M.tuberculosis maar by hoër konsentrasies word ‘n meer bakteriosidiese effek waargeneem. F1082 is effektief teen MDR, XDR en kliniese isolate van M.tuberculosis en teen dieselfde konsentrasie soos vir die M. tuberculosis H37Rv verwysingstam waargeneem is. Dit impliseer dat F1082 dalk ‘n alternatiewe meganisme van werking het in vergelyking met die van die huidige TB teenmiddels. F1082 toon geen antagonistiese werking in kombinasie met die voorste anti- TB middels nie, maar toon wel sinergistiese werking in kombinasie met rifampisien. F1082 toon nog sitotoksiese aktiwiteit teenoor menslike sellyne, maar die sintese van derivate van F1082 toon tot dusvêr groter anti-TB aktiwiteit en verminderde sitotoksisiteit. Die sintese van meer as 40 homoloë het gelei tot die identifisering van vier verbindings met vyf keer hoër anti-TB aktiwiteit en honderd keer verminderde sitotoksisiteit teen menslike sellyne as F1082 self. “Microarray” ontledings het ‘n aantal metabolise paaie geïdentifiseer waar F1082 ‘n effek kan uitoefen. Een stel gene wat die mees uitstaande effek toon kodeer vir siderofore wat betrokke is by yster homeostase in M.tuberculosis. Van hierdie gene was daar sewe van belang omdat hulle in dieselfde groep voorkom en almal betrokke is by ysteropname (mbtB, mbtC, mbtD, mbtE, mbtF, mbtH, bfrB). Weens die rol wat F1082 in ysterhomeostase speel, toon ons ook dat F1082 intrasellulêre oksidatiewe stres bevorder wat yster afhanklik is. Al ons resultate dui daarop dat F1082 ‘n belowende ant-TB voorloper verbinding is met spesifisiteit teen M.tb en unieke teikeneienskappe in M. tuberculosis.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is an independent predictor of cardiovascular morbidity and allcause mortality. Significantly, it is considered a modifiable cardiovascular risk factor as its regression increases overall survival and reduces the frequency of adverse cardiac events. A clear understanding of LVH pathogenesis is thus imperative to facilitate improved risk stratification and therapeutic intervention. Hypertrophic cardiomyopathy (HCM), an inherited cardiac disorder, is a model disease for elucidating the molecular mechanisms underlying LVH development. LVH, in the absence of increased external loading conditions, is its quintessential clinical feature, resulting from mutations in genes encoding sarcomeric proteins. The LVH phenotype in HCM exhibits marked variability even amongst family members who carry the same disease-causing mutation. Phenotypic expression is thus determined by the causal mutation and additional determinants including the environment, epigenetics and modifier genes. Thus far, factors investigated as potential hypertrophy modifiers in HCM have been relatively removed from the primary stimulus for LVH; and the few studies that have been replicated yielded inconsistent results. We hypothesized that the factors that closely interact with the primary stimulus of faulty sarcomeric functioning, have a greater capacity to modulate it, and ultimately the LVH phenotype in HCM. Plausible candidate modifiers would include factors relating to the structure or function of the sarcomere, including known HCM-causal genes; and the enzymes that function in sarcomere-based energetics. Indeed, the literature highlights the relevance of sarcomeric proteins, Ca2+-handling and myocardial energetics in the development of LVH in HCM. This study, therefore, set out to evaluate the hypertrophy-modifying capacity of such factors by means of family-based genetic association testing in 27 South African HCM families in which one of three unique HCM-causing founder mutations segregates. Moreover, the single and combined effects of 76 variants within 26 candidate genes encoding sarcomeric or sarcomere-associated proteins were investigated. The study identified a modifying role in the development of hypertrophy in HCM for each of the candidate genes investigated with the exception of the metabolic protein-encoding gene, PRKAG1. More specifically, single variant association analyses identified a modifying role for variants within the genes MYH7, TPM1 and MYL2, which encode proteins of the sarcomere, as well as the genes CPT1B, CKM, ALDOA and PRKAB2, which encode metabolic proteins. Haplotype-based association analyses identified combined modifying effects for variants within the genes ACTC, TPM1, MYL2, MYL3 and MYBPC3, which encode proteins of the sarcomere, as well as the genes CD36, PDK4, CKM, PFKM, PPARA, PPARG, PGC1A, PRKAA2, PRKAG2 and PRKAG3, which encode metabolic proteins. Moreover, a number of variants and haplotypes showed statistically significant differences in effect amongst the three HCM founder mutation groups. The HCM-modifier genes identified were prioritised for future studies according to the number of significant results obtained for the four tests of association performed. The genes TPM1 and MYBPC3, which encode sarcomeric proteins, as well as the genes PFKM and PRKAG2, which encode metabolic proteins, were identified as stronger candidates for future studies as they delivered multiple significant results for various statistical tests. This study makes a novel contribution to the field of hypertrophy research as it tested the hypothesis that structural or energy-related factors located within the sarcomere may act as modifiers of cardiac hypertrophy in HCM, and succeeded in identifying a modifying role for many of the candidate genes selected. The significant results include substantial single and within-genecontext variant effects; and identified sizeable variation in the risk of developing LVH owing to the compound effect of the modifier and the individual founder mutations. Collectively, these findings enhance the current understanding of genotype/phenotype correlations and may, as consequence, improve patient risk stratification and choice of treatment. Moreover, these findings emphasize the potential for modulation of disease by further elucidation of some of the avenues identified. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is ‘n onafhanklike voorspeller van kardiovaskulêre morbiditeit en van mortaliteit weens alle oorsake. Van belang is dat dit ‘n wysigbare kardiovaskulêre risiko faktor is, aangesien die afname daarvan algehele oorlewing verhoog en die frekwensie van nadelige kardiale voorvalle verlaag. ‘n Duidelike begrip van LVH patogenese is dus noodsaaklik om verbeterde risiko stratifikasie en terapeutiese intervensie te fasiliteer. Hipertrofiese kardiomiopatie (HKM), ‘n oorerflike hart-siekte, is ‘n model-siekte vir die uitpluis van die molekulêre meganismes onderliggend aan die ontwikkeling van LVH. LVH, in die afwesigheid van verhoogde eksterne lading, is die kern kliniese simptoom van HKM en die gevolg van mutasies in die gene wat kodeer vir sarkomeriese proteïene. Die LVH fenotiepe in HKM toon merkbare veranderlikheid selfs in familie-lede wat dieselfde siekte-veroorsakende mutasie dra. Die fenotiepe word dus bepaal deur die siekte-veroorsakende mutasie asook addisionele determinante insluitend die omgewing, epigenetika en modifiserende gene. Potensiële hipertrofie-modifiseerders wat tot dusver bestudeer is, is betreklik verwyder van die primêre stimulus vir LVH en die paar studies wat gerepliseer is, het teenstrydige resultate gelewer. Ons hipoteseer dat die faktore wat in noue interaksie met die primêre stimulus van foutiewe sarkomeriese funksionering is, ‘n groter kapasitieit het om dit en uiteindelik die LVH fenotiepe in HKM, te moduleer. Aanneemlike kandidaat-modifiseerders sou insluit faktore wat betrekking het tot die struktuur en funksie van die sarkomeer insluitend HKM-oorsaaklike gene en die ensieme wat funksioneer in sarkomeer-gebaseerde energetika. Die literatuur beklemtoon inderdaad die relevansie van sarkomeriese proteïene, Ca2+-hantering en miokardiese energetika in die ontwikkeling van LVM in HKM. Hierdie studie het beoog om die hipertrofie-modifiserende kapasiteit van sulke faktore te evalueer deur middel van familie-gebaseerde genetiese assosiasie toetse in 27 Suid-Afrikaanse HKM families waarin een van drie unieke HKM-stigter mutasies segregeer. Verder was die enkel en gekombineerde effekte van 76 variante binne 26 kandidaat gene wat kodeer vir sarkomeer en sarkomeer-geassosieerde proteïene, ondersoek. Hierdie studie het ‘n modifiserende rol in die ontwikkeling van hipertrofie in HKM geïdentifiseer vir elk van die kandidaat gene wat ondersoek is, met uitsluiting van die PRKAG1, wat kodeer vir ‘n metaboliese proteïen. Meer spesifiek, enkel variant assosiasie analises het ‘n modifiserende rol geïdentifiseer vir variante in die gene MYH7, TPM1 en MYL2, wat kodeer vir sarkomeriese proteïene, asook die gene CPT1B, CKM, ALDOA en PRKAB2, wat kodeer vir metabolise proteïene. Haplotipe-gebaseerde assosiasie-analises het gekombineerde modifiserende effekte geïdentifiseer vir variante in die gene ACTC, TPM1, MYL2, MYL3 en MYBPC3, wat kodeer vir strukturele proteïene van die sarkomeer asook die gene CD36, PDK4, CKM, PFKM, PPARA, PPARG, PGC1A, PRKAA2, PRKAG2 en PRKAG3, wat kodeer vir metabolise proteïene. Verder het ‘n aantal variante en haplotipes statisties betekenisvolle verskille in effek tussen die drie HKM-stigter mutasie groepe getoon. Die HKM-modifiserende gene wat geïdentifiseer is, is verder geprioritiseer vir toekomstige studies volgens die aantal beduidende resultate wat vir die vier assosiasie toetse verkry is. Die gene TPM1 and MYBPC3, wat kodeer vir sarkomeriese proteïene, asook die gene PFKM and PRKAG2, wat kodeer vir metaboliese proteïene, is geïdentifiseer as sterker kandidate vir verdere studies omdat veelvuldige beduidende resultate vir die verskeie statistiese toetse deur hulle gelewer is. Hierdie studie maak ‘n nuwe bydrae tot die veld van hipertrofie navorsing omdat dit die hipotese dat strukturele en energie-verwante faktore, wat binne die sarkomeer geposisioneer is, potensieel as modifiseerders van kardiale hipertropfie in HKM kan optree, ondersoek het. Dit slaag ook daarin om ‘n modifiserende rol vir baie van die geselekteerde kandidaatgene te identifiseer. Die beduidende resultate sluit in aansienlike enkel en binne-geen-konteks variant-effekte en aansienlike variasie in die risiko vir LVH ontwikkeling verskuldig aan die gekombineerde effek van modifiseerder en individuele stigter mutasies. Gesamentlik verbeter hierdie bevindinge die huidige begrip van genotipe/fenotipe korrelasies en dit mag tot gevolg hê verbeterde pasiënt risiko stratifikasie en keuse van behandeling. Verder beklemtoon hierdie bevindinge die potensiaal vir siekte modulering deur verdere uitpluis van sekere van hierdie geïdentifiseerde navorsingsrigtings. / National Research Foundation / Dr. Paul van Helden / Stellenbosch University

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: The HIV epidemic in the past couple of decades has spread at an alarming rate throughout Southern Africa. Today the region accounts for roughly one third of all HIV infections, while prevalence rates in other areas of sub-Saharan Africa remain low. In the following study, sampled sequences from Cape Town, spanning over a 21-year period were used to investigate the epidemic history of HIV, which was compared to epidemic trends across Southern Africa. Longitudinal sequence data sets were generated from stored patient samples from Cape Town through standard molecular techniques. Firstly, these sequences were used to estimate the date of origin of the HIV epidemic in Cape Town and to reconstruct a demographic history of the epidemic with advanced Bayesian inference methods. These analyses placed the estimated date of origin of the Cape Town epidemic around the mid 1960‟s with periods of strong epidemic growth observed during the mid 1980‟s and 1990‟s. Secondly, reference strains of HIV from Southern African countries were used to estimate the date of origin of the epidemic in the Southern African region. These analyses placed the date of origin of the epidemic in the Southern African region around the mid 1950‟s roughly ten years before the start of the epidemic in Cape Town/South Africa. These sequences were also used for the reconstruction of the demographic history of the epidemic in the region. A two phased growth in the HIV epidemic in the Southern African region was observed with exponential growth occurring in the mid 1980‟s and 1990‟s. Such findings are also supported by HIV prevalence estimates made by some of the leading HIV research centres and government health departments. Thirdly, a large number of homologous reference strains were used to establish the evolutionary relationship of HIV isolates from Cape Town with those from around the world. A close genetic relationship between Cape Town isolates with other South African and other Southern African isolates was observed in these analyses. Finally, large monophyletic clusters of Cape Town isolates, which was observed during the evolutionary inference, were further investigated. After detailed analyses it appears that these transmission clusters of HIV-1 have been in circulation amongst the infected population of Cape Town for several years or decades. / AFRIKAANSE OPSOMMING: Die MIV-epidemie het in die afgelope paar dekades teen ´n snelspoed deur Suider-Afrika versprei. Een derde van die globale MIV-infeksies kom hiér voor terwyl ander dele van Afrika aansienlik minder infeksies aantoon. Verskeie studies skryf dit toe aan onder andere: manlike besnydenis, seksuele losbandigheid, migrasie en verskeie politike faktore. Die MIV-epidemie in Suider-Afrika word deur ´n enkele subtipe van die virus oorheers (nl. MIV Subtipe C) terwyl ander subtipes sirkuleer deur die res van sub Sahara-Afrika. In die opeenvolgende studie word DNS-monsters uit Kaapstad (wat oor ´n 20 jaar tydperk strek) gebruik om die oorsprong en verloop van die epidemie te bestudeer. Die data van die Kaapstad epidemie word met die geskiedkundige verloop van die epidemie in Suider-Afrika vergelyk. Deur gestoorde bloedmonsters van Kaapstad te gebruik, was DNS-datastelle gegenereer deur middel van standaard molekulêre tegnieke. Die DNS-monsters was eerstens gebruik om die evolusionêre oorsprong en verloop van die epidemie in Kaapstad te bepaal deur Bayesiaanse Markov-ketting Monte Carlo steekproefneming. Volgense die resultate het die epidemie sy oorsprong in die 1960‟s. Klein periodes van epidemiese groei kon waargeneem word gedurende die 1980's en -90's. Die bevindings is toe vergelyk met die geskiedkundige verloop van die epidemie in Suider-Afrika. Die Suider-Afrika epidemie se oorsprong en verloop was afgelei van DNS monsters wat verkry is van publieke databasisse en die gebruik van soortgelyke Bayesiaanse metodes. Die resultate van die ondersoek het bevind dat die epidemie in Suider-Afrika in die 1950‟s ontstaan het. In vergelyking toon dit 'n stadiger liniêre groei met kort periodes van eksponensiële groei. Verder is ´n standard filogenetiese analise onderneem om die evolusionêre verwantskap van die Kaapstad-monsters te bepaal met ander MIV subtipe C isolate. Die filogenetiese steekproef toon dat die Kaapstad-monster baie nou verwant is aan ander isolate van Kaapstad, Suid-Afrika en Suider Afrika. Buiten hierdie bevindings was transmissie-bondels van MIV in Kaapstad ontdek. Na ´n deeglike verdere filogenetiese ondersoek blyk dit of die transmissie bondels al vir ´n paar dekades deur die geïnfekteerde populasie van Kaapstad sirkuleer. / Poliomyelitis Research Foundation (PRF) / Faculty of Medicine and Health of the University of Stellenbosch / National Research Foundation (NRF) of South Africa,

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are conducted on macrovascular endothelial cells. The current dissertation set out to comprehensively investigate the responses of cultured primary adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory cytokine). We were interested to investigate whether this distinct endothelial cell type would develop classical features of ED, and if so, what the underlying mechanisms were. First we aimed to establish a baseline characterization of the CMECs under control conditions. Next, we developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis, hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above measurements were performed by morphological assessment (light and fluorescence microscopy), FACS analysis, western blotting and large-scale proteomic analyses. Data showed that CMECs shared many baseline features with other endothelial cell types, including morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was the high abundance of structural mitochondrial proteins, suggesting that CMECs require mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature, several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways (BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway. In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs shared many responses and mechanisms with more frequently researched endothelial cell types, the data also supplied several novel additions to the literature, particularly with the application of proteomics. We believe that this dissertation has provided more insights into endothelial heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to stimuli typically associated with cardiovascular risk. / AFRIKAANSE OPSOMMING: Blootstelling aan skadelike stimuli kan tot disfunksionaliteit van vaskulêre endoteelselle lei wat deur verlaagde biobeskikbaarheid van stikstofoksied (NO) gekenmerk word. Endoteeldisfunksie (ED) is ‘n omkeerbare voorganger van isgemiese hartsiekte (IHD), en ‘n beter begrip van die onderliggende meganismes van ED kan lei tot die ontwikkeling van kliniese strategieë vir die voorkoming/behandeling van IHD. Baie min is bekend oor die respons wat in kardiale mikrovaskulêre endoteelselle (CMECs) uitgelok word na blootstelling aan pro-ED stimuli, omdat meeste studies op makrovaskulêre endoteelselle uitgevoer word. Die huidige proefskrif het daarna gemik om die respons van primêre kulture van volwasse rot CMECs op bekende skadelike stimuli, nl. hipoksie en tumor nekrose faktor-alfa (TNF-α; pro-inflammatoriese sitokien) in diepte te ondersoek. Ons was veral geïnteresseerd om vas te stel of hierdie spesifieke endoteelseltipe die klassieke kenmerke van ED sou ontwikkel, en indien wel, wat die onderliggende meganismes sou wees. Eerstens het ons beoog om ‘n basislyn karaterisering van CMECs onder kontrole toestande daar te stel. Vervolgens het ons ‘n model van hipoksie-geïnduseerde selskade gevestig en apoptose/nekrose, intrasellulêre NO en reaktiewe suurstofspesies (ROS), sowel as die uitdrukking en aktivering van proteine betrokke by NO-biosintese, hipoksie en apoptose en differensiële regulering van proteine gemeet. Laastens het ons die respons van CMECs op behandeling met TNF-α gekarakteriseer. Ons het apoptose/nekrose, intrasellulêre NO en ROS vlakke, NO-biosintese-seintransduksieproteïene en grootskaalse differensiele regulering van proteïene gemeet. Bg. metings is uitgevoer deur gebruik te maak van morfologiese evaluasie (lig -en fluoressensiemikroskopie), vloeisitometriese analises, western blot analises en proteomiese analises. Data het getoon dat die basislyn eienskappe van CMECs grootliks met dié van ander endoteelseltipes ooreenstem, insluitende morfologiese voorkoms, LDL-opname, NO-produksie en die uitdrukking van eNOS proteïen. In ‘n nuwe waarneming, het die proteomiese data die uitdrukking van 1387 proteïene aangetoon. ‘n Ander nuwe bevinding was die voorkoms van ‘n groot aantal strukturele mitokondriale proteïene, wat daarop dui dat die CMECs mitokondria ook vir nie-respiratoriese doeleindes gebruik. ‘n Hoë uitdrukking van vesikulêre, glikolitiese en RAS-seintransduksie proteïene was ook kenmerkend van die basislyn CMECs. CMECS wat aan hipoksie blootgestel is, het reageer met ‘n verhoging in apoptose / nekrose en verhoogde uitdrukking van die hipoksie merker, HIF-1α. ‘n Interressante bevinding was dat eNOS-NO biosintese sterk toegeneem het in die hipoksiese CMECs wat met verhoogde mitokondriale ROS en verlaagde anti-oksidant sisteme (aanduidend van oksidatiewe stres) gepaardgegaan het. In ooreenstemming met die literatuur, is verskeie glikolitiese proteïene opgereguleer. ‘n Nuwe waarneming was die opregulering van proteïene wat betrokke is by proteïensintese, iets wat nie normaalweg in hipoksie-studies beskryf word nie. Die CMECs het op TNF-α behandeling gerespondeer deur tekens van ED te toon, naamlik ‘n afname in die NO afkomstig van PKB/Akt-eNOS biosintese en die ontwikkeling van uitgesproke reaksie op oksidatiewe stres soos aangedui deur die opregulering van verskeie anti-oksidant sisteme. Die data het ook aangedui dat TNF-α behandeling tot klassieke TNF-reseptor 1 bemiddelde seintransduksie gelei het, wat gekenmerk was deur die tweeledige aktivering van pro-apoptotiese seintransduksiepaaie (BID en kaspase-3) sowel as die beskermende, pro-inflammatoriese IKB-alpha-NF-KB seintransduksiepad. Ten slotte: hierdie is die eerste studie van sy soort wat die kenmerke en response van CMECs onder basislyn en pro-besering omstandighede in diepte beskryf. Alhoewel dit oor die algemeen wil voorkom asof die CMECs baie in gemeen het met ander, beter nagevorste endoteelseltipes, het die data egter ook verskeie nuwe bevindinge tot die bestaande literatuur gevoeg, spesifiek die data afkomstig van die proteomiese analises. Ons glo dat hierdie proefskrif meer insig verleen t.o.v. die heterogeniteit van vaskulêre endoteelselle asook t.o.v. die megansimes wat deur CMECs aangewend word wanneer hulle aan skadelike stimuli (geassosieer met kardiovaskulêre risiko) blootgestel word.

Thesis (PhD)--Stellenbosch Uni versity, 2000. / ENGLISH ABSTRACT: Intracytoplasmic sperm injection (ICSI) introduced a revolutionary way of treatment for male factor infertility. With the exception of some cases of non-obstructive azoospermia, all other male factor infertility cases have the potential to be successfully treated with ICS!. The only prerequisite seems to be the presence of a motile or viable immotile sperm cell for each oocyte. In this study we report on our own experience with the development and implementation of the ICSI method in the Reproductive Biology Unit at Tygerberg Hospita!. An analysis of 5 years of ICSI experience showed that semen parameters, sperm morphology, motility and concentration did not influence fertilization and pregnancy rates adversely. In most cases, patients who could not be treated with in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT), due to poor semen parameters or fertilization failure, were treated successfully with ICS!. Even a case of globozoospermia was treated successfully with ICS!. Testicular spermatozoa, fresh or frozen-thawed, also resulted in excellent fertilization and pregnancy rates. Cryopreservation of testicular samples facilitated the management of the infertile couple, aiding the coordination of the recovery of vital gametes from both partners and also limiting the repetition of testicular biopsies. Incubation (maturation) of testicular spermatozoa also induced an enhancement in pregnancy rates. It can be concluded that ICSI proved to be a treatment method with success similar to that of in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT), in spite of a severe male factor. The study also indicated transfer route and embryo quality (viability) to be very important factors in the success of ICS!. The tubal transfer route was shown to be a significant contributor to the pregnancy success (compared to uterine transfer) as was the transfer of embryos that showed early division to the 2-cell stage, 26 hours post injection. The transfer of early dividing embryos into the fallopian tube resulted in a pregnancy rate of almost 40%, a result similar to that of GIFT with a mild male factor. The role of the oocyte in fertilization and pregnancy success was also revealed indirectly by the introduction of ICS!. Visual observation of denuded oocytes was possible and many morphological features, normal and abnormal, can be observed. Immature oocytes can also be identified and it was shown that they could be successfully matured in vitro before injection. In this study transmission electron microscopy (TEM) was used to study abnormalities in oocyte morphology. The standard method was adapted and modified for single cell TEM. The abnormalities observed included lysosomal and non-lysosomal degeneration (yellowish or darkly coloured oocytes), degeneration and vacuole formation (vacuolated oocytes), large secondary lysosomes filled with multiple small lipid droplets - lipofuscin body (refractile body) and a fragmented oocyte. It was also possible to study at ultrastructural level, possible reasons for fertilization failure in ICS!. Different stages of oocyte activation failure, cytoplasmic immaturity, sperm cell extrusion, abnormal sperm cell decondensation, female spindle abnormalities and technique related factors were observed. TEM was also successfully implemented to elucidate the reason for infertility in a patient with a longstanding, unexplained history of infertility. TEM evaluation of two of the patient's unfertilized oocytes revealed a spindle abnormality with contributing cytoskeletal anomalies at ultrastructural level. The modified TEM technique offers a valuable tool to study this small, but important group of patients with unexplained infertility. ThisTEM study opened up a new, valuable and interesting avenue of research with both diagnostic and prognostic value for patients with unexplained infertility. ICSI is therefore a valuable method in the treatment of especially male factor infertility. It is the most advanced fertilization technique developed in the last decade in this field. Not only can almost all male factor patients be treated, but unexplained female infertility can also be exposed, studied and hopefully in future also be treated with micromanipulation methods. / AFRIKAANSE OPSOMMING: Die ontwikkeling van die mikromanipulasie tegniek "Intracytoplasmic sperm injection" (ICSI) het die behandeling van die manlike faktor in infertiliteit, revolusionêr verander. Met die uitsondering van sommige gevalle van nie-obstruktiewe asoospermia, kan potensieel alle ander manlike infertiliteits faktore suksesvol met ICSI behandel word. Die enigste voorvereiste blyk "n bewegende of "n nie-bewegende, maar bewese lewende spermsel te wees. In hierdie studie word verslag gedoen oor die ontwikkeling en toepassing van die ICSI metode in die Eenhed vir Reproduktiewe Biologie by Tygerberg Hospitaal. 'n Analise van 5 jaar se resultate na die implementering van die ICSI metode het gewys dat die semen parameters, sperm morfologie, motiliteit en konsentrasie, nie "n effek op bevrugting- en swangerskapsyfers gehad het nie. Pasiënte wat, as gevolg van ontoereikende semen parameters, nie met die klassieke metodes, in vitro bevrugting (IVB) of gameet intrafallopiusbuis terugplasing (GIFT) behandel kon word nie, kon suksesvol met ICSI behandel word. Daar was selfs "n geval van manlike infertiliteit as gevolg van globosoospermie, wat suksesvol met ICS behandel is. Die ICSI metode het dit ook moontlik gemaak om uitstekende bevrugting- en swangerskap resultate met testikulêre spermatosoa .(vars en gevries) te bereik. Die bevriesing van testisweefsel het ook bygedra tot beter hantering van sulke pasiënte. Herhaalde testisbiopsies word uitgeskakel en die koórdinasie van die verkryging van die manlike en vroulike gamete, word ook vergemaklik wanneer testisweefsel in gevriesde vorm beskikbaar is. Die studie het verder getoon dat wanneer testikulêre weefsel geïnkubeer word (om spermatosoa te laat matureer), die swangerskapsyfers verhoog was. Dit is dus duidelik dat die ICSI metode net so suksesvol soos die IVB en GIFT metodes toegepas kan word, selfs en veral in gevalle van erge manlike faktor infertiliteit. Die studie het ook verder getoon dat die plek waar embrios teruggeplaas word, asook die embriokwalitiet van teruggeplaasde embrios, belangrike bydraende faktore in die ICSI swangerskapsukses was. Embrioterugplasing in die buis van fallopius en terugplasing van embrios wat vroeë 2-sel deling, 26 uur na ICSI getoon het, is uitgewys as faktore wat ICSI swangerskap betekenisvol verbeter het. Dit was moontlik om "n swangerskapsyfer van ongeveer 40%, sootgelyk aan die van GIFT sonder "n erge manlike faktor, te bereik met die terugplasing van ten minste een vroeë deler embrio in die fallopiese buis. Die ICSI tegniek het ook indirek bygedra tot nuwe insigte met betrekking tot die rol wat die vroulike eiersel (oësief in ICSI bevrugting speel. Oósiete word gestroop van hulomringende selle vir die ICSI proses en kan dan maklik vir hul normale en abnormale morfologiese eienskappe evalueer word. Oësiete wat immatuur is kan ook so geïdentifiseer word en dit is moontlik om hulle suksesvol te matureer voor mikro-inspuiting. Transmissie-elektronmikroskopie (TEM) is in die studie gebruik om die ultrastruktuur van onbevrugde en abnormale oësiete te bestudeer. Hiervoor is "n bestaande tegniek gemodifiseer vir die hantering van "n enkele sel, in hierdie geval die oosiet. Lisasomale en nie-lisosomale degenerasie (oósiete wat geelof donker van kleur voorkom), degeneratiewe tekens en vakuole (oësiete met vakuole), groot sekondêre lisosome gevul met klein lipieddruppels ('refractile body') en 'n gefragmenteerde oosiet was van die morfologies abnormale eienskappe wat ultrastruktureel geïdentifiseer is. Moontlike faktore wat 'n rol kan speel in nie-bevrugting na ICSI kon ook op ultrastrukturele vlak met die tegniek geïdentifiseer word. Hierdie faktore het die volgende ingesluit: die onvermoë van verskillende stadiums van oosiet aktivering, sitoplasmatiese immaturiteit, uitwerping van die spermsel na die periviteliene spasie, abnormale spermsel dekondensasie, vroulike spoelvormings abnormaliteite en tegniekgekoppelde faktore. Die TEM tegniek is ook suksesvol aangewend om die infertiliteitsprobleem van 'n pasiënt wat vir etlike jare aan onverklaarbare infertiliteit gely het, te identifiseer. TEM het op die ultrastrukturele vlak gewys dat daar 'n spoel abnormaliteit in twee van haar onbevrugde oëslete was. TEM kan dus baie vrugbaar gebruik word in hierdie groep pasiënte om onverklaarbare infertiliteit, wat andersins ongeïdentifiseerd sou bly, te verklaar. Die ICSI metode is die mees revolusionêre tegniek wat die afgelope dekade vir die behandeling van veral manlike infertiliteit ontwikkel en baie suksesvol toegepas is. Die metode ook kan 'n bydraende rol speel in die hantering van onverklaarbare infertiliteit veral ten opsigte van die vroulike gameet. In die toekoms is dit moontlik dat selfs hierdie probleem met nuwe mikromanipulasietegnieke opgelos sal kan word.

Dissertation (MD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Acute renal failure still is, with the exception of cardiac deaths, the most important pathological process associated with perioperative mortality in patients operated for abdominal aortic aneurysms. The intraoperative change in renal blood flow (RBF) and glomerular function have been investigated in human and animal models, particularly over the past 15 years. Despite large variation in study populations, measurement techniques and study designs in general, a significant body of evidence has developed which suggests infrarenal aortic clamp-induced renal ischemia to be the cause of postoperative acute renal failure when this complication does occur. It is rather surprizing then that, despite some recent studies which have reported on various pharmacological interventions to prevent intraoperative renal ischemia (with variable success), very little has apparently been done to unravel the pathogenesis and exact pathophysiology of this potentially lethal complication. Although a number of investigators suggest the possibility of hormonal involvement (particularly reninangiotensin, antidiuretic hormone (ADH) and catecholamines) in the process, the exact role of these mediators have not been explored (or reported) in a structured fashion. In an initial human study, renal hemodynamics and function were measured from the preoperative period, during the intraoperative phase and at least until 4 hours after aortic unclamping. To investigate the possibility of a temporal relationship between renal changes and fluctuations in hormonal concentrations, plasma concentrations of relevant hormones were determined at every sampling period where renal parameters were measured. The decrease in RBF and glomerular filtration rate (GFR) which we demonstrated to coincide with infrarenal aortic cross clamping, is consistent with results previously published. We demonstrated persistence of the impairment of these parameters as long as 4 hours into the postoperative phase; which has previously only been reported for the period until immediately after aortic unclamping with the abdomen still open. The persistence of a depressed GFR until the time of discharge of patients is cause for concern, particularly in patients with compromised renal function prior to surgery. Of the measured hormones with a potential influence on RBF and nephron function, renin was the only mediator where changes in plasma concentrations coincided with the depression of RBF and GFR after aortic cross clamping. The design of our study did not allow us to conclude whether the concomitant increase in angiotensin II was primarily responsible for the change in renal hemodynamics, or whether the raised renin (and angiotensin) levels were stimulated by the decrease in RBF induced by another mechanism. In another patient group, we demonstrated that the combination of mannitol and dopamine provided no protection against the deleterious effects of aortic cross clamping. In fact, the high urine volumes produced under the influence of these agents (which did not correlate with RBF at the corresponding periods), is likely to prompt a false sense of security. Given the lack of any objective benefit afforded by these agents, their use in these clinical circumstances should be discouraged. The animal studies were aimed at elucidation of the exact role of angiotensin in the pathogenesis and pathophysiology of the renal changes associated with infrarenal aortic clamping, as well as the interaction of angiotensin with other modulators for which an interactive relationship had been described previously under other experimental and/or clinical circumstances. The first study showed that, although renin (and thus angiotensin) concentrations were high after aortic unclamping, the hormone had no pathogenic or pathophysiological role of significance in the observed renal changes during this period (since blocking angiotensin II activation by the prevention of renin release, or by inhibiting the conversion enzyme, did not prevent a substantial decrease in RBF or GFR during that period). Preventing angiotensin II activation did, however, prevent renal changes during aortic clamping. This beneficial effect did not establish a primary role for angiotensin during that period, since the favourable influence could also (at least partially) be explained by prevention of the permissive influence of angiotensin on other vasoconstrictors and/or other vasodilatory influences of ACE inhibition and [1- blockade which are unrelated to angiotensin. This study did indicate that (at least partially) different mechanisms are responsible for the renal changes seen during aortic clamping, and after aortic unclamping. The second study explored the role of calcium in the renal pathophysiological changes during aortic clamping and after unclamping. The protective influence effected by the administration of a Ca2 + -blocker suggest the dependence of the renal vasoconstrictive and glomerular pathophysiological process( es) on the cellular influx of Ca2 + through voltage-gated channels. It unfortunately provides no definitive insight into the primary instigators of these processes. However, it does offer a clinically useful method of preventing these changes and protecting the kidney against ischemic injury during abdominal aortic surgery. The third component of the animal studies demonstrates the importance of the protective effect of renal prostaglandins during the specific experimental (and probably also the clinical) circumstances. Again, it does not provide definitive information on the mediators responsible for the renal changes, since the deleterious effects of numerous endogenous substances have previously been shown to be counterbalanced by intrarenal synthesis of prostaglandins under various experimental and clinical circumstances. The extent of the pathophysiological and ultrastructural changes which occurred under the influence of a NSAID does, however, suggest that these drugs should not be used under these clinical circumstances. The last component of the study provides evidence that angiotensin only plays a secondary/supplementary role in the renal pathophysiological process even during aortic clamping. This may explain the contradictory evidence regarding the potential beneficial effect of ACE inhibition (on renal hemodynamics and glomerular function) during abdominal aortic surgery (Licker et al. 1996, Colson et al. 1992a). Based on our studies, ACE inhibition can not be supported for this purpose. / AFRIKAANSE OPSOMMING: Akute nierversaking is met die uitsondering van kardiale sterftes, steeds die belangrikste patologiese proses wat geassosieer is met perioperatiewe mortaliteit in pasiënte wat opereer word vir abdominale aorta aneurismes. Die intraoperatiewe veranderinge in renale bloedvloei (NBV) en glomerulêre funksie is die afgelope 15 jaar ondersoek en gerapporteer in pasiënte- sowel as diere-modelle. Ten spyte van groot variasies in studie-populasies, meettegnieke en ontwerp van studies in die algemeen, dui 'n wesenlike hoeveelheid getuienis daarop dat infrarenale klemming van die aorta renale isgemie induseer, wat die oorsaak is van postoperatiewe akute nierversaking wanneer hierdie komplikasie voorkom. Dit is verbasend dat, ten spyte van sommige onlangse studies wat rapporteer oor 'n verskeidenheid farmakologiese ingrepe om intraoperatiewe renale isgemie te voorkom (met wisselende sukses), baie min oënskynlik gedoen is om die patogenese en die presiese patofisiologie van hierdie potensieel dodelike komplikasie te ontrafel. Hoewel verskeie outeurs die moontlikheid van hormonale betrokkenheid (veral renienangiotensien, antidiuretiese hormoon en katekolamiene) in hierdie proses suggereer, is die presiese rol van hierdie mediators nog nie op 'n gestruktureerde wyse ondersoek (of rapporteer) nie. In ons aanvanklike pasiënte-studie is renale hemodinamika en -funksie gemeet vanaf die preoperatiewe periode, gedurende die intra-operatiewe fase en tot minstens vier uur na ontklemming van die aorta. Serumkonsentrasies van relevante hormone is bepaal tydens elke metingsperiode waar renale parameters gemeet is, ten einde die moontlikheid van 'n temporale verwantskap tussen renale veranderinge en variasies in hormoonkonsentrasies te ondersoek. Die vermindering in NBV en glomerulêre filtrasiespoed (GFS) wat ons aangetoon het om saam te val met infrarenale aortaklemming, stem ooreen met resultate wat tevore deur ander navorsers publiseer is. Ons het aangetoon dat die inkorting van hierdie parameters voortduur tot minstens vier uur na aorta-ontklemming. Hierdie veranderinge is tevore slegs rapporteer vir periodes tot kort na aorta-ontklemming voor sluiting van die buikwond. Die feit dat die GFS steeds verlaag is met ontslag van hierdie pasiënte, skep rede tot kommer, veral in pasiënte wat alreeds ingekorte nierfunksie het voor die chirurgiese prosedure. Van die gemete hormone wat moontlik 'n invloed sou kon uitoefen op NBV eh nefronfunksie, was renien die enigste waarvan verandering in plasmakonsentrasies saamgeval het met die onderdrukking van NBV en GFS na aortaklemming. Die ontwerp van ons studie het ons nie toegelaat om 'n besliste uitspraak te maak of die geassosieerde verhoging in angiotensien II primêr verantwoordelik was vir die verandering in renale hemodinamika, of dat die verhoogde renien (en angiotensien) bloedvlakke moontlik sekondêr stimuleer is deur die verandering in NBV wat deur 'n ander meganisme induseer is. In 'n ander pasiëntegroep het ons aangetoon dat die kombinasie van mannitol en dopamien geen beskerming verleen het teen die nadelige effekte van aorta-klemming nie. Die groot volumes uriene wat uitgeskei is onder die invloed van hierdie middels (wat nie korreleer het met NBV tydens ooreenstemmende periodes nie), het inderwaarheid 'n ontoepaslike gerustheid uitgelok. Weens die ooglopende gebrek aan objektiewe voordeel wat verleen word deur hierdie middels, behoort hulle gebruik tydens hierdie kliniese omstandighede ontmoedig te word. Die doel van die diere studies was die identifisering van die presiese rol van angiotensien in die patogenese en patofisiologie van die renale veranderinge geassosieer met infrarenale aortaklemming, sowel as die interaksie van angiotensien met ander modulators waarvoor 'n interaktiewe verwantskap voorheen beskryf is onder eksperimentele en/of kliniese omstandighede. Die eerste studie het getoon dat alhoewel renien (en dus angiotensien) konsentrasies hoog was na aorta-ontklemming, die hormone geen betekenisvolle patogenetiese of patofisiologiese rol in die waargenome renale veranderinge gedurende hierdie periode het nie (aangesien blokkade van angiotensien aktivering deur voorkoming van renien vrystelling, of deur inhibisie van angiotensien omsettingsensiem (AOE), nie 'n daling in NBV of GFS kon voorkom nie). Voorkoming van angiotensien II aktivering het egter wel renale verandering voorkom gedurende aortaklemming. Dié voordelige effek het nie 'n primêre rol vir angiotensien gedurende die periode bevestig nie, aangesien die gunstige invloed ook (ten minste gedeeltelik) verduidelik kon word deur die voorkoming van die fassiliterende invloed van angiotensien op ander vasokonstriktore en/of ander vasodilator-invloede van die onderdrukking van AOE en ïs-blokkers (wat geen verband het met angiotensien of die blokkade daarvan nie). Die studie het aangetoon dat (ten minste gedeeltelik) verskillende meganismes verantwoordelik is vir renale veranderinge wat gesien is gedurende aortaklemming en na -ontklemming. Die tweede studie het die rol van kalsium in die renale patofisiologiese veranderinge gedurende aortaklemming en na ontklemming ondersoek. Die beskermende invloed wat deur die toediening van Ca2 + -blokkers bewerkstellig is, het bevestig dat die renale vasokonstriktoriese en glomerulêre patofisiologiese prosesse afhanklik is van sellulêre influks van kalsium deur spannings-afhanklike kannale. Dit het ongelukkig geen definitiewe insig verleen ten opsigte van die primêre inisieerders van die proses nie. Dit verskaf nogtans 'n bruikbare kliniese metode om daardie veranderinge te voorkom en die niere teen isgemiese besering gedurende abdominale aorta-chirurgie te beskerm. Die derde komponent van die diere-studies demonstreer die belangrikheid van die beskermende effek van renale prostaglandiene tydens die spesifieke eksperimentele (en waarskynlik ook die kliniese) omstandighede. Weereens gee dit nie definitiewe inligting oor die bemiddelaars wat verantwoordelik is vir die renale veranderinge nie, aangesien die skadelike effekte van verskeie endogene stowwe voorheen aangetoon is om beperk of voorkom te word deur die intrarenale vrystelling van prostaglandiene. Die omvang van die patofisiologiese en ultrastrukturele veranderinge wat ontstaan het onder die invloed van nie-steroïed anti-inflammatoriese middels (wat gebruik is om prostaglandien sintese te inhibeer), dui aan dat hierdie middels vermy moet word onder soortelyke kliniese omstandighede. Die laaste komponent van die studie verskaf 'n sterk aanduiding dat angiotensien slegs 'n sekondêre/aanvullende rol speel in die renale patofisiologiese proses, selfs gedurende aortaklemming. Dit mag die weersprekende getuienis oor die potensiële voordeel van AOE onderdrukking (op renale hemodinamika en glomerulêre funksie) gedurende abdominale aortachirurgie (Licker et al. 1996, Colson et al. 1992a) verklaar. Gebaseer op ons studies, kan AOE onderdrukking nie ondersteun word vir hierdie doel nie.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Posttraumatic stress disorder (PTSD) is a multifactorial disorder, with substantial evidence for a genetic contribution. Although genetic association studies have been conducted to identify vulnerability factors in PTSD, the results remain largely inconsistent. Identifying ligands of proteins that are involved in the aetiology of PTSD represents a means of delineating the network of interactions that may play a role in the development of the disorder. Numerous animal studies have identified the Wolframin protein (WFS1) as a putative biomarker for the development of PTSD. However, the function of WFS1 has not yet been fully elucidated. The aim of the present investigation was to identify proteins that interact with the N-terminal domain of WFS1, in order to possibly elucidate the function of the protein, and to subsequently hypothesise on the role that WFS1 may play in the development of PTSD. Yeast two-hybrid (Y2H) methodology was used to identify putative ligands of the N-terminal domain of WFS1 (amino acids 1-300) by screening a human adult brain complementary DNA (cDNA) library. Successive selection stages reduced the number of putative WFS1 N-terminal ligand-containing colonies (preys) from 878 to three. Putative ligands were sequenced and indentified by BLAST-search. Four preys were excluded because they were either out of frame with the vector or the protein they encoded occurred in a subcellular location that was not compatible with the location of the N-terminal domain of WFS1. An interesting putative ligand was identified as carboxypeptidase E (CPE). Colocalisation analyses verified that CPE colocalises with WFS1 in rat hypothalamic GT1-7 cells. Coimmunoprecipitation (Co-IP) further verified a direct interaction between WFS1 and CPE in rat hypothalamic GT1-7 cells, providing conclusive evidence that WFS1 and CPE interact. Both WFS1 and CPE are upregulated in response to fear and both are localised to the secretory granules of the regulated secretory pathway. WFS1 has been detected in both the ER and secretory granules it seems to play an important role in protein biosynthesis, modification, folding, trafficking and the regulation of calcium homeostasis. CPE is involved in neuropeptide processing and trafficking of secreted proteins. The interaction between CPE and WFS1 may thus serve to facilitate an optimal environment in which neuropeptides can be processed and secreted. / AFRIKAANSE OPSOMMING: Posttraumatiese stresversteuring (PTSV) is 'n multifaktoriese siekte, met aansienlike bewyse vir 'n genetiese bydrae. Hoewel genetiese assosiasie-studies uitgevoer word om kwesbaarheidsfaktore in PTSV te identifiseer, is die resultate grootliks teenstrydig. Identifiseering van ligande van proteїene wat betrokke is in die etiologie van PTSV dien as middel om die netwerk van interaksies wat ń moontlike rol in die ontwikkeling van die versteuring kan speel, te oudersoek talle diere studies het die Wolframin proteien (WFS1) geїdentifiseer as 'n moontlike biomerker vir die ontwikkeling van PTSV. Die funksie van WFS1 is egter nog nie ten volle beskryf nie. Die doel van die huidige studie was om proteїene wat interaksie met die N-terminale domein van WFS1 her te identifiseer, om sodoende die funksie van die proteїen uit te lig, en daardeur die rol wat WFS1 kan speel in die ontwikkeling van PTSV te bepaal. Die gis twee-hibried metodologie is gebruik om moontlike ligande van die N-terminale domein van WFS1 te identifiseer, deur die sifting van 'n mens volwasse brein komplementêre DNS biblioteek. Opeenvolgende seleksie stappe het die aantal moontlike WFS1 N-terminale ligand wat moontlike prooi kolonies bevat van 878 tot en met ses verminder. Die DNS volgorde van die moontlike prooi-plasmiede is bepaal en geїdentifiseer deur die BLAST soek-engin. Vier prooi-plasmiede is uitgesluit omdat hulle of nie in die korrekte lees-raam in die vektor was nie of die subsellulêre ligging van die proteїen wat uitgedrukword is nie versoenbaar met die N-terminale domein van WFS1. 'n Interessante moontlike ligand is geїdentifiseer as Karboxypeptidase E (CPE). Ko-lokalisering ontleding bevestig dat CPE ko-lokaliseer met WFS1 in rot hipotalamiese selle (GT1-7). Ko-immunopresipitasie (Ko-IP) toon verder 'n direkte interaksie tussen WFS1 and CPE in rot GT1-7 selle. Wat dus bewys dat WFS1 en CPE wel met mekaar 'n interaksie het. Beide WFS1 en CPE toon 'n verhoogde uitdrukking in respons tot ń vrees-situasie. Beide van hierdie proteїene kom voor in die sekretoriese korrels van die gereguleerde sekretoriese pad. Die WFS1 proteien word bevind in die endoplasmiese retikulum (ER) van die sel, waar dit verantwoordelik is vir proteien biosintese, modifikasie, vouing, vervoer en die reguleering van kalsium homeostase. Die CPE proteїen is verantwoordelik vir die proseseering van neuropeptiede en die vervoer van uitgeskiede proteїene. Dus kan die interaksie tussen CPE en WFS1 dien om 'n optimale omgewing te skep waarin neuropeptiede geproseseer en uitgeskei kan word. / The National Research Foundation (NRF), the Harry Crossley Foundation and the Medical Research Council (MRC)

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Evidence suggests that biological mechanisms, such as energy dependant efflux pumps, in addition to the rpoB gene mutations, define the level of rifampicin (RIF) resistance in drug resistant Mycobacterium tuberculosis (M. tuberculosis) strains with similar genetic backgrounds. Additionally, proteomic studies showed up-regulation of components of F1F0-ATP synthase enzyme, encoded by the atpBEFHAGDC operon which is responsible for ATP production, in response to RIF. The hypothesis of the current study is that the exposure of a multi-drug resistant (MDR) M. tuberculosis strain to RIF leads to the initiation of a signalling cascade of events. This results in the increased expression of F1F0-ATP synthase leading to an increase in energy production and subsequent activation of efflux pumps. RIF will thus be actively extruded from the cell, increasing the level of RIF resistance. This study aims to identify genetic regions responsible for the regulation of expression of the atpBEFHAGDC operon. Additionally we aim to identify other novel mechanisms contributing to the level of RIF resistance in M. tuberculosis. A specialised reporter vector was constructed to monitor the expression of atpBEFHAGDC, with the use of a fluorescent protein. Subsequently a library of random knockouts was created by transposon mutagenesis in order to identify possible regulators, as well as novel mechanisms contributing to RIF resistance. Two hypothetical proteins, Rv2005c and Rv2417c, were identified in M. tuberculosis transposon mutants showing decreased fluorescence correlating to decreased expression of atpBEFHAGDC. Rv2005c encodes a universal stress protein, suggesting its potential role in a signalling cascade initiated upon RIF exposure. In a model pathway of regulation we propose that the product of Rv2005c is responsible for releasing a repressor protein, Rv1049, thereby stimulating a cascade of signalling events resulting in the up-regulation of atpBEFHAGDC. This increase in ATP production thereby fuels the extrusion of RIF from the cell via efflux pumps. In addition, it was found that disruptions in Rv2524c (fatty acid synthase), Rv1048c (hypothetical protein) and Rv3163c (probable conserved secreted protein) resulted in an increase in the level of RIF resistance in a RIF resistant clinical isolate. Interestingly, Rv2524c also showed to have a potential role in regulation of atpBEFHAGDC, whereby it ensures the repression of atpBEFHAGDC. Another gene identified to be involved in the increase in RIF resistance, Rv0260c, is annotated as a possible transcriptional regulator. This study was successful in identifying possible regulatory proteins involved in regulation of the F1F0-ATP synthase in response to RIF, and highlights the complexity of the regulatory events that occur in response to RIF in a MDR M. tuberculosis strain. This study was also successful in identifying candidates for functional analysis to determine novel mechanisms contributing to the level of RIF resistance in M. tuberculosis. Together these findings demonstrate that RIF resistance in M. tuberculosis is more complex than originally thought. Considering that anti-Tuberculosis (TB) drug TMC207 targets the F1F0-ATP synthase, a key enzyme in the production of energy in mycobacteria, the newly identified regulatory genes of F1F0-ATP synthase may represent ideal targets for novel anti-TB drug design. / AFRIKAANSE OPSOMMING: Huidige dogma toon dat mutasies in die rpoB geen vir rifampicin (RIF) weerstandigheid in Mycobacterium tuberculosis (M. tuberculosis) verantwoordelik is. Onlangs is egter bevind dat ander biologiese meganismes, soos energie afhanklike membraanpompe, saam met mutasies in hierdie geen, die verskillende vlakke van RIF weerstandigheid in M. tuberculosis isolate met soortgelyke genetiese agtergrond kan verklaar. Addisionele proteïen studies het gewys dat die komponente van die F1F0-ATP sintase ensiem, wat verantwoordelik vir ATP sintese is en gekodeer word deur die atpBEFHAGDC operon, opgereguleer word na RIF blootstelling. Die hipotese van hierdie studie is dat blootstelling van ‘n multi-middelweerstandige M. tuberculosis isolaat aan RIF aanleiding sal gee tot ʼn aanvanklike sein wat dan verskeie ander biologiese paaie sal aanskakel. Hierdie gebeure sal dan lei tot ‘n verhoging in geenuitdrukking van die F1F0-ATP sintase operon met gevolglike verhoging in energie produksie, wat uiteindelik energie afhanklike membraan pompe sal aanskakel. Die aktiewe uitpomp van RIF uit die sel sal dan ʼn verhoging in die vlak van RIF weerstandigheid veroorsaak. Die eerste doel van hierdie studie is om genetiese areas te identifiseer wat verantwoordelik is vir die regulering van geenuitdrukking van die atpBEFHAGDC operon. Die tweede doel is om nuwe meganismes te identifiseer wat verskille in die vlakke van RIF weerstandigheid in verskillende nou verwante kliniese isolate sal verklaar. ʼn Gespesialiseerde vektor wat die geenuitdrukking van die atpBEFHAGDC operon sal monitor is suksesvol ontwikkel met die gebruik van ʼn fluoresserende proteïen. Daarna is van die transposon mutagenese metode gebruik gemaak om ʼn biblioteek van ewekansige geenuitlatings te maak en hierdie biblioteek is dan gebruik om nuwe meganismes van RIF weerstandigheid te ondersoek. Hierdie studie het twee hipotetiese proteïene, Rv2005c en Rv2417c, in M. tuberculosis transposon mutante geïdentifiseer wat verantwoordelik is vir verlaagde fluoressensie. Dit korreleer met die verwagte verlaagde geenuitdrukking van atpBEFHAGDC. Die geen Rv2005c kodeer vir ʼn universele spanningsproteïen en die resultaat voorspel dat Rv2005c ʼn potensiële rol het om ʼn netwerk van seine in die bakterium aan te skakel direk na blootstelling aan RIF. In ʼn voorgestelde model van regulerende paaie voorspel ons dat die produk van Rv2005c verantwoordelik is vir die vrystelling van ʼn onderdrukker proteïen, Rv1049. Dit lei dan tot die stimulering van ʼn netwerk van intrasellulêre seine wat aanleiding gee tot die opregulering van atpBEFHAFDC. Die opregulering van atpBEFHAFDC sal dan aanleiding gee tot ʼn verhoging in ATP produksie wat die uitpomp van RIF uit die sel sal versnel met die gebruik van energie afhanklike membraan pompe. Dit is verder gevind dat uitskakeling van die gene Rv2524c (vetsuur sintase), Rv1048c (hipotetiese proteïen) en Rv3163c (moontlike konserwatiewe uitskei proteïen) aanleiding gegee het tot die verhoging in die vlakke van RIF weerstandigheid in ʼn RIF weerstandige kliniese isolaat. In die studie is ook bewys dat Rv2524c ʼn potensiële rol in die regulering van atpBEFHAGDC het deurdat dit die onderdrukking van atpBEFHAGDC verseker. Rv0260c is voorheen gelys as ʼn moontlike transkripsionele reguleerder wat betrokke is by die verhoging van RIF weerstandigheid. Hierdie studie was suksesvol in die identifisering van moontlike gene en proteïne wat betrokke is in die regulering van die F1F0-ATP sintase in reaksie tot RIF blootstelling. Dit beklemtoon die kompleksiteit van die regulerende gebeurtenisse wat plaasvind in reaksie tot RIF blootstelling in ʼn multi-middelweerstandige M. tuberculosis isolaat. Verder was daar suksesvol kandidaat gene en ʼn reguleerder geïdentifiseer wat in toekomstige studies ondersoek kan word vir hulle funksionele bydrae om nuwe meganismes te vind wat die varierende vlakke van RIF weerstandigheid in M. tuberculosis sal verklaar. Opsommend demonstreer hierdie studie dat RIF weerstandigheid meer kompleks is as wat voorheen aangeneem was. Die nuwe baie belowende teen-Tuberkulose middel, TMC207, se aanslag is gemik op die belangrike ensiem (F1F0-ATP sintase) wat in hierdie studie ondersoek was. Dus kan nuut geïdentifiseerde proteïene wat betrokke is by die regulering van hierdie ensiem beskou word as ideale kandidate vir die ontwikkeling van nuwe teen -Tuberkulosemiddels. / The National Research Foundation and the Department of Biomedical Sciences

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Anti-tuberculosis drugs target specific essential cellular processes and structural components. The first line drug, rifampicin (RIF) is a RNA polymerase inhibitor which targets the β-subunit and subsequently inhibits the initiation of transcription. Previous proteomic and transcriptomic analyses have shown that exposure to RIF for 24hrs significantly increased the abundance of proteins involved in energy metabolism in clinical isolates. No studies have been done to describe the transcriptional responses to RIF in an in vitro RIF resistant M. tuberculosis isolate. Application of in vitro mutants is novel since it will exclude most of the confounding factors which may be present in clinical isolates obtained from patients where the bacterium may have been incubated for several weeks or even years. This study aimed to determine the effect of prolonged exposure to RIF and the effect of the rpoB Ser531Leu mutation on the expression of energy metabolism genes, sigma factors and a regulator in RIF mono-resistant in vitro mutants with different levels of RIF resistance (minimum inhibitory concentration (MIC): 40μg/ml and 70μg/ml). RIF mono-resistant in vitro mutants were generated from a pan susceptible Beijing cluster 208 progenitor using the Luria Delbruck assay. In vitro RIF mono-resistant mutants harbouring the Ser531Leu rpoB mutation and which displayed different levels of RIF resistance were selected. To assess the effect of prolonged RIF exposure on the expression of candidate genes, the in vitro mutants were cultured in liquid media and exposed to RIF for 1, 7 and 14 days. High quality RNA was extracted from these cultures at each time point and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was done on the selected candidate genes. The results indicate that limited expression of energy metabolism genes and sigma factors was observed after prolonged RIF exposure. In addition, the activity of the regulator (Rv1846c) was down-regulated in the presence of RIF explaining the up-regulated state of energy metabolism genes. To assess the effect of the rpoB Ser531Leu mutation on the candidate genes, RNA was extracted from the RIF unexposed culture at mid-log phase. RT-qPCR was done for each in vitro mutant in addition to the wild-type progenitor isolate. These results show that energy metabolism genes and sigma factors were significantly up-regulated in the RIF resistant mutantss harbouring an rpoB Ser531Leu mutation. This suggests that the mutation had a significant effect on the cellular energy cost due to the up-regulated state of the energy metabolism genes. In addition, an increase in the expression of sigma factors may be required to compensate for the rpoB mutation by enforcing the binding of the RNA polymerase and sigma factors to the promoter for transcription to be initiated. It is therefore important to assess these candidate genes for their potential as novel candidates for future drug design as this is an important aspect to influence tuberculosis control. / AFRIKAANSE OPSOMMING: Teen-tuberkulose middels teiken essensiële sellulêre prosesse en strukturele komponente. Die eerste linie teen-tuberkulose middel, rifampisien (RIF) is ʼn RNS polimerase inhibeerder wat die β-subeenheid teiken en daarna die inisiasie van transkripsie onderdruk. Vorige proteomiese en transkriptomiese analises het getoon dat blootstelling aan RIF vir 24 uur beduidende styging in sekere protiene wat verband hou met energie metabolisme in kliniese isolate veroorsaak. Die huidige studie poog om die effek van langdurige RIF blootstelling, asook die effek van die rpoB Ser531Leu mutasie op die uitdrukking van energie metabolisme gene, sigma faktore en reguleerders op RIF-enkel weerstandige in vitro mutante by verskillende vlakke van RIF weerstandigheid (Minimum Inhiberende Konsentrasie (MIK): 40μg/ml en 70μg/ml) te ondersoek. RIF-enkelweerstandige in vitro mutante isolate is gegenereer van ʼn sensitiewe Beijing 208 stamfamilielid deur die Luria Delbruck metode. In vitro RIF enkelweerstandige mutante met die rpoB Ser531Leu mutasie en verskillende vlakke van RIF weerstandigheid is geselekteer. Om die langdurige effek van RIF blootstelling op kandidaat geen uitdrukking te ondersoek, is in vitro mutante isolate gegroei in vloeibare medium en blootgestel aan RIF vir 1, 7 en 14 dae. Goeie kwaliteit RNS is geëkstraheer van hierdie kulture by elke tydpunt om Werklike-tyd Kwantitatiewe Polimerase Ketting Reaksie (RT-qPCR) op die kandidaat gene uit te voer. Die resultate toon dat ʼn beperkte aantal energie metabolisme en sigma faktor gene uitgedruk was na RIF blootstelling. Verder is die uitdrukking van die reguleerder (Rv1846c) af gereguleer in die teenwoordigheid van RIF en dit verduidelik die op gereguleerde energie metaboliese geen patroon. Om die effek van die rpoB Ser531Leu mutasie op die kandidaat gene te evalueer, is RNS geëkstraheer van ʼn weerstandige en RIF sensitiewe kultuur wat nie blootgestel was aan RIF nie. RT-qPCR is uit gevoer op elke in vitro mutante isolaat asook op ʼn sensitiewe isolaat sonder ʼn mutasie. Hierdie resultate toon dat energie metabolisme gene en sigma faktore beduidend opreguleer word in die isolate met ʼn rpoB Ser531Leu mutasie. Dit dui daarop dat die mutasie ʼn beduidende effek op die sellulêre energie koste het, omdat die energie metabolisme gene op gereguleer is. Verder kan ʼn toename in die uitdrukking van sigma faktore benodig word om die effek van die rpoB mutasie te oorkom deur binding van die RNS polimerase en die sigma faktore aan die promotor om transkripsie inisiasie te forseer. Dit is daarom belangrik om hierdie kandidaat gene verder te ondersoek vir toekomstige ontwikkeling van teenmiddels teen tuberkulose.

Study project (M.Med.) -- University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The main purpose in doing forensic post mortems is to supply information to the judicial system. Medical personnel involved in doing post mortems seldom get feedback regarding efficiency. Numerous allegations have been made regarding the work of district surgeons in terms of forensic post mortems, often suggesting that the work is substandard. In South Africa district surgeons do post mortems mostly in the rural areas, and training centre personnel do forensic post mortems in urban areas. Training centre personnel include specialized forensic pathologists, registrars and medical officers working in the Departments of Forensic Medicine, affiliated to universities. The South African Police Service mostly manages government mortuaries. The South African forensic medico-legal system is unique, and does not correspond in with the four main systems used worldwide. Research was done by sending 200 questionnaires to a representative group from the legal fraternity of the Western Cape (including judges / magistrates, state prosecutors, private lawyers) and also the South African Police Service investigating officers. The judiciary, as the users of the information generated through forensic post mortems, are therefore in a suitable position to determine the efficacy of forensic post mortem. The questionnaire was structured to determine the general perception, as well as comments, regarding 9 different aspects involved with doing forensic post mortems. These include the thoroughness and completeness of reports, standard of academic knowledge, efficacy of verbal testimony in court, length of time in releasing the report, general attitude, efficacy of sketches and diagrams, efficacy of photography, sufficient taking of toxicology samples and sufficient utilization of special laboratory investigations. The last question was an open question to allow for general comments and anecdotes. For each aspect it was also determined whether there was a difference in efficacy noted between the two groups. The effect of this, if any, on the judicial criminal justice system was also assessed. A different questionnaire was sent out to all forensic pathologists in the Western Cape. The standard of work of the district surgeons was hereby assessed. General comment regarding academic knowledge, and findings at post mortem made by district surgeons was assessed. The pathologists were also questioned regarding the general attitude of district surgeons, and imput were asked regarding continued medical education programs. An area for general comment was also supplied. The main findings were as follows: a. The legal fraternity in the Western Cape is generally satisfied with the efficiency of forensic post mortems, except the use of laboratory investigations and also the length of time to release reports. b. The legal fraternity could determine a difference in the efficiency of post mortems done by district surgeons and training centre personnel. Training centre personnel were generally regarded as more effective. c. The difference between the two groups, due to ineffective district surgeon post mortems, had a negative effect on the criminal justice system. The following recommendations were made: a. Training centre personnel: Serious consideration should be given to appointing qualified forensic pathologists in the rural areas. Training centre personnel should also be more involved in training the district surgeons. b. District surgeons: The training, re-training and continued medical education of district surgeons in the Western Cape should be prioritized. The service conditions should also be reviewed. c. Administrative: Audit of post mortem reports. The efficiency regarding court appearances should be audited through the Department of Justice. Administrative power will be necessary to oversee the above-mentioned recommendations. / AFRIKAANSE OPSOMMING: Die hoofdoel met die doen van geregtelike post mortems is om inligting te verskaf aan die regsproses. Medici gemoeid met geregtelike post mortems kry baie seide terugvoer oor die effektiwiteit van werk gelewer in die hof. Daar is ook herhaaldelik suggesties gemaak dat die werk van die distriksgeneeshere met betrekking tot geregtelike post mortems soms suboptimaal is. Regsmediese post mortemdienste in Suid Afrika word verskaf deur distriksgeneeshere in die platteland, en deur personeel verbonde aan opleidingshospitale in die stede. Die opleidingssentra-personeel sluit in gespesialiseerde forensiese patoloe, kliniese assistente en mediese beamptes werksaam in 'n Departement van Geregtelike Geneeskunde verbonde aan 'n universiteit. Staats- Iykshuise word bestuur en beheer deur die Suid-Afrikaanse Polisiediens. Daar bestaan geen soortgelyke model vir die voorsiening van regsmediese dienste in die res van die wereld nie. Navorsing is gedoen deur vraelyste uit te stuur aan 200 verteenwoordigende regslui (wat insluit regters/landdroste, staatsaanklaers, privaat regslui) en aan Suid-Afrikaanse Polisiediens-ondersoekbeamptes in die Wes Kaap. Die reg, as verbruikers van regsmediese dienste is gekies om 'n opinie uit te spreek oor die doeltreffendheid van forensiese post mortems. Die vraelyste is gestruktureer om die algemene tevredenheid en opinies en kommentaar te bekom oor nege verskillende aangeleenthede rakend die forensiese post mortem, nl. deeglikheid en volledigheid van verslae, standaard van akademiese kennis, effektiwiteit van verbale getuienisaflegging in die hof, tydsverloop vir lewering van verslae, houding en gesindheid van medici, doeltreffendheid van sketse en diagram me, effektiewe gebruik van fotografie, effektiewe gebruik van toksikologiese ondersoeke, effektiewe gebruik van spesiale ondersoeke, asook 'n algemene oop vraag vir kommentaar oor probleemareas. Daar word vervolgens vir elke aangeleentheid bepaal of daar 'n verskil in effektiwiteit opgelet word tussen twee mediese subgroepe, en indien wei watter groep meer effektief funksioneer. Verder sal nagegaan word of die regsproses geaffekteer word deur enige van bogenoemde bevindinge. 'n Verskillende vraelys is uitgestuur aan aile geregtelike patoloe in die Wes-Kaap. Hiermee word die standaard van die werk van distriksgeneeshere beoordeel. Kommentaar is gevra oor akademiese kennis met betrekking tot geregtelike post mortems en oor bevindinge gemaak deur distriksgeneeshere by post mortems. Daar word ook gevra oor die algemene houding van distriksgeneeshere, asook vir voorstelle vir voortgesette onderrigsprogramme. 'n Oop vraag is gestel vir kommentaar oor probleemareas. Uit die response is die volgende gevolgtrekkings gemaak: a. Die regslui in die Wes-Kaap is oor die algemeen tevrede met die diens gelewer met betrekking tot geregtelike post mortems, met uitsondering van die effektiwiteit van laboratoriumondersoeke, en ook oor die tydsverloop tussen die doen van post mortem en die vrystel van die verslae. b. Die regslui kon 'n verskil bepaal in die graad van effektiwiteit van post mortems gedoen deur distriksgeneeshere en opleidingssentra-personeel. Opleidingssentra-personeel is deur die meerderheid van respondente identifiseer as meer effektief. c. Die verskil tussen die twee groepe, a.g.v. oneffektiewe distriksgeneesheer post mortems, het 'n negatiewe effek op die regsproses. Aanbevelings is gemaak om die sisteem te verbeter: a. Opleidingssentra-personeel: Die uitplasing van gekwalifiseerde forensiese patoloe in die platteland moet oorweeg word. Opleidingssentrapersoneel kan ook meer betrokke wees by opleiding van distriksgeneeshere. b. Distriksgeneeshere: Aandag moet gegee word aan die opleiding, heropleiding en voortgesette geneeskundige onderrig van distriksgeneeshere in die Wes-Kaap. Die werksomstandighede waaronder hulle diens lewer moet ook aangespreek word. C. Administratief: Ouditering van post mortem verslae. Ouditering van effektiwiteit van hofverskynings, in assosiasie met die Departement van Justisie. Admininistratiewe wilskrag sal ook essensieel wees by implementering van bogenoemde voorstelle.