Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactions between DevS and DevR that are implicated in the regulation of the dormancy response in Mycobacterium tuberculosis. These binding interactions could provide good drug targets for the treatment of persistent tuberculosis, the mechanistic understanding of their binding interactions is important for the development of a validated inhibitor screen. A detailed in silico analysis of the amino acid residues that play a role in the binding of receptor DevR to both kinase DevS and the target DNA was undertaken. A reasonable approximation of the DevS structure was produced using homologous protein structures. In silico docking of DevS to DevR merely produced a set of probable candidate structures, since more than one conformation with similar docked energies was observed. The decision on which one is the more correct form can only be estimated by crystallization of this complex. Therefore, the functional expression and purification of the Dev TCS components were pursued. Denaturing HIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to the production of functional Dev TCS proteins. To understand the binding of DevR to DNA consensus sequences, as well as the nature of these interactions, a model was built of the full length DevR dimer binding to DNA consensus sequences. Based on this model, single mutations were made to DevR in vitro and their effects assessed in order to validate the model built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found that K179I and N183L mutants prevented the binding of DevR to the DNA consensus sequences. If DevR and DevS binding are to be used in a drug development program, it is essential to have the protocols to accurately measure their interaction, in addition to developing a fundamental understanding of how their interactions occur. The binding affinity of DevR to both DevS and the truncated soluble fragment of DevS (DevS201) were explored, using the BIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between a histidine kinase and a response regulator, the KD needs to be in the nM range. The KD was calculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concluded that DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcore could be used to screen small-molecule inhibitors of DevR-DevS interactions. / AFRIKAANSE OPSOMMING: Die Dev twee komponent sisteem (TKS) bestaan uit ‘n histidine kinase naamlik (DevS) en ‘n reaksie reguleerder DevR. DevS en DevR is betrokke by die regulering van die dormante stadium van Mycobacterium tuberculosis. Hierdie meganisme kan ‘n deurbraak dwelm teiken vir die behandeling van sluimerende tuberkulose wees. Die meganisme van hierdie bindings interaksies is van kritieke belang, tesame met die ontwikkeling van 'n erkende inhibeerder toets. ‘n Gedetaileerde in silico analise van die aminosuur volgordes wat 'n rol speel in die binding van die reseptor DevR aan beide DevS sowel as die teiken DNS is voltooi. ‘n Model van die DevS struktuur is saamgstel met behulp van homoloë proteïen strukture. In silico mering van DevS aan DevR het `n stel van die waarskynlike kandidaat strukture verskaf, aangesien meer as een konformasie met soortgelyke merings energieë waargeneem is. Die mees waarskynlike vorm kan alleenlik geïndentifiseer word na kristallisasie van hierdie kompleks. Die funksionele uitdrukking en suiwering van die Dev TKS proteine is gevolglik uitgevoer. Funksionele Dev TKS proteïene is verkry deur denaturerende HIS-select nikkel affiniteit jel suiwering, in die vorm van matriks-geassisteerde hervouing te gebruik. Ten einde die binding te verstaan tussen DevR en DNS konsensus volgordes, sowel as die aard van hierdie interaksies, is 'n model gebou van die volle lengte DevR dimeer binding aan DNS konsensus volgordes. Hierdie model is gevalideer deur punt mutasies in DevR te skep en die gevolge daarvan te beoordeel met elektroforetiese mobiliteits verskuiwing reaksie analises. Dit is bevind dat K179I en N183L mutante, verhoed die binding van DevR aan die DNS konsensus volgordes. Die gebruik van DevR en DevS bindings in ‘n dwelm ontwikkelingsprogram, benodig die fundamentele begrip van hoe die interaksies plaasvind, sowel as akkurate protokolle om die interaksies te meet. Die BIAcore instrument, ’n SPR-gebaseerde biosensor, is ingespan om die bindings affiniteit van DevR aan beide DevS en die fragment van DevS (DevS201) te ondersoek. Om voldoende sterk binding tussen DevS en die DevR te verseker, moet die KD in die nM omgewing wees. Die KD is bepaal as 255 nM en 184 nM vir DevS201 en DevS, onderskeidelik. Die afleiding kan dus gemaak word dat DevS201 sterk genoeg aan DevR bind om in verdere studies gebruik te kan word, en dat die BIAcore gebruik kan word om klein-molekule inhibeerders van DevR-DevS interaksies te toets.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes, in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie, sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro- Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg- Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61 Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang. Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie, informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican; perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante. Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese differensiering en kanker, lê die grondslag vir verdere ondersoeke.

<p>Parkinson’s disease (PD) is a detrimental neurodegenerative disorder characterized by the presence of large protein aggregates in the brain called Lewy bodies, which are primarily composed of the protein α-synuclein (αSyn). Due to the dysregulation of αSyn levels in PD, controlling its levels through the manipulation of protein degradation pathways has been suggested as a therapeutic avenue for the treatment of PD and related diseases. Although αSyn is known to be degraded through the autophagy and proteasome pathways, it is one of only a few known substrates of the ubiquitin-independent proteasome pathway, which utilizes the 20S core particle of the proteasome (20S CP) to degrade proteins. We therefore hypothesize that small molecule stimulation of the 20S CP will enhance αSyn degradation and reduce αSyn pathology, providing a therapeutic benefit in PD models.</p><p>We began our studies by developing a fluorescence resonance energy transfer (FRET) reporter assay to monitor 20S CP activity and screen for small molecule stimulators. This assay provides a greater dynamic range to detect 20S CP stimulation compared to the most commonly used assay to monitor proteasome activity. Using the FRET assay, we were able to identify a number of novel 20S CP stimulators that differ in structure as well as potency and degree of stimulation. We next evaluated the ability of four small molecule stimulators to enhance protein degradation by the 20S CP in a biochemical assay using 15 different purified proteins. These 15 proteins include known substrates of the 20S CP and vary in size and degree of disorder. From this assay, we demonstrate that a 20S CP stimulator is likely to enhance the degradation of highly disordered proteins, such as αSyn, but the effect on other protein levels appears to be distinct for each stimulator. Two of our more potent stimulators, AM-404 and miconazole, were used with the proteasome inhibitor bortezomib for subsequent studies in HEK-293T cells in which we corroborated the results of our biochemical assay. While both AM-404 and miconazole were shown to impact highly disordered proteins, there was not much overlap between the proteins shown to be affected by each stimulator. Due to the distinct effect of each stimulator on protein regulation by the 20S CP, this study indicates the potential of tailoring a small molecule 20S CP stimulator to enhance the degradation of particular substrates.</p><p>Since AM-404 and miconazole were shown to impact 20S CP activity in different ways, we next evaluated whether either stimulator would be able to prevent the αSyn-induced inhibition of the 20S CP. High levels of αSyn have been shown to lead to proteasome impairment in biochemical and cell studies. We confirm 20S CP impairment in the presence of micromolar amounts of αSyn, and we demonstrate that miconazole, but not AM-404, is effective at maintaining 20S CP activity in the presence of increasing concentrations of αSyn. We also show that αSyn-overexpressing PC12 cells (PC12 C4 cells) display reduced proteasome activity compared to the parent cell line. Miconazole and AM-404 increased proteasome activity in PC12 C4 cells, which were more sensitive to 20S CP stimulation than non-transfected PC12 cells, but miconazole was shown to be more effective at modulating αSyn phosphorylated at Ser129 in PC12 C4 cells. </p><p>Our results reveal the dynamic nature of the 20S CP and the ways in which its activity can be modulated to affect protein levels. While AM-404 is effective at stimulating the 20S CP to enhance the degradation of some proteins, miconazole was shown to be more efficient at modulating αSyn levels and impacting αSyn pathology, as it relates to 20S CP impairment. While the results described here mark the beginning of an exciting area of study, we do demonstrate the therapeutic potential of 20S CP stimulation to combat PD. </p><div><br></div>

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: An important next step to Tuberculosis control relies on the translation of basic science and modern diagnostic techniques into primary health care clinics. These assays must be rapid, inexpensive, interpretation of results must be easy and they must be simple so that a healthcare worker with limited training can perform the tests under safe conditions. This study consists of four aims. The first aim was to develop a methodology to sterilize sputum specimens for rapid TB diagnosis and drug resistance testing. Candidate bactericides were identified from the literature, and tested for their bactericidal activity in Mycobacterium tuberculosis. We identified ultraseptin®aktiv as a powerful bactericidal agent which sterilizes sputum specimens for subsequent safe handling prior to light emitting diode microscopy and it also provides a DNA template for PCR-based tests. An algorithm has been proposed for the processing of specimens and rapid diagnosis of TB and drug resistant TB while patients wait for results. Recently, the World Health Organization has endorsed the MTBDRplus test for diagnosis of TB and drug resistant TB. However genotypic tests may have more problems than anticipated. With the HIV pandemic, an increase of non-tuberculous mycobacteria has been reported. The sensitivity of genotypic tests in specimens with underlying non-tuberculous mycobacterial species therefore requires further evaluation. This study therefore also aimed at determining the reliability of the MTBDRplus assay for detection of drug resistant TB where non-tuberculous bacterial load is high. Clinically relevant non-tuberculous mycobacterium DNA and DNA from a multi-drug resistant TB isolate were obtained. Ratios of the different NTM with the MDR-TB DNA were made and subjected to the MTBDRplus assay. Known mix NTM and TB infected clinical isolates and sputum sediments were also evaluated for TB and drug resistance detection on the MTBDRplus assay. Under these conditions, this study provides evidence that the MTBDRplus test cannot reliably detect TB and drug resistance TB in specimens with underlying non-tuberculous mycobacteria. Thirdly, to evaluate the sensitivity of the MTBDRplus assay for detecting drug resistance in hetero-resistant isolates, ratios were made using purified DNA from an MDR and pan-susceptible TB isolate. The MTBDRplus assay was then performed on the different ratios. We report that the MTBDRplus assay can efficiently detect wild type DNA in genes associated with resistance during the early evolution of drug resistance. However, in the later stage during treatment when both the wild type and mutants are present, the detection limit for the mutant DNA was 1:55. Due to these results, the MTBDRplus assay should still be further improved or other tests should be developed to address these limitations. And finally to combat cross amplicon contamination during the final steps of genotypic detection with the MTBDRplus assay, a proof of concept for a patentable closed tube line probe device was proposed on the 4th aim. This device can be improved to enable automated drug resistance genotyping of multiple specimens. The results of this study highlight the need for a sensitive inexpensive point of care drug resistance test that does not require intensive training. / AFRIKAANSE OPSOMMING: 'n Belangrike volgende stap om Tuberkulose te beheer is om basiese wetenskap resultate te gebruik sodat moderne diagnose tegnieke ontwikkel kan word wat in primêre gesondheidsorg klinieke toegepas kan word. Hierdie toetse moet vinnig, goedkoop, en die interpretasie van resultate moet maklik wees. Die toetse moet eenvoudig wees sodat 'n gesondheidswerker met beperkte opleiding die toetse onder veilige omstandighede kan uitvoer. Hierdie studie bestaan uit vier doelwitte, waarvan die eerste was om 'n metode te ontwikkel vir die sterilisasie van sputum monsters vir vinnige TB diagnose en die toesting van middelweerstandigheid. Kandidaat kiemdodende middels was geïdentifiseer vanaf die literatuur en die middels se kiekdodende aktiviteit was getoets op Mycobacterium tuberculosis. Ons het ultraseptin®aktiv geïdentifiseer as 'n kragtige kiemdodende middel wat bakteria in sputum monsters steriliseer vir veilige hantering voordat diagnose met 'n lig uitstralende diode mikroskopie gedoen kan word. Hierdie behandeling met ultraseptin®aktiv bied ook 'n DNA templaat vir PCR-gebaseerde toetse. 'n Algoritme is voorgestel vir die hantering van monsters en die vinnige diagnose van sensitiewe- en middel weerstandige Tuberkulose terwyl die pasiënte by die kliniek wag vir die resultate. Onlangs het die Wêreld Gesondheid Organisasie die genotipiese MTBDRplus toets vir die diagnose van Tuberkulose en middel-weerstandige Tuberkulose onderskryf. Hierdie toets word tans op groot skaal in Suid Afrika gebruik. Dit kan egter wees dat genotipiese toetse baie meer probleme kan he as wat aanvanklik verwag is. Die HIV pandemie gaan toenemend gepaard met n toename van nie-tuberkulose mycobacteria. Die sensitiwiteit van genotipiese toetse op monsters met onderliggende nie-tuberkulose mikobakteriese spesies vereis dus verdere evaluasie. Die doel van hierdie studie was ook om die betroubaarheid van die MTBDRplus-toets te bepaal vir die opsporing van middelweerstandige TB waar die nie-tuberkulose bakteriële lading hoog is. DNA van kliniese relevante nie-tuberkulose mikobakteria en multi-middelweerstige TB isolate was bekom. Verskillende verdunnigs van die spesifieke NTM DNA te same met die van MDR-TB DNA is gemaak en onderwerp aan die MTBDRplus toets. Bekende gemengde NTM- en TB geïnfekteerde kliniese isolate en sputum sedimente was ook geevalueer vir die opsporing van TB en middel weerstandigheid met die MTBDRplus toets. Hierdie studie verskaf bewyse dat die MTBDRplus toets nie betroubaar is met die diagnose van sensitiewe- en middel weerstandige Tuberkulose in monsters met onderliggende nie-tuberkulose mycobacteria nie. Verskillende verdunnings van gesuiwerde DNA van MDR en pan-sensitiewe TB isolate is gemaak om die sensitiwiteit van die MTBDRplus toets vir die opsporing van middelweerstandigheid te bepaal. Die MDRDRplus toets is gebruik met hierdie verdunnings. Resultate in hierdie studie toon dat die MTBDRplus toets effektief is met die identifisering van wilde-tipe DNA (dit beteken middel sensitief) in gene wat geassosieer word met middel weerstandigheid gedurende die vroeë ontwikkeling van weerstandigheid. Hier teenoor toon die resultate dat in die later stadium tydens behandeling, wanneer beide die wilde-tipe (sensitief) en mutante DNA (weerstandig) teenwoordig is, is die opsporingslimiet vir die mutante DNA maar 1:55. As gevolg van hierdie resultate raai ons aan dat die MTBDRplus toets nog verder verbeter moet word of dat ander toetse ontwikkel moet word om hierdie beperkinge aan te spreek. Amplikon kruiskontaminasie kan n groot impak hê op die betroubaarheid van enige genotipiese diagnostiese toets. Die finale stappe van MTBDRplus toets behels die gebruik van 'n oop sisteem sodat kontaminasie maklik kan plaasvind. In die 4de doewit 'n konsep vir 'n patenteerbare geslotebuis toestel ontwikkel en die resultate het getoon dat kontaminasie suksesvol uitgeskakel kan word. Hierdie toestel kan verbeter na 'n outomatiese apparaat verbeter word sodat die module genotipering van verskeie monsters moontlik kan maak. Die resultate van hierdie studie beklemtoon die noodsaaklikheid van 'n sensitiewe goedkoop “point of care” diagnostiese toets wat nie intensiewe opleiding benodig nie. / Medical Research Council of South Africa / University of Stellenbosch, Dept. of Molecular Biology and Human Genetics

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Progressive familial heart block type II (PFHBII) is an inherited cardiac conduction disorder of unknown aetiology, which has been described in a South African family. The disorder was mapped to a 2.9 centimorgan (cM) locus on chromosome 1q32.2-32.3. Clinically, PFHBII manifests cardiac conduction aberrations, that progress to a disease of the heart muscle, dilated cardiomyopathy (DCM). DCM is also reported as an end phase in hypertrophic cardiomyopathy (HCM), another heart muscle disorder. These cardiomyopathies are genetically heterogeneous with some of the genes reported as causes of both disorders. Therefore, genes identified as causes of HCM and DCM were considered plausible candidates for PFHBII mutation analysis. Additionally, this study provided an opportunity to assess potential modifiers of HCM. HCM exhibits marked phenotypic variability, observed within and between families harbouring the same causative mutation. Genes within the PFHBII locus were selected for PCR-SSCP analysis based on homology to genes previously reported as causing conduction system disorders associated with arrhythmias, DCM and/or HCM. Results were confirmed by direct sequencing and association between the detected variants and HCM parameters was assessed using a quantitative transmission disequilibrium test (QTDT). Eleven plausible candidate genes were selected within the PFHBII locus and two of the genes, PFKFB2 and ATF3, that encode for 6-phosphofructo-2,6-bisphosphatase (PFK-2/FBPase-2) and activating transcription factor 3 (ATF3), respectively, were analysed for PFHBII-causing and HCM main locus and/or HCM modifying mutations. Mutation analysis of PFKFB2 and ATF3 in the PFHBII family revealed no PFHBII causal mutation. PFKFB2 and ATF3 were later localised outside the PFHBII locus, and, therefore, were excluded as PFHBII plausible candidates. Further analysis of the two genes for HCM main locus and/or HCM modifying mutations in the HCM panel identified several sequence variants. QTDT analysis of these variants showed no significant association. Completion of the Human Genome Project (HGP) and annotation of new genes within the PFHBII locus allowed the identification of more PFHBII plausible candidate genes. Identification of causal mutations in plausible PFHBII candidate genes will allow molecular diagnosis of PFHBII pathophysiology. Furthermore, identification of both HCM-modifying and HCM-causing genes will give insight into the phenotypic variability noted among South African HCM-affected individuals and into the molecular cause of the disease among individuals with HCM-like clinical features. / AFRIKAANSE OPSOMMING: Progressiewe familiële hartblok tipe II (PFHBII) is ʼn oorgeërfde hart geleidingsiekte van onbekende etiologie wat in ʼn Suid-Afrikaanse familie beskryf is. Die siekte is ʼn 2.9 sentimorgan (cM) lokus op chromosoom 1q32.2-32.3 gekarteer. Klinies presenteer PFHBII met geleidingsfwykings wat uitloop op gedilateerde kardiomiopatie (DCM). DCM word ook gerapporteer as ʼn endfase in hipertrofiese kardiomiopatie (HCM), ʼn ander hartspiersiekte. Die kardiomiopatieë is geneties heterogeen, met ʼn aantal gene wat as oorsaak van altwee siektetoestande gerapporteer word. Daarom is alle gene wat geïdentifiseer is as oorsake van DCM en HCM, as moontlike kandidaatgene vir PFHBII mutasieanaliese beskou. Bykomend het hierdie studie die geleentheid gebied om potensiële modifiseerders van HCM te assesseer. HCM toon beduidende fenotipiese variasie binne en tussen families wat dieselfde siekteveroorsakende mutasie het. Gene binne die PFHBII-lokus is geselekteer vir PCR-SSCP-analiese gebaseer op homologie met gene wat voorheen gerapporteer is om betrokke te wees by geleidingsiesisteemsiektes, geassosieerde arritmieë, DCM en/of HCM. Resultate is bevestig deur volgordebepaling. Assosiasie tusssen ontdekte variante en die siekteparameter is bepaal met ʼn kwantitatiewe transmissie disekwilibrium toets (QTDT). Elf moontlike kandidaatgene in die PFHBII-lokus is geselekteer en twee van die gene, PFKFB2 en ATF3, wat kodeer vir 6-fosfofrukto-2,6-bifosfatase (PFK-2/FBPase-2) en aktiveringstranskripsiefaktor 3 (ATF3) respektiewelik, is vir PFHBII-oorsakende en HCMhooflokus en/of HCM-modifiseerende mutasies ondersoek. Mutasie-analiese van PFKFB2 en ATF3 in die PFHBII-familie het nie ʼn siekteveroorsakende mutasie onthul/uitgelig nie. PFKFB2 en ATF3 is later buite die PFHBII-lokus geplaas en dus ook as moontlike PFHBII-kandidate uitgesluit. Verdere ondersoek van díe twee gene vir HCM-hooflokus en/of HCM-modifiserende mutasies in die HCM-paneel het ʼn aantal volgorde variante geïdentifiseer. QTDT-analiese van die variante het geen beduidende assosiasies aangetoon nie. Voltooiing van die Menslike Genoom Projek (HGP) en annotasie van nuwe gene in die PFHBIIlokus het tot die identifikasie van verdere moontlike PFHBII-kandidaatgene gelei. Identifikase van siekte-veroorsaakende mutasies in die moontlike PFHBII-kandidaatgene sal die molekulêre diagnose van PFHBII toelaat en insig in die patofisiologie van die siekte gee. Verder, identifikasie van beide HCM-veroorsakende of HCM-modifiserende gene kan insig gee in die fenotipiese varieerbaarheid wat onder Suid-Afrikaanse HCM-geaffekteerde individue waargeneem word en ook in die molekulêre oorsake van die siekte in individue met HCMsoortige kliniese kenmerke.

Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: The genome of the pathogen Mycobacterium tuberculosis contains five copies of the ESAT-6 (ESX) gene cluster region, which encodes for a novel type VII secretion system. These gene cluster regions, which are directly involved in pathogenicity and phagosomal escape, contain genes encoding exported T-cell antigens ESAT-6 and CFP-10. The mechanism of action of the ESX secretion system however, remains largely unknown. This study focused on ESX gene cluster region 4 (ESX-4), which has been shown to be the most ancestral region and is also present in other species of Mycobacteria and even in other high G+C Gram-positive bacteria, such as Corynebacterium diptheriae and Streptomyces coelicolor. This project aimed to investigate the protein-protein interactions of ESX-4 of M. tuberculosis in the model organism Mycobacterium smegmatis by means of Mycobacterial Protein Fragment Complementation (M-PFC). M-PFC is a two-hybrid technique which employs two cloning vectors, pUAB300 (conferring resistance to hygromycin B) and pUAB400 (conferring resistance to kanamycin). Genes of interest are cloned into these vectors and co-transformed into the model organism M. smegmatis after which it is expressed as fusion proteins. Interaction of the proteins allows selective growth on a medium containing the antibiotic trimethoprim. Various interactions were identified throughout this region, including selfinteractions as well as the expected interaction between the ESAT-6 and CFP-10 protein family members esxT and esxU. Since this region is ancestral, ESX-4 provides the basic model of the mechanism of secretion of the type VII secretion system. Many similarities were apparent when the interactions identified for ESX-4 were compared to the interactions previously identified in ESX-3. Interactions identified by means of M-PFC provide a basis for the further study of the structure of this secretion system, and should be confirmed by means of other techniques, such as co-immunoprecipitation. Despite the ability of M-PFC to identify protein-protein interactions in a mycobacterial system, and thus overcoming some of the limitations of the classical yeast two-hybrid model, it must still be regarded as a fishing experiment for potential interactions. A further aim of the project was to construct a knock-out of ESX-4 in the model organism M. smegmatis, which contains three ESX regions, namely ESX-1, -3 and -4. Homologous recombination proved to be an effective technique for the construction of the knock-out, also indicating that ESX-4 is not essential for in vitro growth of M. smegmatis. The knock-out strain showed no morphological differences to the wild type strain of M. smegmatis. The knock-out strain will in future be compared to the wild type strain in various functional studies in order to determine the function of the ancestral ESX region. / AFRIKAANSE OPSOMMING: Die genoom van die patogeen Mycobacterium tuberculosis bavat vyf kopieë van die ESAT-6 geen groep gebiede wat kodeer vir ‘n unieke tipe VII sekresie sisteem. Die geen groep gebiede, wat direk betrokke is by patogenisiteit en fagosomale ontsnapping, bevat gene wat kodeer vir die gesekreteerde T-sel antigene ESAT-6 en CFP-10. Die meganisme van die ESX sekresie sisteem is egter steeds tot ‘n groot mate onbekend. Hierdie studie het gefokus op die ESX geen groep gebied 4 (ESX-4), wat voorheen bepaal is om die vroegste kopie van die gebied te wees en wat ook in ander species van Mikobakterieë en hoë G+C Gram-positiewe bakterieë, soos Corynebacterium diptheriae en Streptomyces coelicolor, voorkom. Hierdie projek was daarop gemik om die proteïen-proteïen interaksies van ESX-4 van M. tuberculosis in die model organisme Mycobacterium smegmatis te ondersoek deur middel van Mikobakteriële Proteïen Fragment Komplementasie (M-PFK). M-PFK is ‘n twee-hibried tegniek wat van twee kloningsvektore, naamlik pUAB300 (wat weerstand teen hygromycin B bied) en pUAB400 (wat weerstand teen kanamycin bied) gebruik maak. Gene van belang word in die vektore ingekloneer en in die model organisme, M. smegmatis geko-transformeer, waarna dit as fusieproteïene uitgedruk word. Indien ‘n interaksie tussen die proteïene plaasvind, sal selektiewe groei op ‘n medium wat die antibiotikum trimethoprim bevat, waargeneem word. Verskeie interaksies is in hierdie gebied geïdentifiseer, insluitende self-interaksies, sowel as die verwagte interaksie tussen die ESAT-6 en CFP-10 proteïen familielede esxT en esxU. Aangesien hierdie gebied die vroegste kopie is, bied ESX-4 die basiese model vir die meganisme van sekresie van die tipe VII sekresie sisteem. Wanneer interaksies wat vir ESX-4 geïdentifiseer is met die wat voorheen vir ESX-3 geïdentifiseer is vergelyk word is daar heelwat ooreenkomste. Interaksies wat deur middel van M-PFK geïdentifiseer is, verskaf ‘n basis vir die vêrdere studie van interaksies van hierdie gebied, en sal bevestig moet word deur gebruik te maak van aanvullende tegnieke, soos ko-immunopresipitasie. Ten spyte van die vermoë van M-PFK om proteïen-proteïen interaksies in ‘n mikobakteriële sisteem, wat dus sommige van die beperkings van die klassieke gis twee-hibriedmodel oorkom, te bestudeer, behoort dit steeds as ‘n voorlopige metode van identifikasie beskou te word. ‘n Vêrdere doel van die projek was om ‘n uitslaanmutant van ESX-4 in die model organisme M. smegmatis, wat drie van die ESX gebiede, naamlik ESX-1, -3 en -4 bevat, te skep. Homoloë rekombinasie is bewys om ‘n effektiewe tegniek te wees vir die skep van ‘n uitslaanmuntant en het daarop gedui dat ESX-4 nie essensieel is vir die in vitro groei van M. smegmatis nie. Die uitslaanstam het ook geen morfologiese verskille getoon teenoor die oorspronklike stam nie. Die uitslaanmutant sal in die toekoms gebruik word in ‘n verskeidenheid funksionele studies waar dit vergelyk sal word met die oorspronklike stam, ten einde die funksie van die vroegste ESX-gebied te bepaal. / Medical Research Council of South Africa / National Research Foundation of South Africa / Ernst and Ethel Eriksen Trust

Thesis (MScMedSc)--University of Stellenbosch, 2011. / Please refer to full text to view abstract.

Heat shock proteins function as molecular chaperones which help protein folding and prevent protein aggregation. My study shows that celastrol, a pharmacological compound capable of up-regulating the levels of heat shock proteins, inhibits cell death and protein aggregation caused by expanded polyglutamine containing protein, and the protective effects of celastrol are dependent on heat shock factor 1. These results suggest the potential of celastrol as a therapeutic agent in the treatment of polyglutamine diseases. Sumoylation is a protein modification which plays diverse roles in regulating the target proteins. My study shows that lamin A is a target of protein sumoylation, and two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. My results also indicate that sumoylation is important for the normal localization of lamin A, and support a role for altered sumoylation in the underlying molecular mechanism of cardiomyopathies associated with the E203G/E203K lamin A mutations. In the third project, my results show that amyloid precursor protein is another target of SUMO modification, and sumoylation of amyloid precursor protein reduces the levels of amyloid β aggregates, which are the primary causative factor for Alzheimer’s disease. My results provide a new mechanism for the generation of amyloid β, and indicate the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer’s disease. My results also provide the first demonstration that SUMO E2 enzyme exists in the lumen of the endoplasmic reticulum, extending the sub-cellular reach of sumoylation to include the regulation of proteins in secretory pathways.

Anti-tumor therapeutic strategies based on combinations of chemotherapeutic agents with a death inducing ligand such as TNF-α Related Apoptosis Inducing Ligand (TRAIL), are directed towards selective and effective cancer cell apoptosis and enhanced therapeutic response. We previously demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of initiator caspase-8. The present study investigated the functional link between caspase-8 and the proteasome, by analyzing the impact of caspase-8 ubiquitination and proteasomal degradation on the outcomes of the extrinsic apoptosis pathway in cancer cells. Caspase-8 ubiquitination status was assessed by polyubiquitin immunoprecipitation (IP) and fluorescent microscopy. Apoptosis induction in response to death receptor stimuli or proteasome inhibitor was evaluated using the Annexin V/Propidium iodide staining (PI). To determine the consequences of proteasome inhibition on caspase-8 stability, trafficking, and activity following death receptor activation, we used the TRAIL-resistant human prostate cancer LNCaP cells, and the caspase-8 deficient Neuroblastoma 7 (NB7) cells, as cellular models for reconstituting the non-cleavable mutant forms of caspase-8. Our findings demonstrate that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8 upon treatment with proteasome inhibitor and GST-TRAIL. Furthermore, caspase-8 processing into its active subunits preceded caspase-8 polyubiquitination, implicating caspase-8 processing as a potential regulatory mechanism, rather than a requirement for caspase-8 activation in apoptosis induction. The mechanistic control of caspase-8 by ubiquitination in cancer cells may have significant significance in bypassing mechanisms of therapeutic resistance in human tumors and optimization of anti-cancer treatment strategies in human tumors and optimization of anti-cancer treatment strategies.

SUMOylation and ubiquitination are important post-translational modifications. While ubiquitination is well known for targeting proteins for degradation, SUMOylation often regulates the intracellular localization of substrates. In the first project of this dissertation, we developed proteomic strategies to identify novel SUMOylated proteins in mammalian cells. In the second project, we investigated the regulation of protein ubiquitination in the NF-κB signaling pathway in the context of Paget’s disease of bone (PDB). Identification of SUMOylated proteins has been a challenge because of low abundance of SUMOylation substrates. Here, we utilized a mass spectrometry (MS)-based proteomic approach to identify novel SUMOylated proteins in mammalian cells. Seventy-four unique proteins were commonly identified in the collection of four SUMO-1 plasmids, thus considered candidate SUMOylated proteins. Many of these proteins are associated with the nucleus. The results were validated by confirming SUMOylation of a novel substrate Drebrin and a well known substrate Ran-GAP1. Furthermore, the potential SUMOylation sites in Drebrin have been identified and confirmed using site-directed mutagenesis. PDB is a disorder characterized by increased bone turnover containing hyperactive osteoclasts. Mutations in Sequestosome 1 (p62) are associated with 40% of familial PDB. P62 is a scaffold protein and plays a critical role in regulating ubiquitination of TRAF family signaling molecules and mediating the activation of NF-κB by RANK and TNFα ligands. P62 also plays a critical role in shuttling substrates for autophagic degradation. The objective of this project is to determine the effects of PDB-associated p62 mutants on NF-κB signaling and autophagy. We compared the effect of wild-type (WT) p62 and PDB mutations (A381V, M404V and P392L) on the TNFα-induced NF-κB signaling using an NF-κB luciferase assay. Our results show that these p62 mutations increased the NF-κB signaling. In addition, we found that the PDB mutations did not change the interaction between p62 and the autophagy marker protein LC3. In summary, the PDB mutations in p62 are likely gain-of-function mutations that can increase NF-κB signaling and potentially contribute to disease progression. Based on the results, we proposed a model to speculate the synergetic role of p62 PDB mutant on NF-κB signaling and autophagy.