Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / Water is used for various purposes and contamination can have severe implications if untreated. One of the most common and cost effective water disinfectants, especially used in developing countries, is chlorine. However, microorganisms have developed different mechanisms in response to environmental stress conditions, such as the viable but nonculturable (VBNC) effects possibly displayed in this study, enabling them to survive. Chlorine may also exert several effects on microorganisms, such as the expression of multi-substrate efflux pumps, decreased membrane permeability and transport inhibition that may lead to chlorine tolerance and antimicrobial resistance. In a descriptive and comparative study, the molecular characteristics of E. coli strains isolated from environmental waters in the Western Cape and the possible relationship between chlorination and antimicrobial resistance were investigated. Water and biofilm samples were exposed to chlorine, as well as efflux pump inhibitor (EPI) concentrations, and surviving E. coli strains were tested for their phenotypic characteristics including antimicrobial susceptibility profiles and morphological types. Candidate genes possibly involved in resistance to antimicrobials, disinfection and efflux pumps were detected with polymerase chain reaction (PCR) and sequenced. Sequencing analysis and homology searches were done and E. coli strains were typed as either Enteropathogenic E. coli strains (EPEC) or Enterotoxigenic E. coli strains (ETEC) on the presence of virulence genes. All water and biofilm sources examined were heavily polluted with E. coli, and a high enumeration level of this indicator organism of faecal contamination was recorded. Chlorine tolerance was found to be associated with antimicrobial resistance. Addition of EPI with exposure to chlorine decreased enumeration levels of these organisms, suggesting that efflux pumps may play a role in tolerance to chlorine. Several morphological patterns were described amongst the E. coli strains and a change in this was recorded after exposure to chlorine. Highly resistant antibiograms displayed by the isolated strains included ampC β-lactamase producing E. coli strains and extended spectrum β-lactamases (ESBLs). Amplification of the candidate genes selected for heatshock, oxidative stress genes and efflux pump were most frequently detected while the structural genes involved in fluoroquinolones (FQs) resistance were detected less frequently in the selected strains. Sequencing of these amplified candidate genes demonstrated various changes in amino acid sequences, including one common mutational pathway taken by E. coli when exposed to stress conditions. Further homology searches of the sequenced candidate genes illustrated similarities in 19 pathogenic and 14 non-pathogenic E. coli as well as 3 Shigella strains. Detection of virulence genes found three EPEC strains (bfpA, eaeA), two EPEC (eaeA), ten EPEC (bfpA) and one ETEC strain (st) amongst the isolates. This study underlines the need for monitoring our water sources, which poses a public health risk due to incomplete chlorination, antimicrobial resistance and the spread of clinically relevant pathogenic strains.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: BACKGROUND: The emergence of mycobacterium tuberculosis resistance to first line drugs has renewed interest in second-line anti-tuberculosis drugs. Generally, Paraaminosalicylic acid (PAS) is less potent and frequently more toxic than the first line drugs. Furthermore, the pharmacokinetics of PAS in children has not been well characterized. AIMS: The aims of the present study were (1) to determine the pharmacokinetics of PAS in pediatric patients, (2) to describe the discrepancy between children and adult pharmacokinetics and the appropriate dosing regimen of PAS and (3) to investigate the potential of the second-line anti-tuberculosis drugs PAS, terizidone and ethionamide (often used as first-line drug in children) to inhibit the catalytic activities of CYP450 1A2 and 2C9. PATIENTS: Twenty two patients with drug resistant tuberculosis were included in the study. Ten patients were children with mean age of 4.2 years (range: 1 to 12 years). Twelve patients were adults with mean age of 31.3 years (range: 18 to 53). 4 children (40%) and 4 adults (33.3%) were HIV positive and were on ART. METHODS: Children received 75 mg/kg twice daily on the first visit and after two weeks they received 150 mg/kg once. Adults received a standard 4 g twice daily. Blood samples were taken at different time points after the dose. In the additional study, the inhibitory effects of PAS, ethionamide and terizidone on phenacetin O-deethylation, a marker substrate of CYP1A2 and diclofenac 4’-hydroxylation, a marker substrate of CYP2C9, were studied using human liver microsomes. RESULTS: For the 75 mg/kg dose, the mean AUC was 233.3 =g•h/ml and the mean CL was 10.4 l/h/kg. The mean of the observed Cmax of the drug was 45.4 =g/ml and the mean Tmax was 4.8 hrs. For the 150 mg/kg dose, the mean AUC of PAS was 277.9 =g•h/ml and the mean CL was 47.1 l/h/kg. The mean of the observed Cmax of the drug was 56.5 =g/ml and the mean Tmax was 4.8 hrs. On the first visit the mean AUC was 368 =g•h/ml and the mean CL was 13.2 l/h/kg. The mean of the observed Cmax of PAS was 51.3 =g/ml and the mean Tmax was 5.2 hrs. On the second visit the mean AUC was 230 =g•h/ml and the mean CL was 23.9 l/h/kg. The mean of the observed Cmax of PAS was 37.6 =g/ml and the mean Tmax was 5.2 hrs. The comparisons between pharmacokinetics profile of PAS and patients characteristics e.g. age, indicated no statistically significant differences between children (both treatment regimens) and adult patients as well as HIV positive and negative patients. In the in vitro study, all drugs demonstrated no inhibition potency towards the investigated CYP450 enzymes. CONCLUSIONS:The dose of 75 mg/kg twice daily in children appears to be appropriate to achieve serum concentration above the PAS minimum inhibitory concentration of approximately 1 =g/ml. PAS, ethionamide and terizidone are unlikely to affect the metabolism of concomitantly administered medications that are metabolized by either CYP450 1A2 and/or 2C9 isoenzymes. / AFRIKAANSE OPSOMMING: AGTERGROND: Die opkoming van eersteliniemiddel-weerstandige mycobacterium tuberculosis het opnuut belangstelling in tweedelinie-antituberkulosemiddels aangewakker. Oor die algemeen is para-aminosalisielsuur (PAS) minder kragtig en dikwels ook meer toksies. Verder is die farmakokinetika van PAS in kinders nog nie goed vasgestel nie. DOELSTELLINGS: Die doelstellings van hierdie studie was (1) om die farmakokinetika van PAS in pediatriese pasiënte vas te stel, (2) om die diskrepansie tussen kinder- en volwasse-farmakokinetika, sowel as die toepaslike doseringskedule, van PAS te beskryf en (3) om die potensiaal van die tweedeline-antituberkulosemiddels PAS, terisidoon en etioonamied (gereeld gebruik as eerste linie middels in kinders) te ondersoek wat betref hul vermoë om die katalitiese werking van CYP450 1A2 en 2C9 te inhibeer. PASIËNTE: Twee-en-twintig pasiënte met middelweerstandige tuberkulose is in hierdie studie ingesluit. Tien pasiënte was kinders met ‘n gemiddelde ouderdom van 4.2 jaar (reeks: 1 tot 12 jaar). Twaalf pasiënte was volwassenes met ‘n gemiddelde ouderdom van 31.3 jaar (reeks: 18 tot 53 jaar). 4 kinders (40%) en 4 volwassenes (33.3%) was MIV positief en was op TRM’s. METODES: Kinders het 75 mg/kg twee maal daaliks gedurende die eerste besoek ontvang en 150 mg/kg een maal ná twee weke ontvang. Volwassenes het ‘n standaarddosis van 4 g twee maal daagliks ontvang. Bloedmonsters is op verskillende tye ná die dosering geneem. In die addisionele studie is in die inhiberende effekte van PAS, etioonamied en terisidoon op fenasetien-O-deëtilering, ‘n merkersubstraat van CYP1A2 en diklofenak-4’-hidroksilasie, ‘n merkersubstraat van CYP2C9, ondersoek deur gebruik te maak van menslike lewermikrosome. RESULTATE: Vir die 75 mg/kg dosis was die gemiddelde area-onder-die-kurwe (AOK) 233.3 =g•h/ml en die gemiddelde middelopruiming (CL) 10.4 l/h/kg. Die gemiddelde geobserveerde Cmaks van die middel was 45.4 =g/ml en die gemiddelde Tmaks was 4.8 h. Vir die 150 mg/kg dosering was die gemiddelde AOK van PAS 277.9 =g•h/ml en die gemiddelde CL 47.1 l/h/kg. Die gemiddelde geobserveerde Cmaks van die middel was 56.5 =g/ml en die gemiddelde Tmaks was 4.8 h. Gedurende die eerste besoek was die AOK 368 =g•h/ml en die gemiddelde CL was 13.2 l/h/kg. Die gemiddelde geobserveerde Cmaks van PAS was 51.3 =g/ml en die gemiddelde Tmaks was 5.2 h. Gedurende die tweede besoek was die gemiddelde AOK 230 =g•h/ml en die gemiddelde CL 23.9 l/h/kg. Die gemiddelde geobserveerde Cmaks van PAS was 37.6 =g/ml en die gemiddelde Tmaks was 5.2 h. Die vergelyking van PAS-farmakokinetika en eienskappe van die pasiënte het geen statisties beduidende verskille in die gemiddelde AOK tussen kinders (op albei doserings) en volwassenes getoon nie. Met die in vitrostudie het geen van die middels inhibisie-werking teenoor die CYP450-ensieme wat ondersoek is, getoon nie. GEVOLGTREKKINGS: Die gevolgtrekking kan gemaak word dat die dosering van 75 mg/kg twee maal daagliks voldoende is om serumkonsentrasies wat bo PAS se minimum inhiberende konsentrasie van 1 =g/ml te bereik. Dit is onwaarskynlik dat PAS, etioonamied en terisidoon die metabolisme van gelyktydig-toegediende medikasies, wat op hul beurt deur die CYP240-isoënsieme 1A2 en/of 2C9 gemetaboliseer word, sal affekteer. / Division of Pharmacology, Stellenbosch University / National Research Foundation (NRF) grant generously offered by Professor Donald Grant

Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: Since the South African public sector antiretroviral roll-out programme started in 2004, the success of antiretroviral combination therapy (cART) has been experienced in terms of survival, prevention of mother-to-child transmission (PMTCT) and quality of life. However, as the programme matures, viral resistance to the constituent drugs will increase. Monitoring antiretroviral drug resistance (ARVDR) should therefore be a priority in the public health approach to HIV treatment. Methods: A cross-sectional investigation of genotypic antiretroviral drug resistance in: a) HIV-infected mothers who were exposed to a PMTCT regimen of short course azidothymidine (AZT) with single dose nevirapine (NVP) during labour. b) HIV-infected adults and children who were cART-naïve (transmitted or initial resistance). c) HIV-infected adults and children who were failing cART (drug-induced or acquired resistance). In case of adults, this includes patients on a first-line, non-nucleoside reverse transcriptase (NNRTI)-based regimen, or on a second-line, protease inhibitor (PI)-based regimen, and in case of children, this includes patients on a first-line PI-based regimen. Results: In mothers who received a PMTCT-regimen that combined AZT and NVP the prevalence of NNRTI resistance mutations was 17.1% (95% CI: 8.7-25.6%). The prevalence of transmitted ARVDR in adults was low, as was initial ARVDR in young children (mostly PMTCT-exposed), except for NNRTI resistance in children who had received NVP as part of PMTCT. Drug-induced resistance was found in adults failing first-line NNRTI-based cART, with 83% having resistance to ≥1 drug. In contrast, adult patients failing second-line PI-based cART had a low prevalence of PI resistance; the predominant reason for failure was poor drug exposure, as detected by measuring lopinavir concentrations in blood plasma and hair samples. In contrast, PI resistance in children was not rare, largely due to historic exposure to un-boosted PIs. This resulted in extensive resistance to PIs and reverse transcriptase inhibitors (RTI) in some children. Conclusions: A combined regimen of short course AZT with intrapartum NVP for PMTCT may, in addition to reducing the risk of neonatal infection, also reduce the risk of NVP resistance in the mothers compared to a regimen of NVP only. In South Africa, the prevalence of transmitted ARVDR remains low relative to industrialised countries, probably as comparatively little time has elapsed since the scale-up of cART. Adults failing first-line cART are likely to respond to second-line cART, without failure due to resistance. However some children with PI and RTI resistance cannot be adequately treated with drugs currently available through the roll-out programme. This emphasizes the urgent need for a rational and science-based approach to managing cART-experienced children, including access to additional drugs to form a third-line paediatric cART regimen. / AFRIKAANSE OPSOMMING: Inleiding: Sedert die begin van die Suid Afrikaanse publieke sektor antiretrovirale uitrol program in 2004 is die sukses van antiretrovirale kombinasie-behandeling (k-ARB) ervaar in terme van oorlewing, voorkoming van moeder na kind oordrag (VMKO) en lewenskwaliteit. Nietemin, sal weerstandigheid teen die middels wat in die antiretrovirale program gebruik word toeneem soos wat die program gevestig raak. Die monitoring van antiretrovirale middel-weerstandigheid is derhalwe ‘n prioriteit in gemeenskap-gesondheid benadering tot MIV behandeling. Metodes: ‘n Deursnit ondersoek van genotipiese antiretrovirale middel-weerstandigheid in: a) MIV-geïnfekteerde moeders wat blootgestel is aan VMKO regimen bestaande uit ‘n kort kursus AZT met ‘n enkeldosis nevirapien (NVP) tydens kraam. b) MIV-geïnfekteerde volwassenes en kinders wat komibinasieterapie-naïef (oorgedraagde of inisiële weerstandigheid) is. c) MIV-geïnfekteerde volwassenes en kinders wat k-ARB faal (middel-geïnduseerde weerstandigheid). In geval van volwassenes, sluit dit pasiënte op ‘n eerste-linie, non-nucleosied tru-transkriptase inhibitor (NNRTI)-regimen, en tweede-linie protease inhibitor (PI)-gebaseerde regimen, en in geval van kinders, sluit dit pasiënte in op ‘n eerste-linie PI-gebaseerde regimen. Resultate: In moeders wat ‘n gekombineerde AZT en NVP VMKO-regimen ontvang het, was die voorkoms van NNRTI weerstandigheid 17.1% (95%-vertrouensinterval: 8.7-25.6%). Die voorkoms van oorgedraagde ARVMW in MIV-geïnfekteerde volwassenes en kinders wat kombinasieterapie-naïef is, was laag, so ook ARVMW in jong kinders (meestal VMKO-blootgestel), behalwe vir non-nukleosied tru-transkriptase inhibitor (NNRT) weerstandigheid in kinders wat NVP ontvang het deur VMKO. Middel-geïnduseerde weerstandigheid was gevind in volwassenes wat die eerste-linie NNRTI-gebaseerde k-ARB gefaal het, met 83% wat weerstandigheid teen ≥1 middel het. Volwassenes wat ‘n tweede-linie protease inhibitor (PI) –gebaseerde k-ARB gefaal het , het ‘n lae voorkoms van PI weerstandigheid, met die oorwegenede oorsaak, swak middel-bloostelling, soos bepaal deur van lopinavir-konsentrasies in bloed plasma en hare. In teenstelling hiermee was PI weerstandigheid nie skaars in kinders nie, hoofsaaklik weens historiese blootstelling an ongeskraagde PI-behandeling. Dit het tot uitgebreide weerstandigheid tot PIs en tru-transkritptase inhibitors (RTI) in sommige kinders gelei. Gevolgtrekkings: ‘n Gekombineerde regimen van ‘n kort kursus AZT met NVP tydens kraam vir VKMO, mag bykomend tot die vermindering die risiko van pasgebore infeksie, ook die kans vir weerstandigheid teen NVP in die moeders verlaag in vergelyking met ‘n regimen van NVP-alleen. Die voorkoms van oorgedraagde ARVMW is tans laag in vergelyking met geïndustrialiseerde lande, waarskynlik aangesien daar nog betreklik min tyd verloop het sedert k-ART wyd beskikbaar gemaak is. Volwassenes wat eerstelyn kombinasie terapie faal sal waarskynlik goed reageer op tweede-linie terapie, sonder terapie faling weens middelweerstandigheid. Daarenteen kan sommige kinders met protease inhibitor en tru-transkriptase weerstandigheid nie voldoende behandel word met die huidig-beskikbare middels in die uitrol program nie. Dit beklemtoon die dringende noodsaaklikheid van ‘n rasionele en wetenskaplike benadering tot k-ART in kinders, met ‘n lang terapie geskiedenis, wat toegang tot bykomende medikasie behels om `n derde-linie regimen saam te stel.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The tuberculosis (TB) incidence has increased in Southern Africa and the situation is worsened by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular biological techniques have been used to understand the disease dynamics of TB. In a series of studies we describe the use of these techniques to understand the disease dynamics of TB in Southern Africa. Using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) to characterize M. tuberculosis strains from TB patients in Zimbabwe, we identified a genotype causing a disproportionate number of TB cases. The genotype belonged to the Latin American Mediterranean (LAM) lineage and we named it the Southern Africa1 (SAF1) family and later renamed it SAF1/RDRio, also reflecting its predominance in South America. To establish if this family of strains was predominant elsewhere in Southern Africa, genotypes were compared to those from Western Cape, South Africa and Zambia. The SAF1/RDRio strains were highly prevalent in Zambia but were only a minor fraction of the strains in South Africa. The geographical distribution of SAF1/RDRio strains was determined in Gweru, Zimbabwe, and was found to be spread in high incidence areas. From these two studies it was hypothesized that certain host and bacterial factors were associated with disease due to SAF1/RDRio. Subsequently potential risk factors and clinical outcomes of disease due to SAF1/RDRio strains were explored. An association was found with smoking and cavitary pulmonary disease suggesting that SAF1/RDRio caused a more severe and highly transmissible formof TB Using IS6110-RFLP, principal genetic grouping, spoligotyping, IS6110 insertion-site mapping and variable-number tandem repeats (VNTR) typing, low IS6110 copy clade (LCC) identified in Zimbabwe were characterized and compared to the strains from Cape Town, South Africa and other regions. The LCC strains from Cape Town, South Africa, were found to have close evolutionary relationship with strains from Zimbabwe and other regions and were widely distributed suggesting they play an important role in the global TB epidemic. Observations from these studies and those from other studies led to the hypothesis that specific genotypes of M. tuberculosis predominate in regions of Southern Africa. To gain an insight on the population structure of M. tuberculosis strains in Southern Africa, spoligotyping and/or IS6110-RFLP data from eight countries were compared. This is the first study to describe the M. tuberculosis population structure in Southern Africa. Distinct genotypes were associated with specific geographic regions. These findings have important implications for TB diagnostics, anti-TB drug and vaccine development. The population structure of multidrug-resistant (MDR), pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) M. tuberculosis isolates from provinces in South Africa was also determined. This is again the first study to describe the population structure of drug-resistant M. tuberculosis in South Africa. The results also showed geographic localization of genotypes and an association with resistance class. However, decreasing strain diversity was observed as the isolates evolved from MDR-TB to XDR-TB suggesting selection for the specific genotypes. These findings highlight the importance of identifying genetic markers in drug-resistant strains, to enhance early detection of those at risk of developing XDR-TB. / AFIKAANSE OPSOMMING: Die voorkoms van tuberkulose (TB) in Suider Afrika word vererger deur stamme van Mycobacterium tuberculosis wat weerstandig is teen die beskikbare anti-tuberkulose middels. Molekulêre tegnieke word gebruik om in hierdie reeks studies die dinamika van TB in Suider Afrika te ondersoek Deur spoligotipering en IS6110 restriksie fragment lengte polimorfisme (RFLP) tegnieke te gebruik om M. tuberculosis stamme van pasiente in Zimbabwe te beskryf, het ons ‘n genotipe gevind wat ‘n buitengewone aantal TB gevalle veroorsaak het. Hierdie genotipe is deel van die internasionaal beskryfde Latyns Amerikaase en Meditereense (LAM) stam familie. Ons het dit die Suider Afrikaanse Familie1 (SAF1) genoem, maar later hernoem na SAF1/RDRio, omdat dieselfde genotipe in ook volop is in Suid Amerika. Om vas te stel of hierdie familie ook oorheesend is in die res van Suider Afrika, is dit vergelyk met beskikbare databasisse van die Wes-Kaap, Suid-Afrika en Zambië. Alhoewel SAF1/RDRio in die Wes-Kaap gevind is, dra dit slegs tot ‘n mindere mate by tot die plaaslike TB epidemie. Aan die anderkant kom SAF1/RDRio baie algemeen in Zambië voor. ‘n Verdere studie wys ook dat die SAF1/RDRio familie eweredig en wyd verspreid voorkom in hoë insidensie gebiede in Gweru, Zimbabwe. Vanuit die bevindings van hierdie 2 studies, kan ons aflei dat sekere gasheer- en bakteriële eienskappe geassosieer is met SAF1/RDRio-TB-infeksie. Hierna is potensiële risiko faktore en kliniese uitkomste van siekte as gevolg van infeksie met SAF1/RDRio ondersoek. ‘n Assosiasie met rook en kaviterende pulmonale infeksie is gevind,wat daarop dui dat SAF1/RDRio erger vorm van TB veroorsaak en hoogs oordraagbaar is. Deur gebruik te maak van IS6110- (RFLP), hoof groep groepering, spoligotipering, IS6110 invoegings kaartering en veranderlike getal tandem herhaling (VNTR) tipering kon lae IS6110 invoeginsgetal (LCC) stamme van Kaapstad, Zimbabwe en ander gebiede vergelyk word. Al die LCC stamme in die studie is evolusionêr naby verwant aan mekaar en is wyd verspreid, wat dui op hulle belangrike rol in die wêreldwye TB epidemie. Waarnemings in hierdie asook ander studies het tot die hipotese gely dat spesifieke genotipes van M. tuberculosis dominant is in verskillende gebiede van Suider Afrika. Om meer insig tot die populasie samestelling van M. tuberculosis stamme in Suider Afrika in te win is spoligotipes en RFLP-data van 8 lande vergelyk. Hierdie is die eerste studie om die populasie samestelling van M. tuberculosis in Suider Afrika te beskryf en is belangrike fir toekomstige ontwikkeling van nuwe TB diagnose tegnieke, anti-TB middels en TB entstowwe. Die populasie samestelling van multiweerstandige (MDR), pre-ekstreme weerstandige (pre-XDR) en ekstreme weerstandige (XDR) M. tuberculosis van verskillende provinsies in Suid-Afrika is ook bepaal. Hierdie studie is ook die eerste wat die populasie samestelling van weerstandige M. tuberculosis in Suid-Afrika beskryf. Die resultate wys geografiese lokalisering van genotipes en ‘n assosiasie met weerstandigheidsklas. ‘n Afname in stam diversiteit soos die isolate van MDR-TB tot XDR-TB ontwikkel, dui op seleksie van spesifieke genotipes. Hierdie bevinding lê die klem op die belangrikheid van die identifisering van genetiese merkers in weerstandige stamme om die risiko vir die ontwikkeling van XDR-TB te verminder deur vroë deteksie.

<p>Molecular interactions lie at the heart of myriad biological processes. Knowledge of molecular recognition processes and the ability to model and predict interactions of any biological molecule to any chemical compound are the key for better understanding of cell functions and discovery of more efficacious medicines.</p><p>This thesis presents contributions to the development of a novel chemo-bioinformatics approach called proteochemometrics; a general method for interaction space analysis of biological macromolecules and their ligands. In this work we explore proteochemometrics-based interaction models over broad groups of protein families, evaluate their validity and scope, and compare proteochemometrics to traditional modeling approaches.</p><p>Through the proteochemometric analysis of large interaction data sets of multiple retroviral proteases from various viral species we investigate complex mechanisms of drug resistance in HIV-1 and discover general physicochemical determinants of substrate cleavage efficiency and binding in retroviral proteases. We further demonstrate how global proteochemometric models can be used for design of protease inhibitors with broad activity on drug-resistant viral mutants, for monitoring drug resistance mechanisms in the physicochemical sense and prediction of potential HIV-1 evolution trajectories. We provide novel insights into the complexity of HIV-1 protease specificity by constructing a generalized IF-THEN rule model based on bioinformatics analysis of the largest set of HIV-1 protease substrates and non-substrates.</p><p>We discuss how proteochemometrics can be used to map recognition sites of entire protein families in great detail and demonstrate how it can incorporate target variability into drug discovery process. Finally, we assess the utility of the proteochemometric approach in evaluation of ADMET properties of drug candidates with a special focus on inhibition of cytochrome P450 enzymes and investigate application of the approach in the pharmacogenomics field.</p>

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.

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

Genetic polymorphism in Plasmodium falciparum is a considerable obstacle to malaria intervention. Parasites have repeatedly evolved to overcome every front-line antimalarial deployed throughout history, and artemisinin resistant populations are expanding in Southeast Asia. Promising vaccine candidates routinely fail when challenged by the genetic diversity of natural parasite populations, and a recent trial using a blood-stage antigen showed immunity was allele specific. Modern sequencing technologies have revolutionized our understanding of parasite genomics and population genetics by providing access to single nucleotide variation, but characterizing more complex polymorphism remains a key challenge. Solving this problem is important because the selective pressures from drugs and host immunity often create complex polymorphism in the most clinically relevant genes that is missed using standard genotyping methods. In three sections, this thesis is a narrative about 1) encountering complex variation, 2) overcoming it with novel tools, and then 3) innovatively applying those tools to old and new questions. I first show examples of complex variation in a vaccine candidate (EBA-175) and a drug resistance gene (pfcrt) while reporting SNP based analyses of Kenyan and Tanzanian field isolates. While introducing this complex variation I also describe biological insights discovered in these populations. In Kenya I show evidence that chloroquine resistance selects for parasites that are primaquine sensitive, use a GWAS approach to discover new drug resistance loci, and catalogue variation in known resistance genes. In Tanzania I describe the population structure and allele frequencies of parasites from two geographic regions. In the second section of the thesis I develop methods for accessing complex variation and demonstrate their utility by producing de novo assemblies of eba-175, pfcrt, ama1, and msp3.4 from thousands of sequenced samples. Finally, in the third section I apply these tools in depth to eba-175. I comprehensively characterize the SNP and structural variation in eba-175 using an alignment of 1419 de novo assemblies. I use this resource to illustrate the profiles of positive selection across the gene, and corroborate these signals of balancing selection by showing the geographic distribution of the F/C indels and a lesser known 6bp indel positioned between the DBL domains. I then use the alignments to design Sequenom genotyping assays that facilitate a genome wide association study, testing for human associations with the eba-175 indels in the infecting parasite. I close by reporting a potential association on human chromosome 14 with the 6bp indel in eba-175.

Les mutations de résistance génotypiques constituent un problème majeur pour l’optimisation du traitement antirétroviral chez les patients infectés par le VIH-1 naïfs au traitement ou prétraités. Cependant, l’analyse de l’impact des mutations sur la réponse au traitement est compliquée par i) le nombre élevé de mutations, ii) la colinéarité possible entre ces mutations, iii) le faible nombre de patients inclus dans les études et iv) la définition du critère de jugement. Les objectifs de cette thèse sont 1) de donner une vue d’ensemble et de discuter, en collaboration avec le réseau européen NEAT (European AIDS treatment network), les critères de jugement utilisés dans les essais cliniques récents et ceux utilisés lors de l’analyse des mutations de résistance, 2) d’évaluer l’impact des mutations génotypiques sur la réponse au traitement chez les patients naïfs dans le cadre d’une grande collaboration Européenne (EuroCoord-CHAIN) et 3) de comparer des méthodes adaptées pour les données à haute-dimension dans le but de construire un score génotypique pour la prédiction de la réponse virologique chez les patients prétraités. Les critères de jugement composites sont les plus utilisés dans les essais cliniques récents mais un critère purement virologique devrait être utilisé pour l’analyse de l’impact des mutations génotypiques. Les mutations de résistance transmises impactent sur la réponse à la première ligne de traitement si le traitement antirétroviral n’est pas adapté au génotype du virus du patient. L’analyse en composantes principales et l’analyse partial least square avaient une bonne capacité à prédire la réponse virologique mais n'étaient guère meilleures que le score génotypique. Nous allons continuer à travailler sur la comparaison de ces méthodes utilisant des critères de jugement différents dans le cadre de notre collaboration avec le Forum for collaborative HIV research. / Genotypic resistance mutations are a major concern for antiretroviral treatment optimisation in HIV-1 infected treatment naïve and treatment experienced patients. However, the analysis of the impact of genotypic mutations on treatment outcome is hampered by methodological issues such as the i) high number of possible mutations, ii) the potential collinearity between mutations, iii) the low number of patients included in those studies and iv) the definition of a virological endpoint. The objective of this thesis are 1) to give an overview and to discuss endpoints used in recent clinical trials in collaboration with European AIDS treatment network (NEAT) and those used in the context of drug resistance analysis, 2) to investigate the impact of genotypic resistance mutations on treatment outcome in treatment naïve patients in a huge European collaboration EuroCoord-CHAIN and 3) to compare methods adapted for high-dimensional data in order to construct a genotypic score to predict treatment outcome in treatment experienced patients. We saw that most of the endpoints used in recent clinical trials are composite endpoints but pure virological outcomes should be used for the evaluation of drug resistance mutations. Transmitted drug resistance mutations impact on virological outcome of initial antiretroviral therapy if the treatment of the patient is not adapted to the viral genotype the patient is harbouring. Principal component analysis and partial least square showed a good performance but had only a slightly better predictive capacity for a virologal outcome compared to the genotypic score. We continue working on the comparison of these and other methods using different endpoints in the context of a collaboration with the Forum for collaborative HIV research.