Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Familial hypercholesterolaemia (FH) contributes significantly to the high death rate from cardiovascular disease worldwide. FH is a common autosomal co-dominant disease characterised by raised cholesterol levels and premature coronary heart disease (CHD). Whilst these features usually are very prominent in homozygotes the clinical diagnosis of heterozygotes is complicated by variable phenotypic expression. Specific founder genes in the low-density lipoprotein receptor (LDLR) gene have increased the prevalence of FH in South African Afrikaners, Indians, Jews and Coloureds, and screening for these known mutations allows unequivocal diagnosis of FH-affected individuals. The systematic molecular analysis of FH resulted in the identification of at least ten founder-type LDLR gene mutations among the 56 different gene defects described to date in the diverse South African population. DNA screening of 792 at-risk family members for the FH-related mutations identified in 379 index cases, allowed accurate disease diagnosis in an additional 340 relatives and exclusion of the relevant mutation in 452 individuals. This effort forms part of the MED PED FH initiative, a collaborative project to "Make Early Diagnosis and Prevent Early Deaths in MEDical PEDigrees with FH". Evaluation of clinical criteria versus DNA diagnosis of three founder-related mutations (D154N, D206E and V408M) in the South African population demonstrated that the sensitivity and specificity of diagnoses, based on total cholesterol values measured in family members of index cases recruited for this study, were 88% and 77%, respectively. A population-directed DNA diagnosis of FH is therefore justified in South Africa on a routine basis, since expression of the defective gene measured in biochemical tests does not allow accurate diagnosis of FH in all cases. The application of mutation detection was illustrated by prenatal diagnosis of FH performed for a couple who are both heterozygous for the most common Afrikaner mutation, D206E. The mutation was absent in the foetus and a normocholesterolaemic infant was born. Prenatal diagnosis of FH, aimed at the detection of homozygous cases, is particularly applicable in populations and families with molecularly defined LDLR gene mutations. The MED-PED approach resulted in accurate diagnosis and subsequent treatment of FH in more patients, and referral to lipid clinics where they could receive the intensive care their condition justifies. Molecularly diagnosed FH patients will be the first to benefit from future treatment approaches based on mutation type. / AFRIKAANSE OPSOMMING: Familiële hiprcholesterolemie dra grootliks by tot die wêreldwye hoë sterftesyfer van kardiovaskulêre siekte. FH is 'n algemene outosomale ko-dominante siekte wat gekenmerk word deur verhoogde cholesterolvlakke en vroeë koronêre hartsiekte. Terwyl hierdie kenmerke prominent is in homosigote, word die kliniese diagnose van heterosigote bemoeilik deur variasie in fenotipiese uitdrukking. Spesifieke stigtergene in die lae-digtheids lipoproteien reseptor (LDLR) geen het die voorkomssyfer van FH verhoog in Suid Afrikaanse Afrikaners, Indiërs, Jode en Kleurlinge. Sifting vir hierdie bekende mutasies maak akkurate diagnose van FH geaffekteerde individue moontlik. Die sistematiese molekulêre analise van FH het aangetoon dat ten minste tien van die 56 verskillende geen defekte wat tot dusver beskryf is in die Suid-Afrikaanse populasie stigtertipe LDLR geen mutasies is. DNA sifting van 792 familielede vir die FH-verwante mutasie in 379 indeksgevalle geïdentifiseer is, het akkurate diagnose moontlik gemaak in 340 addisionele familielede, en uitsluiting daarvan in 452 individue. Hierdie poging vorm deel van die MED-PED FH ("Make Early Diagnosis and Prevent Early Deaths in MEDical PEDigrees with FH) inisiatief. Evaluering van kliniese kriteria teenoor DNA diagnose van drie stigter verwante mutasies (D154N, D206E en V408M) in die Suid Afrikaanse populasie het getoon dat die sensitiwiteit en spesifisiteit van die diagnose, wat gebasseer is op totale cholesterol waardes in familielede van indeksgevalle, onderskeidelik 88% en 77% was. 'n Populasie gerigte DNA diagnose van FH is dus geregverdig in Suid-Afrika op "n roetine basis, omdat die defektiewe geen nie altyd in biochemiese toetse uitgedruk word nie. Die waarde van mutasie opsporing is geillustreer deur 'n voorgeboortelike diagnose van FH wat aangevra is vir ouers wat beide heterosigoties is vir die mees algemene Afrikaner mutasie, D206E. Die mutasie was afwesig in die fetus en 'n normocholesterolemiese baba is gebore. Voorgeboortelike diagnose van FH, wat gemik is op die opsporing van homosigotiese gevalle, is veral van toepassing in populasies en families met bekende LDLR geen mutasies. Die MED-PED benadering het gelei tot akkurate diagnose en daaropvolgende behandeling van FH in meer pasiënte, en verwysings na lipiedklinieke waar hulle intensiewe aandag kan geniet. Molekulêre gediagnoseerde FH pasiënte sal die eerste wees om baat te vind by toekomstige behandeling wat moontlik gebasseer sal word op mutasie status.

Thesis (PhDAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Maize is undoubtedly South Africa's most important field crop. The identification of markers and genes for traits of interest is important to sustain the improvement of maize cultivation. Northern corn leaf blight (NClB) is a disease that occurs worldwide and can dramatically reduce yield. A number of single dominant resistance genes have been identified for NClB and some have been mapped. Currently there are no simple PCR markers for any of these resistance genes, making markerassisted selection (MAS) difficult. The aim of this study was to develop PCR markers for the NClB resistance genes Ht1, Ht2, Ht3 and Htn1 in maize. To accomplish this, the AFlP (amplified fragment length polymorphism) technique was first optimised. The results indicated that the Mlul/Msel restriction enzyme combination produces a higher percentage of polymorph isms when compared to the PstllMsel enzyme combination. It was also shown that the enzyme combination plays an important role in the percentage of polymorphic fragments observed, whereas the number of restriction enzymes used in AFlP analysis only significantly affects the total number of fragments scored. Populations segregating for the different resistance genes were not available for this study. Nearly-isogenic lines (Nils) were used in combination with AFlP technology to identify markers that map close to the genes. AFlP markers common in at least two resistant or susceptible lines were cloned and converted to PCR markers. Two commercially available recombinant inbred line (Ril) populations were then used to map the identified markers. For Htn1 fifteen polymorphic fragments were present in both resistant lines. They were selected for sequence specific marker conversion. Seven of the fifteen sequence characterized amplified region (SCAR) markers were polymorphic on the Nil pairs and five mapped to one region of maize chromosome 8.05/06. Twenty-one AFlP markers were identified for Ht1 and four SCAR markers were polymorphic In the Ht1 Nils. Three of these were mapped to chromosome 2.07. Three AFlP markers were identified for Ht2 of which two were converted to SCAR markers. Both SCAR markers were polymorphic on the Ht2 Nils and mapped to chromosome 8.05/06. On the Ht3 NILs, four AFLP markers were identified and two converted SCAR markers and one microsatellite marker (bnlg1666) were polymorphic. One of the SCAR markers and the microsatellite marker were mapped to chromosome 7.04 using a RIL population. This reports the first tentative mapping position for the Ht3 locus. The next step was to determine if a set of marker alleles could be used in a number of Htn 1 resistance lines to identify a common donor region selected by the breeders. Nine markers consisting of five SCAR markers, three converted RFLP markers and one microsatellite marker were used on 16 Htn1 resistant lines. The marker allele of us3 was in 12 of the 16 lines in coupling with Htn1 resistance. Second was the marker us5 in 11 of the 16 lines. Using this data 14 of the 16 lines shared a common introgressed region between the markers us3 and us5. A further common introgressed region between 11 of the inbred lines was found between the markers us14 and asg17. The last aim of this study was to propose a new marker technique that might be more successful than the AFLP technique in the identification of markers closely linked to genes. A new marker approach was identified where a MITE (Hbr) primer was used as an anchor primer in combination with resistance gene analog primers. This was found to be a highly polymorphic marker technique that could be used to identify markers and possibly candidate genes. It is a robust technique, which is affordable since amplifications occur from undigested genomic DNA and the primers mainly amplify fragments from genic regions. / AFRIKAANSE OPSOMMING: Mielies (Zea mays) is ongetwyfeld Suid Afrika se belangrikste lanbou gewas. Vir volgehoue opbrengs verbetering is die identifisering van merkers en gene vir belangrike eienskappe noodsaaklik. Noordelike blaarskroei (NBS) kan opbrengs wesenlik kan beïnvloed. Tans is daar reeds "n aantal enkel weerstandsgene geïdentifiseer, maar geen PKR-merkers is beskikbaar vir merker gebaseerde seleksie nie. Die doelwit van hierdie studie was om PKR-merkers te ontwikkel vir vier enkel weerstands gene (Ht1, Ht2, Ht3 en Htn1) teen NBS in mielies. Om die doelstelling te bereik is die AFLP-tegniek eers geoptimiseer. Op grond van waargenome aantal polimorfismes, was Mlul/Mse/"n beter restriksie ensiem kombinasie as Pstl/Msel. In die studie is ook bewys dat die aantal (meer as twee) restriksie ensieme wat gebruik word slegs die aantal fragmente, en nie die persentasie polimorfismes, wesenlik beïnvloed nie. Geen segregerende populasie was vir die verskillende gene beskikbaar nie. Naby isogeniese lyne (NILe) is daarom in kombinasie met die AFLP-tegniek gebruik om merkers te identifiseer wat naby die gene karteer. Alleenlik polimorfiese merkers wat in ten minste twee weerstand biedende of vatbare lyne voorgekom het, is gekloneer en omgeskakel na PKR-merkers. Daarna is twee kommersiële rekombinante ingeteelde lyn populasies gebruik om die gene te karteer. Vyftien fragmente is gevind wat gekoppel was met die Htn1 weerstand. Sewe van hierdie merkers is omgeskakel in polimorfiese SCAR-merkers waarvan vyf gekarteer is in een gebied op chromosoom 8.05/06. Een-en-twintig AFLP-merkers is geïndentifiseer vir Ht1 en vier is omgeskakel na polimorfiese SCAR-merkers. Drie hiervan is gekarteer op chromosoom 2.07. Drie AFLP-merkers is geïndetifiseer vir Ht2 waarvan 2 omgeskakel is na polimorfiese SCAR-merkers. Altwee hierdie merkers is gekarteer op chromosoom 8.05/06. Op die Ht3 lyne is vier AFLP-merkers geïdentifiseer waarvan twee omgeskakel is na polimorfiese SCAR-merkers. Een mikrosatelliet merker (bnlg1666) is ook gevind wat die selfde polimorfiese patroon wys op die Ht3 lyne. Die mikrosateliet en een van die SCAR-merkers het gekarteer op chromosomale posisie 7.04. Hierdie is die eerste tentatiewe posisie vir die Ht3 lokus. Die volgende stap was om te bepaal of "n stel polimorfiese merker-allele gebruik kan word om die donor DNA-segment te identifiseer wat die plantteiers geselekteer het. Nege PKR-merkers wat bestaan het uit vyf SCAR-merkers, 3 omgeskakelde RFLP merkers en een mikrosateliet is gebruik op 16 Hnt1 weerstandslyne. Us3 was die merker alleel wat in die meeste gevalle gekoppel was met die Htn1 weerstandslyne (12/16). Tweede was die merker us5 (in 11 van die 16 lyne). Uit die data blyk dit dat 14 van die 16 lyne "n donor segment het wat beide merkers us3 en us5 bevat. Merkers us14 en asg17 het in 11 van die 16 bestande lyne saam voorgekom. Die laaste doelstelling van hierdie studie was om "n nuwe tegniek te ontwikkel wat dalk meer suksesvol as AFLPs kan wees om merkers te identifiseer nabyaan gene. "n Nuwe tegniek word voorgestel waar "n MITE (Hbr) inleier gebruik kan word in kombinasie met weerstandgeen-analoog inleiers. Dit is gevind dat hierdie kombinasie van inleiers "n hoogs polimorfiese band patroon gee en dat die merkers ook dalk kandidaat-gene kan wees. Die tegniek is maklik uitvoerbaar, relatief goedkoop en maak gebruik van onverteerde genomiese DNA. Die fragmente wat geamplifiseer word is hoofsaaklik vanaf geenryke areas.

Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: This study exploits the Mycobacterium tuberculosis H37Rv genome sequence data in the context of M. tuberculosis clinical isolates, to elucidate genetic variation, and examine the phenotypic and molecular epidemiological implications thereof. The study was initiated by investigation of the insertion sequence IS6110, the primary DNA fingerprinting probe for the molecular epidemiology of tuberculosis. The transposable element is present in variable copy number and chromosomal location in clinical isolates of M. tuberculosis strains, giving rise to extensive genetic diversity. At the inception of this study, little was known about this element in terms of the genetic identity of its surrounding regions, its chromosomal distribution, and the mechanisms contributing to genetic diversity. These shortcomings were therefore addressed by a number of approaches. Firstly, to establish their genetic identity and chromosomal distribution, IS6110 insertion sites from clinical isolates of M. tuberculosis were cloned and sequenced. This data was examined in conjunction with available genome sequence data. The results demonstrated that the majority of insertions occurred within coding regions. Furthermore, the element was shown to have a non-random chromosomal distribution, and a number of preferential integration sites were identified. Secondly, the stability of chromosomal domains flanking IS611 0 elements was investigated by utilizing the insertion site clones as hybridization probes against clinical isolates. This allowed the identification of extensive genetic variation associated with these chromosomal domains, arising from IS6110 transpositions, deletions and point mutations. These events were expressed in terms of a phylogenetic tree which demonstrated ongoing genome evolution associated with IS6110. Thirdly, to investigate the hypothesis that IS6110-mediated deletions occur via homologous recombination between adjacent elements, deletion junctions were mapped and sequenced in clinical isolates representing predecessor and descendant strains. While these results support the involvement of IS6110 as a mediator of genetic deletion, they suggest either alternative mechanisms or the existence of unidentified intermediates. The investigation of IS6110 flanking regions identified the disruption of a number of members of the PPE gene family, leading to the second main area of investigation. The PPE gene family was newly identified as a result of the M. tuberculosis genome sequencing project, and its products are speculated to be of antigenic importance. However, at the commencement of this study very little data was available regarding the biological role of PPE proteins. Therefore, to explore the phenotypic implications of PPE gene disruption, various aspects of the gene family were investigated. Firstly, phylogenetic relationships between members of the PPE family were elucidated, which suggested an evolutionary progression, and highlighted the possibility that there may be functional subdivisions within the gene family. Secondly, the extent and mechanisms of PPE gene variation were analyzed by a combination of hybridization, peR and sequence analysis. This approach revealed extensive variation associated the gene family, although different members of the family exhibit different levels of variation. Of special interest was the discovery that long tandem repeat regions (~69 bp) found within 3 members of the gene family demonstrate variation in the numbers of these tandem repeats. A third avenue of investigation focused on in vitro and in vivo PPE gene expression profiles. RT- , peR was utilized to demonstrate in vitro expression of PPE genes, while RNA:RNA in situ hybridization demonstrated the expression of PPE genes in human tissue samples. Intriguingly, in situ hybridization suggests that there is variable PPE gene expression within the human granuloma. The final approach reported here focused on the subcellular localization of one member of the PPE family, Rv1917c. A combination of cell fractionation and whole-cell antibody binding experiments suggest that the Rv 1917c protein is a cell wallassociated, surface exposed molecule. In summary, the results obtained have potential implications for the interpretation of molecular epidemiological data, support the role of IS6110 as an agent of genome evolution, and emphasize the potential for IS6110 to impact on strain phenotype. Investigation of the PPE family demonstrated that this gene family contributes to genetic variation, is expressed in vitro and in vivo and that at least one protein encoded by the gene family is cell wall associated. Together, the results obtained support the hypothesis that selected members of the PPE gene family may encode products involved in antigenic variation. / AFRIKAANSE OPSOMMING: Dié studie maak gebruik van die Mycobacterium tuberculosis H37Rv genoom volgorde data in die konteks van M. tuberculosis kliniese isolate, om genetiese variasie toe te lig en die fenotipiese en molekulêre epidemiologiese implikasies daarvan te ondersoek. Die studie het 'n aanvang geneem deur die ondersoek van die inset-volgorde /S6110, wat die primêre DNS vingerafdruk pylfragment vir die molekulêre epidemiologie van tuberkulose is. Hierdie transponerende element is in wisselende kopiegetal en chromosomale posisies teenwoordig in kliniese isolate van M. tuberculosis stamme, en gee so oorsprong aan omvangryke genetiese afwisseling. Met die aanvang van hierdie studie was min bekend omtrent hierdie element betreffende die genetiese identiteit van die areas wat die insetsels omring, die chromosomale distribusie van insetsels, asook die meganismes wat bydra tot genetiese afwisseling. Hierdie gebreke is dus deur 'n aantal benaderings aangespreek. Eerstens is IS6110 insettingsetels van kliniese M. tuberculosis isolate gekloneer en hul nukleotiedvolgorde bepaal om sodoende hul genetiese identiteit en chromosomale verspreiding vas te stel. Hierdie data is in oorleg met beskikbare genomiese volgorde data geanaliseer. Die resultate het gewys dat die meerderheid van insetsels binne koderende gebiede plaasgevind het. Verder is gewys dat hierdie element nie na willekeur deur die chromosoom versprei is nie, en 'n aantal gebiede waar insetting by voorkeur plaasvind, is geïdentifiseer. Tweedens is die stabiliteit van die chromosomale gebiede wat IS6110 elemente flankeer ondersoek deur die insettingsetel klone as pylfragmente te gebruik in hibridisasie van kliniese isolate. Dit het die identifisering van omvangryke genetiese afwisseling binne hierdie chromosomale gebiede, wat ontstaan deur IS611 0 transposisies, delesies en puntmutasies, tot gevolg gehad. Hierdie afwisselings is uitgedruk as 'n filogenetiese boom waarin die voortdurende genomiese evolusie wat geassosieer word met IS6110 gewys word. Derdens, om die teorie dat IS6110-gedrewe delesies deur middel van homoloë rekombinasie tussen naasliggende elemente plaasvind te ondersoek, is die grense van delesies gekarteer en die nukleotiedvolgorde daarvan bepaal in kliniese isolate wat voorganger- en afstammelingstamme verteenwoordig. Alhoewel die resultate die betrokkenheid van IS6110 as 'n bemiddelaar van genetiese delesie ondersteun, stel dit ook die bestaan van of alternatiewe meganismes of van onbekende intermediêre vorme voor. Ondersoek van die IS6110-flankerende gebiede het gelei tot die ontdekking van ontwrigting van 'n aantal gene wat behoort tot die PPE geenfamilie, en het so gelei tot die tweede hoof ondersoek tema. Die PPE geenfamilie is ontdek as gevolg van die M. tuberculosis genoomprojek, en dit word gespekuleer dat die produkte van hierdie gene van antigeniese belang mag wees. Daar was egter met die aanvang van hierdie studie baie min data beskikbaar omtrent die biologiese rol van die PPE proteïene. Om die fenotipiese implikasies van ontwrigting van PPE gene te ondersoek is daar dus ondersoek ingestel na verskeie aspekte van hierdie geenfamilie. Eerstens is filogenetiese verwantskappe tussen lede van die PPE familie bepaal, wat gedui het op 'n evolusionêre progressie en wat ook aangedui het dat daar moontlik funksionele onderverdelings binne hierdie geenfamilie mag bestaan. Tweedens is die omvang en meganismes van PPE geenvariasie geanaliseer deur 'n kombinasie van hibridisasie, PKR en nukleotiedvolgorde analise. Hierdie benadering het omvangryke afwisseling binne hierdie geenfamilie getoon, alhoewel verskillende lede van die familie verskillende vlakke van afwisseling demonstreer. Wat veral interessant was, was die ontdekking dat lang tandem herhalingsvolgordes (~69 bp) wat in 3 lede van hierdie geenfamilie voorkom, variasie toon in die getalle van hierdie tandem herhalingsvolgordes. 'n Derde been van ondersoek het gefokus op in vitro en in vivo PPE geen uitdrukkingsprofiele. RT-PKR is gebruik om te toon dat PPE gene in vitro uitgedruk word, terwyl RNA:RNA in situ hibridisasie getoon het dat PPE gene ook in menslike weefsel uitgedruk word. Interessant genoeg dui in situ hibridisasie daarop dat daar wisselende PPE geen uitdrukking binne die menslike granuloom voorkom. Die laaste benadering wat hier gerapporteer word fokus op die sub-sellulêre lokalisering van een lid van die PPE familie, Rv1917c. 'n Kombinasie van selfraksionering en heel-sel antiliggaam-bindingseksperimente dui daarop dat Rv1917c 'n selwand-geassosieerde molekuul is wat aan die oppervlak blootgestel word. Ter opsomming het die resultate wat bereik IS potensiële implikasies vir die interpretasie van molekulêr-epidemiologiese data, dit ondersteun die rol van IS6110 as 'n bemiddelaar van genoom evolusie en beklemtoon die potensiaal vir IS6110 om 'n invloed te hê op die fenotipe van die stam. Ondersoek van die PPE familie het getoon dat hierdie geenfamilie bydra tot genetiese afwisseling, dat dit uitgedruk word beide in vitro en in vivo en dat ten minste een lid van hierdie geenfamilie geassossieer word met die selwand. Tesame ondersteun hierdie resultate die teorie dat geselekteerde lede van die PPE geenfamilie wel produkte enkodeer wat betrokke is by antigeniese variasie.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Current knowledge suggests that MS is associated with autoimmunity and that infectious agents and hereditary factors may be involved. The demonstration of a higher recurrence risk of MS in families (4-5%) compared with the general population (0.1%) provides strong evidence for a genetic basis. Extensive analyses of the entire human genome to identify new genes that may underlie MS have indicated that several genes may contribute to disease susceptibility, but these remain largely unidentified. In this study candidate genes involved in iron metabolism and immunology have been analysed for the first time within the context of both autoimmune and infectious disease susceptibility, in order to investigate the role of genetic and viral factors implicated in the pathogenesis of MS. The Z-DNA forming repeat polymorphism in the promoter region of the solute carrier family 11 (proton-coupled divalent ion transporters) member 1 (SLC11A1) gene was found to be significantly associated with MS (P<0.01) in the genetically homogeneous Afrikaner population of South Africa, but not in the German and French populations using a case-control study and transmission linkage disequilibrium approach, respectively. However, significant differences were observed in genotype distribution between German MS patients with a primary- and secondary progressive disease course (P<0.05), and between the German patients with relapsing remitting and primary progressive MS (P<0.05). These findings provide further evidence that the SLC11A1 gene is associated with MS, most likely due to its role in iron homeostasis. In order to investigate the influence of viruses in the apparent multi-step aetiology of MS, serum and peripheral blood mononuclear cells (PBMCs) of MS patients, close relatives and unrelated controls were screened for the presence of MS-associated retrovirus (MSRV) and two herpes virus (HHV-6 and EBV) sequences. Detection of the pol gene expression of MSRV in the serum RNA of 69% of South African MS patients and in 70% of their unaffected close relatives, whilst absent in the serum of 39 unrelated healthy control individuals (P<0.001), indicated that virus infections affect the population risk but not the familial risk in MS. HHV-6 sequences were also present at a significantly lower frequency (P<0.04) in the PBMCs of unrelated controls (5%) compared to MS patients (22.5%). A point mutation (77C^G) in the gene encoding protein-tyrosine phosphatase, receptortype C (PTPRC), which is essential for activation of T and B cells, was found to be associated with MS in the German population. Analysis of the Afrikaner and German study populations included in our study did not indicate a causative role for the PTPRC gene in MS. However, it seems likely that this mutation may contribute to disease expression, since in one of the South African families with two MS affected sibs, the most severely affected sister was heterozygous for the 77C-»G mutation. The PTPRC mutation may therefore be of significance in disease prognosis. The multidisciplinary study approach has led to a stepwise accumulation of scientific information, which forever changed our understanding of the disease process underlying MS. / AFRIKAANSE OPSOMMING: Veelvoudige sklerose (VS) is ‘n kroniese inflammatoriese siekte van die sentrale senuweestelsel. Oor die algemeen word aanvaar dat VS geassosieerd is met outoimmuniteit en dat infektiewe agente en oorerflike faktore ’n rol speel. Die hoër herhalingsrisiko van VS in families (4-5%) in vergelyking met die voorkoms in die algemene populasie (0.1%) dui op 'n genetiese basis. Alhoewel volledige analise van die mensgenoom om gene onderliggend aan VS te identifiseer aangedui het dat verskeie gene waarskynlik bydra tot vatbaarheid vir die siekte, is die aard van die gene wat betrokke is grootliks onbekend. In hierdie studie is kandidaatgene betrokke by ystermetabolisme en immunulogie vir die eerste keer geanaliseer binne die konteks van beide outoimmuun en infektiewe siekte vatbaarheid, ten einde die rol van genetiese en virale faktore in die patogenese van VS te ondersoek. Die Z-DNS herhalingsvolgorde polimorfisme in die promotor area van die SLC11A1 geen was betekenisvol geassosieerd met VS (P<0.01) in die geneties homogene Afrikaner populasie van Suid-Afrika. ’n Soortgelyke assosiasie kon egter nie aangetoon word in die Duitse en Franse populasies deur gebruik te maak van onderskeidelik ‘n gevalle-kontrole studie en transmissie-koppelings-disekwilibrium benadering nie. Betekenisvolle verskille in die genotipe verspreiding is egter tussen Duitse VS pasiente met ‘n sekonder- en primer progressiewe verloop van die siekte (P<0.05), en tussen die Duitse pasiente met terugvallende en primere progressiewe VS aangetoon (P<0.05). Hierdie bevinding verskaf verdere bewyse dat die SLC11A1 geen geassosieerd is met VS, heel waarskynlik weens die rol van die geen in yster-homeostase. Ten einde die invloed van virusse in die etiologie van VS te ondersoek is serum en witbloedselle van VS pasiente, naby-verwante familielede en nie-verwante kontroles getoets vir die teenwoordigheid van die VS-geassosieerde retrovirus (MSRV) en twee herpesvirus (HHV-6 en EBV) geenvolgordes. Die pol geen uitdrukking van MSRV was teenwoordig in die serum RNA van 69% van die Suid-Afrikaanse VS pasiente en in 70% van hul ongeaffekteerde naby-verwante familielede, terwyl dit afwesig was in 39 nieverwante kontrole individue (P<0.001). Dit dui daarop dat virusse waarskynlik die risiko vir VS meer in die populasie verhoog as in families. HHV-6 was ook teenwoordig teen ‘n beduidende laer frekwensie (P<0.04) in nie-verwante kontroles (5%) in vergeleke met VS pasiente. ‘n Puntmutasie (77C-G) in die geen wat kodeer vir die proteien tirosien fosfatase reseptor tipe C (PTPRC), wat belangrik is vir aktivering van T- en B-helperselle, is vroeer gevind om geassosieerd te wees met VS in die Duitse populasie. Analise van die Afrikaner en Duitse populasies in ons studie het egter geen bewyse gelewer dat die PTPRC geen ‘n rol speel in VS nie. Dit egter is moontlik dat hierdie mutasie bydra tot die uitdrukking van VS, aangesien die mees geaffekteerde VS pasient in een van die Suid- Afrikaanse families met twee geaffekteerde susters positief getoets het vir die mutasie. Die mutasie mag dus van belang wees in die prognose van VS. Die multidissiplinere studie-benadering en stapsgewyse insameling van wetenskaplike inligting het gelei tot ’n nuwe perspektief ten opsigte van die siekteproses onderliggend aan VS.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Parkinson’s disease (PD) is a progressive and debilitating neurodegenerative disorder, characterized by a distinct motor phenotype and the selective loss of dopaminergic neurons in the substantia nigra. While the etiology of PD is not fully understood, it is thought to involve a combination of different genetic, cellular and environmental factors that independently or concurrently contribute to neurodegeneration. To date, several PD-causing genes have been identified, and investigations of their function have provided novel insights into the pathobiology of disease. Particularly interesting among the known PD genes is parkin, mutations in which are the most common genetic cause of early onset PD. Parkin is an E3 ligase that ubiquitinates protein substrates and targets such substrates for degradation via the ubiquitin proteasome system (UPS). Therefore, the loss of parkin may result in the deleterious accumulation or dysregulation of parkin substrates and neurotoxicity. Parkin’s enzymatic activity has also been implicated in the maintenance of mitochondrial health, and mitochondrial dysfunction is commonly reported in cellular and animal models of parkin deficiency. This study aimed to investigate parkin and its role in PD on various levels. Initially, genetic screening approaches were used to assess the contribution of parkin mutations to PD in a group of 229 South African patients. It was concluded that parkin mutations are rare in the South African PD population, being present in only seven (3.1%) patients in the study group. Interestingly, this study identified two of only three Black African PD patients with mutations in a known PD-causing gene to date. The low frequency of known PD genes raises the interesting possibility that the unique South African ethnic groups may harbor mutations in novel PD-causing genes. Although many parkin-interacting proteins have been identified in the literature, it is anticipated that novel, pathologically-relevant parkin substrates remain to be discovered. Hence, this study used a yeast two-hybrid (Y2H) approach to identify novel parkin interactions. This yielded 29 putative parkin interactors, of which four, namely ATPAF1, SEPT9, actin and 14-3-3η, were prioritized for verification by co-localization and co-immunoprecipitation experiments. Interestingly, two of the parkin interactors (ATPAF1 and SEPT9) were found to accumulate in the absence of parkin, supporting their role as authentic parkin substrates. The identification of these two intriguing proteins implicates parkin in the regulation of mitochondrial ATP synthase assembly and septin filament dynamics, which may be of significant relevance to our understanding of processes underlying neurodegeneration. Moreover, it was aimed to assess various markers of mitochondrial function in a parkin-deficient cellular model, as previous studies had reported conflicting results regarding mitochondrial impairments in patient-derived cells with parkin mutations. Hence, dermal fibroblasts were obtained from PD patients with homozygous parkin mutations, after which cell growth and viability, mitochondrial membrane potential, respiratory rates and the integrity of the mitochondrial network were assessed. Surprisingly, it was found that cell growth was significantly higher in the parkin-mutant fibroblasts compared to wild-type controls fibroblasts under basal conditions (p=0.0001), while exhibiting a greater inhibition of cell growth in the presence of the mitochondrial toxin CCCP (p=0.0013). Furthermore, whereas the mitochondrial networks of patient-derived fibroblasts were more fragmented than controls (p=0.0306), it was found that mitochondrial respiratory rates were paradoxically higher in the patients (p=0.0355). These unanticipated findings are suggestive of a compensatory response to the absence of parkin. The parkin-deficient cellular model was also used in a pilot study of the functional effects of vitamin K2 treatment, which has recently been identified as a promising PD therapeutic modality. It was found that treatment with vitamin K2 resulted in more interconnected mitochondrial networks (p=0.0001) and enhanced respiratory rates (p=0.0459) in both parkin-mutant and wild-type control cells. While these results need to be studied further, it suggests that vitamin K2 supplementation may be of use as a general promoter of mitochondrial integrity and function. In conclusion, this dissertation highlights some novel interactions of the parkin protein and some interesting phenotypes of parkin deficiency. It is hoped that further investigation of parkin and its role in PD will, ultimately, aid in the development of therapeutic strategies to treat this debilitating and poorly-understood disorder. / AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is 'n progressiewe en aftakelende neurodegeneratiewe kondisie, wat gekarakteriseer word deur 'n kenmerkende bewegingsfenotipe en die selektiewe afsterwing van dopaminergiese neurone in die substantia nigra. Terwyl die etiologie van PS nie ten volle verstaan is nie, behels dit waarskynlik 'n kombinasie van verskillende genetiese, sellulêre en omgewings-faktore wat onafhanklik of gelyktydig lei tot senuwee-afsterwing. Tot op hede is daar al verskeie PS-veroorsakende gene geïdentifiseer, en die bestudering van hul funksie het nuwe insigte in die patobiologie van hierdie siekte verskaf. Onder meer hierdie PS gene is parkin van besondere belang, aangesien mutasies in parkin die mees algemene genetiese oorsaak van vroeë-aanvang PS is. Parkin is 'n E3 ligase-ensiem wat proteïen substrate ubiquitineer en teiken vir degradasie via die ubiquitien proteasoomstelsel (UPS). Dus kan die verlies van parkin lei tot die beskadigende opeenhoping of wanregulasie van parkin substrate en senuwee-afsterwing. Parkin se ensiematiese aktiwiteit is ook betrokke by die instandhouding van mitokondriale gesondheid, en mitokondriale afwykings word dikwels gerapporteer in sellulêre en diermodelle van parkin tekort. Hierdie studie het gepoog om parkin en sy rol in PS op verskillende vlakke te ondersoek. Aanvanklik is genetiese siftingsbenaderinge gebruik om die bydrae van parkin mutasies tot PS in 'n groep van 229 Suid-Afrikaanse pasiënte te evalueer. Die gevolgtrekking is bereik dat parkin mutasies skaars is in die Suid-Afrikaanse PS bevolking, aangesien dit teenwoordig is in net sewe (3.1%) pasiënte in die studie groep. Interessant genoeg, hierdie studie het twee van slegs drie gevalle van Swart Afrika-pasiënte met mutasies in 'n bekende PS geen to op datum geïdentifiseer. Die lae frekwensie van bekende PS gene versterk die stimulerende moontlikheid dat die unieke Suid-Afrikaanse sub-populasies dalk mutasies in nuwe PS-veroorsakende gene mag koester. Alhoewel baie parkin proteïen-interaksies reeds in die literatuur geïdentifiseer is, word daar verwag dat nuwe, patologies-relevante parkin substrate nog wag om ontdek te word. Dus het hierdie studie 'n gis twee-hibried (G2H) benadering gebruik om nuwe parkin interaksies te identifiseer. Hierdie het 29 vermeende parkin interaktors opgelewer, waarvan vier, naamlik ATPAF1, SEPT9, aktien en 14-3-3η, geprioritiseer is vir verifikasie deur mede-lokalisering en mede-immunopresipitasie eksperimente. Interessant genoeg, daar is gevind dat twee van die parkin interaktors (ATPAF1 en SEPT9) ophoop in die afwesigheid van parkin, wat hul rol as werklike parkin substrate ondersteun. Die identifisering van hierdie twee interessante proteïene impliseer parkin in die regulering van mitokondriale ATP sintase vervaardiging en septienfilament dinamika, wat moontlik van beduidende belang is vir ons begrip van die onderliggende prosesse wat senuwee-afsterwing veroorsaak. Verder is daar daarop gemik om verskeie aanwysigings van mitokondriale funksie in 'n parkin-gebrekkige sellulêre model te evalueer, aangesien vorige studies teenstrydige resultate rapporteer rakende mitokondriale afwykings in pasiënt-selle met parkin mutasies. Dus is daar dermale fibroblaste verkry van PS pasiënte met homosigotiese parkin mutasies, waarna sel-groei en lewensvatbaarheid, mitokondriale membraanpotensiaal, respiratoriese tempo en die integriteit van die mitokondriale netwerk geëvalueer is. Daar is verbasend gevind dat sel-groei aansienlik hoër is die parkin-mutante fibroblaste in vergelyking met wilde-tipe kontrole fibroblaste onder basale kondisies (p=0.0001), terwyl hulle 'n groter inhibisie van sel-groei in die teenwoordigheid van die mitokondriale toksien CCCP ondergaan (p=0.0013). Verder, terwyl die mitokondriale netwerke van pasiënt fibroblaste meer gefragmenteer is as die van kontroles (p=0.0306), is daar gevind dat mitokondriale respiratoriese tempo’s, paradoksaal-gewys, hoër is in die pasiënte (p=0.0355). Hierdie onverwagte bevindinge is suggestief van die aanskakeling van 'n vergoedende respons-proses in die afwesigheid van parkin. Die parkin-gebrekkige sellulêre model is ook gebruik in 'n voorlopige studie van die funksionele effekte van vitamiene K2 behandeling, wat onlangs geïdentifiseer is as 'n belowende terapeutiese moontlikheid vir PS. Daar is gevind dat sel-behandeling met vitamiene K2 lei tot meer geïnterkonnekteerde mitokondriale netwerke (p=0.0001) en verbeterde respiratoriese fuksie (p=0.0459) in beide parkin-mutante en wilde-tipe kontrole selle. Terwyl hierdie resultate verder bestudeer sal moet word, dui dit daarop dat vitamiene K2-aanvulling moontlik gebruik kan word as 'n algehele promotor van mitochondriale integriteit en funksie. Ten slotte, hierdie verhandeling beklemtoon ‘n paar nuwe interaksies van die parkin proteïen en 'n paar interessante fenotipes van parkin tekort. Daar word gehoop dat verdere ondersoek van parkin en parkin se rol in PS sal, uiteindelik, steun in die ontwikkeling van terapeutiese strategieë om hierdie aftakelende en swak-verstaande wanorde beter te behandel.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy