Thesis (PhD)--University of Stellenbosch, 2004. / 426 Leaves printed single pages, preliminary pages i-xxiv and i-xxvii and 399 numberd pages. Includes bibliography. List of figures, List of tables, List of abbreviations. / ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease. The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and an increased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring in thirteen genes, have been identified. As the vast majority of HCM-causing mutations occur in components of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere. Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed that HCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausing mutations in the gamma two subunit of adenosine monophosphate activated protein kinase highlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports a hypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion. Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second most prevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomeric thick filament, where it acts as a regulator of thick filament structure and cardiac contractility. Although cMyBPC has been studied extensively, the mechanisms through which it fulfill these functions have remained elusive, largely due to a lack of a comprehensive understanding of its interactions with other sarcomeric components and its quaternary structure. The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in a panel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC in which HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individuals was screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)- based single-strand conformation polymorphism method, as well as restriction enzyme digestion, DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands of domains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandand library-assays were performed. Three novel and two previously described putative HCM-causing mutations were identified in MYBPC3. Data generated in this and other studies, however, suggest that two of these “mutations” are likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausing mutations. Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This finding identified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specific C7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 and domain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no known sarcomeric ligands of the N-terminal region of cMyBPC. Identification of the ligands of specific domains of cMyBPC led to the development of detailed models of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fully phosphorylated. The integration of these models into an existing model of thick filament quaternary structure allows new insights into the functioning of cMyBPC as a regulator of both thick filament structure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HKM) is ‘n outsosomaal dominante primêre hartsiekte. Die primêre kenmerke van HKM is linker ventrikulêre hipertrofie, miokardiale wanorde, fibrose en ‘n verhoogde risiko van skielike dood. Tot dusver is 260 HKM-veroorsakende mutasies in 13 gene geïdentifiseer. Aangesien die oorgrote meerderheid van HKM-veroorsakende mutasies in komponente van die kardiale sarkomeer voorkom, is HKM as ‘n siekte van die kardiale sarkomeer beskryf. Funksionele analise van HKM-veroorsakende mutasies in sarkomeriese protein-koderende gene het aan die lig gebring dat hierdie mutasies ‘n wye spektrum van gevolge op kontraktiele funksie het. Die ontdekking van HKM-veroorsakende mutasies in die gamma-twee subeenheid van adenosien monofosfaat-geaktiveerde proteïen kinase het die feit dat mutasies nie-sarkomeriese proteïene ook HKM kan veroorsaak onderstreep en ondersteun ‘n hipotese dat HKM-veroorsakende mutasies energievermorsing en energie uitputting tot gevolg het. Mutasies in die kardiale miosien-bindingsproteïen C (kMiBPC) geen (MYBPC3) is die tweede mees algemene oorsaak van HKM. kMiBPC is ‘n modulêre protein wat ‘n integrale deel van die sarkomeriese dik filament vorm, waar dit die struktuur van die dik filament en kardiale kontraktiliteit reguleer. Nieteenstaande die feit dat kMiBPC intensief bestudeer is, word die meganismes hoe hierdie funksies vervul word swak verstaan, grotendeels weens die afwesigheid van ‘n in diepte begrip van sy interaksies met ander komponente van die sarkomeer asook sy kwaternêre struktuur. Die doelstellings van hierdie studie was, eerstens, om MYBPC3 vir HKM-veroorsakende mutasies in ‘n paneel van HKM-geaffekteerde individue te deursoek en tweedens, om die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is te identifiseer.‘n Paneel van deoksiribonukleïensuur (DNS) monsters verkry van onverwante HKM-geaffekteerde individue is deursoek vir HKM-veroorsakende mutasies in MYBPC3, deur middel van die polimerase ketting-reaksie (PKR)-gebasseerde enkelstrand konformasie polimorfisme metode, sowel as restriksie ensiem vertering, DNS volgordebepaling en terugtranskripsie PKR tegnieke. Die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is, is geïdentifiseer deur middel van gis twee-hibried (G2H) kandidaat-ligand en biblioteek-siftings eksperimente. Drie onbeskryfde en twee voorheen beskryfde vermeende HKM-veroorsakende mutasies in MYPBC3 is geïdentifiseer. Data gegenereer in hierdie en ander studies dui daarop dat twee van hierdie “mutasies” eerder polimorfismes, of siekte-modifiserende faktore, as hoof-lokus HKMveroorsakende mutasies is. Onlangse bevindings het ‘n spesifieke interaksie tussend die C5 en C8 domeine van kMiBPC getoon. Hierdie bevindings het óf domein C6, óf C10, as kandidaat-ligande van domein C7 geïdentifiseer. G2H eksperimente het ‘n spesifieke interaksie tussen domains C7 en C10 getoon. Addisionele G2H eksperimente het ook C-zone titin as ‘n ligand van domein C7 sowel as domein C10 as ‘n ligand van domein C3 geïdentifiseer. Verdere G2H eksperimente het egter geen sarkomeriese ligande van die N-terminale gedeelte van kMiBPC geïdentifiseer nie. Die identifikasie van ligande van spesifieke domeins van kMiBPC het gelei tot die ontwikkelling van ‘n gedetaileerde model van kMiBPC kwaternêre struktuur wanneer kMiBPC beide ongefosforileerd en ten volle gefosforileerd is. Die intergrasie van hierdie modelle in bestaande modelle van dik filament kwaternêre struktuur werp nuwe lig op die funksionering van kMiBPC as ‘n reguleerder van beide dik filament struktuur en kardiale kontraktiliteit, sowel as die patofisiologie van kMiBPCgeassosieerde HKM.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Setting Study conducted in Tygerberg, Cape Town in South Africa. Hypothesis Host biomarkers associated with the antimycobacterial immune response during active infection with M. tuberculosis and during anti-tuberculosis chemotherapy are indicative of bacterial killing in the host and can be used in models to predict eventual treatment outcome. Objectives 1. To investigate immune parameters that were selected in a biological context as biomarkers of the extent of disease and early response to anti-tuberculosis treatment. 2. To use selected immune parameters to characterise fast and slow responders to anti-tuberculosis therapy. Findings Evaluation of cytokine multiplex fluorescent bead-based immunoassays as a screening tool in the search for biomarkers The data showed that cytokine multiplex fluorescent bead-based immunoassays achieved acceptable recoveries to detect antigen-specific IFN- responses in whole blood supernatant making it attractive for biomarker screening. However, proper optimisation needs to be done and proper controls included when using these kits. Markers of extent of disease High levels of CRP at diagnosis were found to be associated with the presence of multiple cavities on chest X-rays. A high level of suPAR and sICAM-1 at diagnosis were associated with the extent of alveolar disease. Also significant were the associations between the level of granzyme B, LAG-3 at diagnosis and the size of the cavities. No significant associations were observed between sTNFRs or DR5 with the chest X-ray grading of tuberculosis disease. Early classification of fast and slow responders to anti-tuberculosis treatment After cross-validation classification, discriminant analysis (DA) and support vector machine (SVM) analysis of selected immune parameters (sICAM-1 CRP, granzyme B, suPAR, sTNFRs, LAG-3 and CD3dim/CD56+ (% of CD45+) resulted in a 75% to 100% correct classification of the fast responders and a 82% to 100% correct classification of the slow responders when using DA. For SVM, the correct classification of the fast responders ranged from 88% to 100%, and that for the slow responders ranged from 95% to 100%. Differential gene expression in fast and slow responders to treatment Direct comparison of fast and slow responders showed that IL-4 transcripts were significantly higher in the fast responders at week one after initiation of treatment when compared to slow responders. IL-42 was also differentially expressed. Although IL- was significantly up-regulated in both fast and slow responders after one week of treatment compared to diagnosis, IL- expression was more than two folds higher in slow responders than in fast responders. No significant differences between the fast and slow responders were observed in the expression of TGF-, TGF-RII, Foxp3 and GATA-3. Conclusion Predictive models for differential anti-tuberculous treatment responses combining host proteins are promising and should be included in larger prospective studies to find the optimal markers for inclusion into clinical trials of new drugs and for implementation into clinical practice. / AFRIKAANSE OPSOMMING: Ligging Studie onderneem in Tygerberg, Kaapstad, Suid-Afrika. Hipotese Gasheerbiomerkers wat verband hou met die antimikobakteriële immuunrespons tydens aktiewe infeksie deur M. tuberculosis en tydens teentuberkulose chemoterapie dui op bakteriële doding in die gasheer en kan in modelle gebruik word om die uiteindelike uitkoms van die behandeling te voorspel. Doelwitte 1. Om gekose immuunparameters in ’n biologiese konteks as biomerkers van die omvang van siekte en vroeë reaksie op behandeling te ondersoek. 2. Om gekose immuunparameters te gebruik om vinnige en stadige reageerders op teentuberkulosebehandeling te karakteriseer. Bevindings Evaluering van die sitokien veelvuldige fluoresseer-pêrelbaseerde immuuntoets (cytokine multiplex fluorescent bead-based immunoassays) as ’n siftingsinstrument in die soeke na biomerkers Die data het getoon dat die sitokien veelvuldige fluoresseer-pêrelgebaseerde immuuntoets in staat was om antigeenspesifieke IFN--respons te meet wat dit aanloklik maak vir biomerkersifting. Sorgvuldige optimering moet egter gedoen word en behoorlike beheer moet ingesluit word wanneer hierdie stelle gebruik word. Merkers van omvang van siekte Hoë vlakke van CRP by diagnose is getoon om verband te hou met die teenwoordigheid van veelvoudige holtes op die pasiënte se borskas x-strale. Hoë vlakke van suPAR en sICAM-1 by diagnose was assosieer met die omvang van alveolêre siekte. Die assosiasie tussen die vlakke van granzyme B, LAG-3 by diagnose en die grootte van die holtes was ook betekenisvol. Daar was geen betekenisvolle assosiasies toe sTNFRs of DR5 en die borskas x-straalgradering van tuberkulosesiekte nie. Vroeë klassifikasie van vinnige en stadige reageerders op teentuberkulosebehandeling Ná klassifikasie op grond van kruisstawing het diskriminant-analise (DA) en ondersteuningsvektormasjiene (SVM) van geselekteerde immuunparameters (sICAM-1 CRP, gransiem B, suPAR, sTNFRs, LAG-3 en CD3dim/CD56+ (% van CD45+)) gelei tot ’n 75% tot 100% korrekte klassifikasie van die vinnige reageerders met DA en ’n 82% tot 100% korrekte klassifikasie van stadige reageerders. Vir SVM het die korrekte klassifikasie van vinnige reageerders gewissel van 88% tot 100%, en vir stadige reageerders het dit gewissel van 95% tot 100%. Differensiële geenuitdrukking in vinnige en stadige reageerders op behandeling In vergelyking met die vlak by diagnose is die uitdrukkingsvlak van IL-4 in die vinnige reageerders betekenisvol opgereguleer met ’n faktor van 9.2 teen die eerste week ná die aanvang van behandeling, in kontras met die stadige reageerders. Daar was geen verskille tussen die vinnige en die stadige reageerders met betrekking tot die uitdrukking van TGF-, TGF-RII, Foxp3 en GATA-3 nie. Gevolgtrekking Voorspellende modelle vir differensiële tuberkulose behandelingsresponse wat gasheerproteïene kombineer, hou belofte in en behoort in groter prospektiewe studies ingesluit te word om die mees geskikte merkers te vind vir insluiting in kliniese proewe van nuwe middels en vir implementasie in kliniese praktyk.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Sequence analysis showed that the essential glnA1 gene of Mycobacterium tuberculosis might be closely related to an actinomycetes progenitor sequence and that this sequence may have undergone duplication into other non-essential GS encoding genes in some Actinobateria, notably the mycobacteria. Also, the M. tuberculosis glnA1 sequence remains conserved throughout the evolution of M. tuberculosis. It was also shown that glnA1 is widely expressed in M. tuberculosis infected human pulmonary tissue, where M. tuberculosis might be present in altered phenotypes. These results imply that glnA1 is under selective pressure against evolutionary change. At transcriptional level it was shown that M. tuberculosis glnA1 might be expressed from two alternate promoter sites, but that these promoter sites may not be controlled by environmental nitrogen (in the form of ammonium) variation. We also showed that M. tuberculosis GS is effectively exported by M. smegmatis to the cell wall, but that GS secretion into the exogenous environment does not occur. Also, evidence has been presented which suggested that M. tuberculosis GS might be modified at the C-terminus, probably as part of a mechanism that retains GS in the cytosol. This hypothesis was further substantiated where it was demonstrated that two GS isoforms of different size (short isoform in cytosol, longer isoform in cell wall) are present in M. bovis BCG. It is unknown what causes this modification, since it couldn’t be observed in M. smegmatis, but it was suggested that it might be through the action of a cis- or trans-acting element present in proximity of the M. tuberculosis glnA1 gene. It was also shown that a cluster of genes found immediately downstream of the M. tuberculosis glnA1 sequence might be regulated in an operonic fashion under conditions of elevated environmental nitrogen concentrations. Two of the genes (glnE and glnA2) in this operon arrangement have been previously shown to be involved in nitrogen and glutamine metabolism. The function of the other gene, Rv2223c, is unknown. It was shown that Rv2223c homologs are mostly found in the mycobacteria and that it may encode an exported protease. It was hypothesised that this sequence and its adjacently located progenitor sequence, Rv2224c, might be involved in M. tuberculosis GS mediated metabolism. It was showed that over-expression of Rv2223c and Rv2224c may be toxic to E. coli and mycobacterial hosts, such as M. smegmatis, but that inhibition of transcription of these genes may be fatal to M. bovis BCG and M. tuberculosis H37Rv. It was also shown that Rv2223c is widely expressed in M. tuberculosis infected human tissue, which was comparable to that of glnA1. The results presented in this study shed more light on the distribution and transcriptional regulation of GS in mycobacteria and has identified new genes that might be involved in GS regulation. These results may present new approaches to tuberculosis control and thereby contribute to alleviate the burden of the disease. / AFRIKAANSE OPSOMMING: Genetiese en proteien volgorde analise het aangedui dat die glnA1 (kodeer vir glutamien sintetase (GS), ‘n essentiele protein) geen van Mycobacterium tuberculosis die naaste verwant is aan ‘n actinomycetes voorloper volgorde wat duplikasie ondergaan het om die ander nieessensiele GS koderende gene in sommige Actinobakterieë te vorm, veral in die mikobakterieë. Voords het die glnA1 geen geneties gekonserveerd gebly gedurende die evolusie van M. tuberculosis. Dit is ook aangetoon dat volop transkribasie van die glnA1 geen voorkom in die M. tuberculosis geïnfekteerde pulmonêre weefsel waar M. tuberculosis moontlik mag voorkom. Op transkriptionele vlak is dit aangetoon dat die M. tuberculosis glnA1 geen vanaf twee onderskeie promotors uitgedruk mag word, maar dat hierdie twee promotors nie deur variasies in die konsentrasie van stikstof (in die vorm van ammonium) in die omgewing beïnvloed word nie. Daar is ook aangedui dat M. tuberculosis GS effektief deur M. smegmatis oor die selmembraan na die selwand vervoer word, maar dat daar nie GS sekresie na die ekstrasellulêre omgewing geskied nie. Ook is bewyse gevind dat M. tuberculosis GS modifikasie aan die C-terminus mag ondergaan wat waarskynlik dien om GS beweging uit die sitosol te verhoed. Hierdie hipotese is versterk deurdat twee isoforms van verskillende grootte (klein in sitosol en groter in die selwand) gevind is in M. bovis BCG. Dit modifikasie meganisme is onbekend, maar vind moontlik nie plaas in nie-patogeniese mikobakterieë soos M. smegmatis nie en mag moontlik deur cis- of trans-werkende elemente gefasiliteer word. Daar is aangedui dat ‘n groepering van vier gene lanksaan die glnA1 lokus in ‘n operoniese meganisme gereguleer mag word onder variërende konsentrasies van stikstof in die omgewing. Dit is bekend dat twee van die gene in die operon (glnE en glnA2) betrokke in stikstof en gultamien metabolisme is. Die funksie van die ander twee gene (Rv2223c en Rv2224c) is onbekend. Daar is aangetoon dat volgordes soortgelyk aan Rv2223c beperk is tot die mikobakterieë en dat die geen ‘n protease, wat moontlik gesekreteer word vanuit die sitosol, kodeer. Daar is aangetoon dat die oor-produksie van die Rv2223c en Rv2224c proteine toksies is vir E. coli en mikobakterieë, soos M. smegmatis, maar dat transkripsie inhibisie hierdie gene dodelik is vir M. tuberculosis H37Rv en M. bovis BCG. Daar is ook angedui dat die ekspresie van hierdie gene volop verspreid is in M. tuberculosis geïnfekteerde menslike weefsel, soortgelyk aan diè van glnA1. Die resultate vervat in hierdie studie werp meer lig op die verspreiding en transkiptionele regulasie van GS in mikobakteriee en nuwe gene is ontdek wat betrokke by GS regulasie mag wees. Hierdie resultate mag bydra tot nuwe maniere om tuberkulose te bekamp en daardeur die voorkoms van die siekte te beperk.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Healthcare workers (HCWs) wear latex gloves to protect themselves and their patients against the transmission of microbial, viral and bloodborne diseases. These individuals are primarily exposed to latex via cutaneous (direct contact) and mucocutaneous (inhalation of airborne allergens on glove powder) routes. Repeated exposure leads to the formation of circulating latex-specific IgE and subsequent sensitisation with varying clinical expression. The airconditioning system of the Tygerberg Hospital (TBH) complex was investigated for the presence of aerosolised cornstarch glove powder and proteins. Dust samples were collected from 14 areas with different levels of latex glove usage. Dust samples were spectrophotometrically compared to a calibration graph of pure glove powder. The detection of starch and proteins in all the dust samples confirmed the presence of glove powder and possibly airborne latex allergens in the airconditioning ducts. As expected, the high exposure areas showed the highest concentrations of both starch and proteins. It is possible that other proteins than latex were involved, but the confirmed high level of protein contamination should be a cause for concern. Correlation between starch and protein levels was highly significant (p<0.01) in all instances. A total of 500 questionnaires were circulated for completion by HCWs from TBH. The response rate was 69.8%. After considering specific inclusion criteria, a study group of 152 individuals was compiled (28 males, 124 females). All subjects had current latex exposure and suffered from at least three pre-defined symptoms. Serum was collected from all subjects and dermal fluid from 31 subjects. Total IgE and latex specific IgE analysis were done on all serum and dermal fluid samples. Latex-specific IgE was positive (>0.35 IU/ℓ) in 23 serum and six dermal fluid samples. Skin prick tests (SPTs)for latex were done on 59 subjects with negative serum latex-specific IgE and 34 had positive results. Twelve subjects with negative latex-specific IgE and latex SPTs underwent patch tests with the European Standard Series, a piece of latex glove and glove powder in petrolatum. Three subjects had positive results to one or more of these allergens. Western blot analysis for latex was done on all positive sera and dermal fluid collected from these subjects. Western blot analysis for latex proved to be more sensitive than the capRAST, because it was able to identify specific bands in samples with negative capRAST results. All subjects showed a band for Hev b 1, which has been confirmed as a powder-bound airborne allergen. Hev b 6.01 is associated with HCWs with cutaneous symptoms and this band was recognised by 81% of the subjects. These findings confirmed that airborne and cutaneous routes are the major routes of exposure in HCWs. According to their laboratory results, subjects were divided into the following subgroups and compared statistically: Group A (serum positive, n=23), Group B (SPT positive, n=34) and Group C (negative, n=25). Group D (withdrawn, n=70) could not be used for statistical comparisons, due to incomplete results. An overall latex allergy prevalence of 38% was found. Group A differed significantly from Group B and Group C for most clinical and special investigations. Group A and B were also combined to represent all subjects with positive results (Cohort AB). The Allergy Score and Class were highly significant when Cohort AB was compared to Group C. The selection of clinical symptoms was confirmed to be relevant and work-related deterioration on any of the symptoms should bear a high index of suspicion in the evaluation of latex allergy. Numerical indices and specific symptoms showed high positive predictive values and the Allergy Score produced statistical significance in the positive subgroups when compared to the negative subgroup. Paired statistical significance was confirmed between the Allergy Score and occupational exposure (number of years, hours and pairs per week). The areas with the highest occupational latex exposure in HCWs are the face and hands. Different occupations also have different levels of exposure and two subgroups of HCWs (16 laboratory technologists and 13 theatre staff) were investigated for sebum content on different facial areas and the palms and dorsal areas of both hands. Baseline measurements were done before putting on gloves. In 21 subjects follow up measurements were done following three to four hours of occupational exposure, but before washing their hands. Baseline and follow up values were compared for all the different anatomical regions. Levels on the forehead and cheeks increased over time, while the level on the nose decreased. All hand regions decreased significantly during occupational exposure, suggesting that glove powder contributes to dryness of the skin. In conclusion, the problem posed by latex allergy will not be solved overnight and will probably remain a major occupational hazard for years to come. It is currently not possible to avoid exposure to latex, but it is imperative to institute safety measures to prevent further sensitisation in predisposed individuals and manage those already affected. / AFRIKAANSE OPSOMMING: Gesondheidswerkers dra lateks handskoene om hulleself en hulle pasiënte te beskerm teen die oordrag van mikrobiale, virale en bloed-gedraagde siektes. Die lateks blootstelling vind hier hoofsaaklik plaas via kutane (direkte velkontak) en mukokutane (inaseming van lug-gedraagde allergene op hanskoen poeier) roetes. Herhaalde blootstelling veroorsaak sirkulerende lateksspesifieke IgE en sensitisasie met variërende kliniese beelde. Die lugreëlingstelsel van die Tygerberg hospitaalkompleks is ondersoek vir die teenwoordigheid van handskoenpoeier (stysel) en lateks proteïene. Stofmonsters is versamel in 14 areas wat verskillende blootstellingsvlakke verteenwoordig het. Die stofmonsters is spektrofotometries vergelyk met "n kalibrasiekurwe van suiwer hanskoenpoeier. Stysel en proteïene kon in al die stofmonsters aangetoon word en het die teenwoordigheid van handskoenpoeier en moontlike luggedraagde lateks proteïene in die lugreëlingstelsel bevestig. Soos verwag kon word, het die hoogste stysel en proteïen waardes in hoë blootstellingsareas voorgekom. Hoogs beduidende statistiese korrelasies (p<0.01) tussen die stysel en proteïenvlakke kon aangedui word in alle monsters. "n Totaal van 500 vraelyste is gesirkuleer vir voltooiing deur TBH gesondheidswerkers, waarvan 69.8% voltooide vraelyste terugontvang is. Na evaluering van insluitingskriteria, is "n studiegroep van 152 individue saamgestel (28 mans, 124 vrouens). Almal het huidige lateks blootstelling en ten minste drie het vooraf gedefinieerde simptome gerapporteer. Serum is van die hele groep versamel en dermale vog van 31 proefpersone. Totale IgE en lateks-spesifieke IgE vlakke is op alle serum en dermale vog bepaal. Positiewe resultate (>0.35 IU/ℓ) is verkry in 23 serum en ses dermaIe vog monsters. Velpriktoets vir lateks is op 59 proefpersone uitgevoer en 34 daarvan het positiewe resultate opgelewer. Twaalf proefpersone met negatiewe lateks-spesifieke IgE en velpriktoets resultate het kutane plaktoetse ondergaan met die Europese Standaard Reeks, "n stukkie lateks handskoen en handskoenpoeier in petrolatum. Drie proefpersone het positiewe resultate teen een of meer van die allergene gehad. Westerse kladanalise vir lateks is op alle positiewe serum gedoen, asook die dermale vogte van hierdie proefpersone. Westerse kladanalise vir lateks blyk baie meer sensitief te wees as die capRAST, aangesien dit spesifieke bande kon identifiseer in monsters capRAST resultate. Alle monsters het "n band getoon vir Hev b 1, "n poeier-gebinde, luggedraade allergeen. Hev b 6.01 is geassosieer met gesondheidswerkers met velsimptome en hierdie band is gevind in 81% van die monsters. Hierdie resultate bevestig dat die belangrikste blootstelling aan lateks in gesondheidswerkers deur die vel en inaseming plaasvind. Proefpersone is in die volgende drie groepe verdeel volgens laboratorium resultate en statisties vergelyk: Groep A (positiewe serum, n=23), Groep B (positiewe velpriktoetse, n=34) en Groep C (negatief, n=25). Groep D (onttrek, n=70) kon nie vir betekenisvolle statistiese vergelykings aangewend word nie, as gevolg van onvolledige resultate. 'n Finale lateks allergie prevalensie van 38% is gevind. Groep A het hoogs beduidend verskil van Groep B en C vir die meeste van die kliniese en spesiale laboratoriumondersoeke. Groep A en B is gekombineer om alle proefpersone in te sluit met positiewe resultate (Kohort AB). Die Allergie Telling en Klas van Kohort AB was hoogs beduidend in vergelyking met Groep C. Die gekose simptome is bevestig as relevant en enige werksverwante verergering van simptome moet met 'n hoë mate van agterdog bejeën word in lateks allergie. Numeriese indekse en spesifieke simptome het hoë positiewe voorspellingswaardes gelewer en die Allergie Telling was hoogs beduidend in die positiewe subgroep in vergelyking met die negatiewe subgroep. Gepaarde statistiese beduidenheid is ook gevind tussen die Allergie Telling en beroepsblootstelling (jare van blootstelling, uur en paar handskoene per week). Die meeste beroepsblootstelling aan lateks in gesondheidswerkers vind plaas op die hande en gesig. Verskillende beroepe het ook verskillende blootstellingsvlakke en two subgroepe gesondheidswerkers (16 laboratorium tegnoloë en 13 teater personeel) is ondersoek vir die sebumgehalte op veskillende areas van die gesig en hande. Basislynvlakke is gemeet voordat handskoene aangetrek is en in 21 gevalle is opvolgvlakke gemeet na drie tot vier uur beroepsblootstelling, maar voor die hande gewas is. Basislyn en opvolgvlakke is met mekaar vergelyk vir al die anatomiese areas. Die voorkop en wange het 'n toename in sebumgehalte getoon, terwyl dié van die neus afgeneem het. AI die areas op die hande toon 'n hoogs beduidende afname tydens beroepsblootstelling, wat impliseer dat hanskoenpoeier moontlik bydra tot droogheid van die vel. In samevatting, die lateks allergie probleem sal nie oornag opgelos word nie en sal waarskynlik 'n belangrike beroepsrisiko bly vir die aansienlike toekoms. Totale vermyding van lateks is tans onmoontlik en daarom is dit van uiterste belang om voorsorgmaatreëls in plek te stel om verdere sensitisasie in blootgestelde individue te verhoed en die wat reeds geaffekteer is, effektief te hanteer.

Thessis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase-3 activation or the apoptotic index. EPA and ARA (20 jiM, present before and after SI) significantly reduced caspase-3 activation, PARP-cleavage and the apoptotic index during reperfusion. This was associated with increased ERK- and decreased p38 phosphorylation. Vanadate (a tyrosine phosphatase inhibitor), but not okadaic acid (a serine-threonine phosphatase inhibitor), significantly reduced ARAinduced inhibition of p38 phosphorylation, suggesting involvement of tyrosine phosphatases during Sl/R. MKP-1, a dual-specificity phosphatase, was targeted and a significant induction of MKP-1 by ARA and EPA was observed. An in vitro dephosphorylation assay confirmed that this phosphatase might be responsible for the inhibition of p38 activation. It was also demonstrated that the protective actions of ARA are PI3-K dependent. The results suggest that p38 has a pro-apoptotic role while JNK phosphorylation is protective and that these kinases act via caspase-3 to prevent or promote cell survival in response to SI/R-induced injury. It was demonstrated for the first time that EPA and ARA protect neonatal cardiac myocytes from ischaemia/reperfusion-induced apoptosis through induction of a dual-specific phosphatase, MKP-1, causing dephosphorylation of the proapoptotic kinase, p38. These beneficial effects of ARA and EPA were also reflected by improvement in functional recovery during ischaemia/reperfusion of the isolated perfused rat heart model. / AFRIKAANSE OPSOMMING: Dit word algemeen aanvaar dat lang-ketting poli-onversadigde vetsure teen kardiovaskulere siektes beskerm, maar hul meganisme van aksie sowel as hul invloed op intrasellulere seinoordragpaaie is egter onbekend. Die doelwitte van hierdie studie is dus tweevoudig: (i) om die belang van mitogeen-geaktiveerde proteien kinases (MAPKs) en protein kinase B (PKB/Akt) in isgemie/herperfusie-geinduseerde apoptose vas te stel en (ii) om te bepaal of lang-ketting poli-onversadigde vetsure die hart, deur manipulering van hierdie kinases, beskerm. Rot neonatale ventrikulere miosiete, blootgestel aan gesimuleerde isgemie en herperfusie (Sl/H), is gebruik om die aktivering van ekstrasellulere seingereguleerde kinase (ERK), p38, c-Jun NH2-terminale protein kinase (JNK) asook PKB/Akt tydens apoptose, te karakteriseer. Die effek van ‘n omega-3 vetsuur (eikosapentaenoSsuur - EPA) en ‘n omega-6 vetsuur (aragidoonsuur - ARA) op die respons van bogenoemde kinases in neonatale kardiomiosiete tydens Sl/H, is ondersoek. Blootstelling van miosiete aan SI (energie-uitputting gemduseer deur kaliumsianied en 2-deoksi-D-glukose) het ‘n afname in die vermoe van die sel om te oorleef, soos gemeet deur die MTT (3-[4,5-dimetieltiazol-2-yl]-2,5- difeniel tetrazolium bromied) bepaling, tot gevolg gehad. ‘n Toename in apoptose (kaspase-3 aktivering en poli(ADP-ribose) polimerase (PARP) kliewing) is ook waargeneem. Morfologiese bewyse van apoptose (Hoechst 33342 kleuring) was egter eers tydens herperfusie sigbaar. SI is gekenmerk deur vinnige aktivering van p38 en PKB/Akt Ser473, terwyl ERK en JNK fosforilering slegs tydens herperfusie waargeneem is. Vooraf-behandeling met SB203580, ‘n p38 inhibitor, het ‘n beduidende toename in sellewensvatbaarheid asook ‘n afname in die apoptotiese indeks tydens Sl/H teweeggebring, terwyl SP600125, ‘n spesifieke JNK inhibitor, apoptose bevorder het. PD98059, ‘n ERK inhibitor, het geen invloed op apoptose tydens Sl/H gehad nie. Wortmannin, ‘n PI3-kinase inhibitor, het Thr308 (nie Ser473) fosforilering onderdruk, gepaargaande met ‘n toename in PARP kliewing, maar dit het geen invloed op kaspase-3 aktivering of die apoptotiese indeks gehad nie. EPA en ARA (20 (iM, teenwoordig voor en na SI) het kaspase-3 aktivering en PARP kliewing asook die apoptotiese indeks tydens herperfusie beduidend verminder. Beide vetsure het ook ‘n beduidende toename in ERK en afname in p38 fosforilering veroorsaak. Vanadaat (‘n serien-threonien fosfatase inhibitor), maar nie “okadaic” suur (‘n tirosien fosfatase inhibitor), kon die ARA-gemduseerde inhibisie van p38 ophef nie. Induksie van MKP-1, ‘n tweeledige-spesifieke fosfatase, is beduidend deur ARA en EPA tydens herperfusie verhoog. 'n In vitro defosforileringbepaling het bevestig dat hierdie fosfatase wel betrokke by die inhibisie van p38 kan wees. Daarbenewens is gevind dat die beskermende aksie van ARA PI3-K afhanklik is. Hierdie resultate wys dat fosforilering van p38 pro-apoptoties is, terwyl JNK beskermend is en dat hierdie kinases via kaspase-3 seldood of oorlewing tydens SI/H-geinduseerde beskadiging bemiddel. In hierdie model is daar vir die eerste keer getoon dat EPA en ARA neonatale kardiale miosiete teen isgemie/herperfusie-geinduseerde apoptose beskerm deur induksie van MKP- 1, wat defosforilering van die pro-apoptotiese kinase, p38 teweegbring. Hierdie voordelige effekte van EPA en ARA is ook sigbaar in die funksionele herstel tydens isgemie/herperfusie van die geisoleerde rothart model.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: In this study the author presents new data that will shed light on the fairly nebulous knowledge of intracellular pathways involved in the physiologically induced acrosome reaction and the subsequent events leading to fertilization. The zona pellucidainduced acrosome reaction, sperm-zona interaction as well as various sperm motion characteristics were investigated. The first part of the study focussed on the role of extracellular signal regulated kinase (ERK), a member of the family of mitogen activated protein kinases, during the zona pellucida-induced acrosome reaction and sperm-oocyte interaction. It was shown that the inhibition of ERK significantly reduced the zona pellucida-induced acrosome reaction as measured by fluorescent microscopy. This suggests that ERKs are directly or indirectly involved in the signal transduction pathway through which the human sperm acrosome reaction is induced by the zona pellucida. In an attempt to provide further proof that ERK was involved in human acrosome signalling hemizona assays were employed to test sperm-oocyte binding. From these sperm-oocytebinding experiments it was clear that the inhibition of ERK leads to increased binding. These results support the idea that the zona pellucida-induced acrosome reaction, and therefore the physiologically relevant exocytotic event, is regulated by an ERKmediated signal transduction process. In the second part of the study the significance of phosphatidylinositol 3-kinase (PI3K) in the process of human sperm motility, acrosome reaction and sperm-oocyte binding, was investigated by employing the specific PI3K, LY294002. PI3K inhibition increased the percentage motility and percentage progressive motility in asthenozoospermia patients. Under the present laboratory conditions PI3K inhibition furthermore did not influence the acrosome reaction, whilst it enhanced sperm-oocyte binding. These results therefore imply that PI3K negatively affect sperm motility and zona-binding. In the last part of the study the possible effects of intracellular cGMP accumulation via acute in vivo sildenafil citrate (ViagraTM) administration was investigated on seminal parameters, induction of the acrosome reaction, sperm-oocyte binding and sperm motility. As was expected no changes in the macro- and microscopically seminal parameters were caused by sildenafil citrate when compared to placebo. Furthermore the acrosome reaction was also not initiated or potentiated by sildenafil citrate at concentrations of 50mg orally. Sperm-oocyte binding, smooth path velocity, straight line velocity and the percentage rapid cells all increased after sildenafil citrate treatment. From these results it appear that there are various role players in the zona pellucidainduced acrosome reaction intracellular signalling system. A thorough understanding of such signal transduction systems and the crosstalk in-between will ultimately yield information regarding the nature of receptors to which these signal transduction pathways are coupled in human spermatozoa as well as the intracellular effectors that ultimately regulate sperm function. Moreover, an understanding of these regulatory pathways will be essential for the future development of clinical approaches designed to enhance or preclude fertilization. / AFRIKAANSE OPSOMMING: Die outeur lê in hierdie studie nuwe data voor ten einde meer lig te werp op die relatief vae veld van intrasellulêre seintransduksie paaie betrokke by die fisiologiesgeïnduseerde akrosoomreaksie en die daaropvolgende gebeure wat aanleiding gee tot bevrugting. Die zona pellucida-geïnduseerde akrosoomreaksie, sperm-zona interaksie sowel as spermmotiliteitseienskappe is ondersoek. Die eerste gedeelte van die studie fokus op die rol van ekstrasellulêreseingereguleerde- kinase (ERK), 'n lid van die familie van mitogeen-geaktiveerde proteïenkinases, tydens die zona pellucida-geïnduseerde akrosoomreaksie en sperm-oosiet interaksie. Daar word aangetoon dat die inhibisie van ERK die zona pellucida geïnduseerde akrosoomreaksie, soos gemeet met behulp van fluorosensie mikroskopie, betekenisvol verminder. Dit suggereer dat ERK direk of indirek betrokke is by die seintransduksie paaie waardeur die akrosoomreaksie van die menslike sperm deur die zona pellucida geïnduseer word. In 'n poging om onomwonde te bewys dat ERK betrokke is by menslike akrosoom-seintransduksie, is hemizona essais gebruik om sperm-oesiet binding te bepaal. Van hierdie sperm-oosiet bindingeksperimente is dit duidelik dat die inhibisie van ERK aanleiding gee tot verhoogde binding. Hierdie resultate ondersteun dus die idee dat die zona pellucidageïnduseerde akrosoomreaksie en dus die fisiologies relevante eksositotiese gebeurtenis gereguleer word deur 'n ERK-gemediëerde seintransduksie proses. In die tweede gedeelte van die studie is die belang van fosfatidielinositol 3-kinase (PI3K) in die proses van menslike spermmotiliteit, akrosoomreaksie en sperm-oesiet binding ondersoek deur van die spesifieke PI3K inhibitor LY294002, gebruik te maak. Pl3K-inhibisie het die persentasie motiliteit en progressiewe motiliteit by astenozoospermiese pasiënte verhoog. Onder hierdie laboratoriumtoestande het Pl3K-inhibisie nie die akrosoomreaksie beïnvloed nie, terwyl sperm-oosiet binding verhoog is. Hierdie resultate beteken dus dat PI3K spermmotiliteit en zona-binding negatief beïnvloed. In die laaste gedeelte van die studie is die effekte van intrasllulêre cGMP akkumulasie deur akute in vivo sildenafil sitraat (ViagraTM) toediening op seminale parameters, induksie van die akrosoomreaksie, sperm-oesiet binding en spermmotiliteit ondersoek. Soos verwag is geen veranderinge in die makro- en mikroskopiese seminale parameters deur sildenafil sitraat in vergelyking met plasebo veroorsaak nie. Verder is die akrosoomreaksies ook nie deur 50mg orale sildenafil sitraat geïnisieer of potensieer nie. Sperm-oosiet binding, geplaneerde snelheid, reguitlyn snelheid en persentasie vinnigbewegende selle was almal vehoog na sildenafil sitraat behandeling. Uit hierdie resultate blyk dit dat daar verskeie rolspelers in die zona pellucidageïnduseerde akrosoomreaksie is. 'n Deeglike insig van al hierdie seintransduksiepaaie en die onderlinge kruiskontak tussen mekaar sal uiteindelik die nodige inligting rakende die reseptore waaraan hierdie seintransduksie paaie gekoppel is, verskaf sowel as die intrasellulêre effektore wat uiteindelik spermfunksie beheer. Nog te meer sal die begrip van hierdie regulatoriese paaie verder noodsaaklik wees vir die toekomstige ontwikkeling van kliniese benaderings om bevrugting te bevorder of te voorkom.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis. The present study initially focused on the characterization of a family of membrane anchored, cell wall associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high levels of gamma interferon secretion by T-cells in response to these proteases were observed in Mantoux positive individuals. The absence of any detectable protease activity lead to a protein sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein processing proteases. To identify possible substrates for these proteases, the genome sequence regions surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part of a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M. tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigen family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In silico analysis of finished and unfinished genome sequencing data revealed the presence of orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have established the duplication order of the gene clusters and demonstrated that gene cluster region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could be found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple duplications of this cluster have occurred and have been maintained only within the genomes of members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene family (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins (secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putative ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the observation that members of the ESAT-6 family are secreted without the normal sec-dependent secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins function together to form a transport system for the secretion of the members of the ESAT-6 protein family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was also identified and its activity characterized. Subsequent secretion analyses results have shown that secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a number of features of quorum sensing and lantibiotic operons, and an extensive review of the literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as signaling molecules and may be involved in the regulation of expression of genes during intracellular residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE gene families (members of which is found situated in the ESAT-6 gene clusters) were investigated. This investigation revealed that the expansion of these families are linked to the duplications of the ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences distinguishing these two groups of mycobacteria. In conclusion, this study has highlighted numerous important aspects of mycobacterial genomics and has greatly contributed to the current body of knowledge concerning the role of proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Children contribute little to disease transmission and the maintenance of the tuberculosis epidemic, but they constitute a significant proportion of the total tuberculosis (TB) caseload and experience considerable morbidity and mortality in endemic areas, despite the availability of cheap and effective treatment. The difficulty of diagnosing childhood tuberculosis is one of the major obstacles that hinder the provision of antituberculosis treatment to children in endemic areas. The diagnosis of childhood tuberculosis is complicated by the lack of a practical gold standard, as bacteriologic specimens are difficult to collect and the yield is low. In non-endemic countries the diagnosis of childhood tuberculosis is based on the triad of: 1) exposure to an adult index case, 2) a positive tuberculin skin test, and 3) suggestive radiographic signs. However, the triad has limited value in endemic areas where exposure to and/or infection with Mycobacterium tuberculosis is common, and chest radiography is rarely available. The objective of this dissertation was to address the diagnostic dilemma faced by health professionals in endemic areas with limited resources, where children currently have poor access to chemoprophylaxis and antituberculosis treatment. We first clarified basic disease concepts, through a critical review of the pre-chemotherapy literature that documented the natural history of childhood tuberculosis. Three central concepts were identified; 1) the importance of accurate case definition, 2) the relevance of risk stratification, and 3) the diverse spectrum of disease, which necessitates accurate disease classification. The importance of accurate case definition is illustrated by the fact that isolated hilar adenopathy, considered the principal radiographic sign of primary tuberculosis, becomes transiently visible in the majority of children following recent primary infection. Our analysis of the natural history of childhood tuberculosis allowed accurate quantification of the risk to progress to disease following primary infection with M.tuberculosis. This demonstrated that the risk depends mainly on the age and/or immune-status of the child, the time since primary infection occurred and the presence or absence of symptoms. After analysing these historic studies, we proceeded to document the burden of childhood tuberculosis in an endemic area. We first conducted a retrospective study to describe current diagnostic practices and demonstrated almost exclusive reliance on chest radiography. We then calculated the burden of childhood tuberculosis in a prospective descriptive study. The corrected tuberculosis incidence rate in children was 407/100 000/year and children with severe forms of disease, such as disseminated (miliary) tuberculosis and/or tuberculous meningitis, were rarely recorded in the TB treatment register used for routine community-based surveillance. An additional obstacle to progress in the field of childhood tuberculosis has been the lack of standard descriptive terminology. Following a careful review of the literature, we proposed a radiological classification of childhood intra-thoracic tuberculosis and explored the different pathologic mechanisms that underlie these diverse disease manifestations. We then conducted a prospective descriptive study to document the disease spectrum in children treated for tuberculosis in an endemic area. The disease patterns observed were consistent with those described in the pre-chemotherapy literature. In addition, we demonstrated that bacteriologic confirmation may be achieved in the majority of children with intra-thoracic tuberculosis, in highly endemic settings. Finally we developed a novel symptom-based approach to diagnose pulmonary tuberculosis in children from endemic areas with limited resources. We followed a step-wise approach by first conducting a community-based survey to document the prevalence of symptoms traditionally associated with tuberculosis in a random selection of children from an endemic area. The survey demonstrated that poorly defined symptoms offer poor diagnostic value. The second step was to evaluate the diagnostic value of well-defined (persistent, non-remitting) symptoms in a small prospective study. Well-defined symptoms demonstrated good diagnostic value, but these promising results required further validation. As a final step, we validated the diagnostic value of a novel symptom-based approach in a large prospective, community-based study. In this study, a simple symptom-based approach diagnosed childhood pulmonary tuberculosis with a remarkable degree of accuracy, particularly in HIV-uninfected children older than 3 years of age. This novel diagnostic approach offers the exciting prospect of extending antituberculosis treatment to children in endemic areas with limited resources, where current treatment access is poor. / AFRIKAANSE OPSOMMING: Tuberkulose beheer programme plaas feitlik geen klem op die behandeling van kinders nie, omdat kindertuberkulose selde aansteeklik is en die persepsie bestaan dat kinders slegs in raar gevalle ernstig siek word. Tuberkulose lewer egter ‘n betekenisvole bydrae tot kindermorbiditeit en mortaliteit in endemiese areas, terwyl dit ‘n maklik behandelbare siekte is. Kindertuberkulose is moeilik om te diagnoseer en dit is ‘n belangrike faktor wat daartoe bydra dat kinders dikwels nie antituberkulose behandeling ontvang wanneer hulle dit benodig nie. Die diagnose van kindertuberkulose is moeilik, omdat die organisme selde aangetoon kan word. In nie endemiese areas word kindertuberkulose dikwels gediagnoseer na aanleiding van: 1) blootstelling aan ‘n volwasse indeks geval, 2) ‘n positiewe tuberkulien veltoets, en 3) die teenwoordigheid van radiologiese tekens suggestief van tuberkulose. Hierdie benadering het defnitiewe tekortkominge in endemiese areas, waar blootstelling aan en infeksie met Mycobacterium tuberculosis algemeen is. Gevolglik berus die diagnose van kindertuberkulose hoofsaaklik op die subjektiewe interpretasie van die borskasplaat, wat welbekende tekortkominge het en verder is radiologiese toetse dikwels nie beskikbaar in hierdie areas nie. Die doel van die navorsingsprojek was om die dilemma rondom die diagnose van kindertuberkulose in endemiese areas aan te spreek. Eerstens is basiese siektekonsepte uitsorteer deur ‘n kritiese oorsig van studies uit die pre-chemoterapie era. Hierdie kosbare studies het die natuurlike verloop van tuberkulose in kinders beskryf, nog voordat antituberkulose middels beskikbaar was. Drie sentrale konsepte is geidentifiseer; 1) die belang van akkurate siekte definisie, 2) die relevansie van risiko stratifikasie en 3) die diverse spektrum van patologie wat akkurate siekte klassifikasie noodsaak. Die belang van akkurate siekte definisie word geïllustreer deur die feit dat geïsoleerde hilêre adenopatie ‘n verbygaande verskynsel is in die meerderheid van kinders kort na primêre infeksie. Ons analise het daarop gefokus om die risiko om siekte te ontwikkel nadat primêre infeksie met M.tuberculosis plaasgevind het, te kwantifiseer. Die hoof risiko faktore was; 1) die ouderdom en/of immuunstatus van die kind, 2) die tydsverloop sedert infeksie, en 3) die teenwoordigheid van simptome al dan nie. Hierna het ons die siektelas wat tuberkulose vandag op kinders in endemiese areas plaas gedokumenteer. Ons het eers die huidige diagnostiese praktyke geëvaluaeer in ‘n retrospektiewe studie en toe ‘n prospektiewe beskrywende studie gedoen om die siektelas so akkuraat as moontlik te meet. Die insidensie van kindertuberkulose was hoog (>400/100 000/jaar), selfs na korreksie vir kinders wat ontoepaslik behandeling ontvang het. Verder is gevind dat die meerderheid van kinders met ernstige siekte toestande soos miliêre tuberkulose en/of meningitis, nie in roetine moniterings data reflekteer word nie. ‘n Bykomende struikelblok in kindertuberkulose is die gebrek aan standaard beskrywende terminologie. Om dit te bevorder ontwikkel ons ‘n nuwe radiologiese klassifikasie van intra-torakale kindertuberkulose en beskryf ons die verskillende patologiese meganismes onderliggend tot hierdie uiteenlopende siektebeelde. Daarna dokumenteer ons die volledige spektum van kindertuberkulose in ‘n endemiese area en demonstreer dat die siektepatrone wat ons vandag observeer soortgelyk is aan die wat in die pre-chemoterapie literatuur beskryf is. Ons toon ook dat bakteriologiese bevestiging moontlik blyk te wees in die meerderheid van kinders wat vir intra-torakale tuberkulose behandel word in endemiese areas. Nadat ons duidelikheid verkry het oor die basiese siektekonsepte, siekte klassifikasie en die siektelading in ons omgewing, kon ons op die ontwikkeling van ‘n simptoom-gebaseerde benadering tot die diagnose van kindertuberkulose fokus. Ons het ‘n stapsgewyse benadering gevolg. Die eerste stap was om die voorkoms van simptome wat gebruiklik met tuberkulose vereenselwig word te dokumenteer in ‘n ewekansige groep kinders. Die gemeenskapsopname het getoon dat swak gedefiniëerde simptome swak diagnostiese waarde bied. Die tweede stap was om vas te stel of verbeterde simptoom definisie die diagnostiese waarde kan verbeter. ‘n Klein prospektiewe studie het getoon dat goed gedefiniëerde simptome (persisterende simptome van onlangse aankoms) goeie diagnostiese waarde bied. Die finale stap was om hierdie belowende benadering formeel te toets in ‘n groot prospektiewe, gemeenskapsgebaseerde studie. Hierdie studie het getoon dat ‘n eenvoudige simptoom-gebaseerde benadering pulmonale tuberkulose met goeie akkuraatheid kan diagnoseer, veral in HIV-ongeïnfekteerde kinders wat ouer is as 3 jaar. Hierdie nuwe diagnostiese benadering bied die moontlikheid om antituberkulose behandeling te voorsien aan kinders in endemiese areas wat tans feitlik geen behandeling ontvang nie.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Male fertility has for many years been defined in vitro as the ability of sperm to fertilize oocytes and to obtain early cleavage-stage embryos. Spermatozoa comprise of an extraordinary high percentage of polyunsaturated fatty acids in their plasma membrane. Due to an extremely low content of cytoplasm, sperm cells have a particularly low potential to scavenge reactive oxygen species (ROS), and are therefore highly sensitive to oxidative processes, which lead to sperm nucleus DNA damage/fragmentation. Normally, DNA fragmentation occurs in every ejaculate and can be induced by an excessive ROS production of active leukocytes or the spermatozoa themselves. Under distressed conditions, DNA fragmentation may also occur in the testis as a result of oxidative processes in the apoptotic cascade. These DNA fragmentations can be regarded as late signs of programmed cell death (apoptosis). Clinically, DNA fragmentation in spermatozoa results in significantly decreased implantation and pregnancy rates especially in patients with oligo- and/or teratozoospermia. The p-pattern normal sperm morphology has been shown to give poorer fertilization rates in vitro than the g- and n-patterns. In this study there is reported on the significant correlation found between the p-pattern normal sperm morphology and sperm DNA fragmentation as measured with the terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling (TUNEL) assay. This finding further explains the lower fertility potential of patients presenting with p-pattern normal sperm morphology. In addition, this study explores the intricate relations between ROS in the semen, DNA fragmentation of the spermatozoa, as measured with the TUNEL assay and the sperm chromatin structure assay (SCSA ), spermatozoa apoptotic status and sperm parameters as measured with a standard semen analysis. Positive correlations were found between ROS and the apoptotic status of the sperm, as well as between sperm with non-fragmented DNA and sperm concentration and percentage motility. The results emphasize the importance of sperm selection especially when the treatment of choice is intracytoplasmic sperm injection (ICSI). An early sign of programmed cell death, also known as apoptosis, is the externalization of phosphatidylserine (PS) from the inner membrane leaflet to the outer leaflet. PS shows a high affinity to Annexin V. Apoptotic spermatozoa are able to fertilize oocytes, but embryo senescence may occur at the time when the paternal genes are activated. In this study there is reported on a novel method whereby spermatozoa can be separated on the basis of their apoptotic status through flow cytometry. Results showed that the normal sperm morphology, according to strict criteria, of the resultant nonapoptotic sperm fraction is significantly higher than that of the apoptotic counterpart. With refinement of this technique, it will be possible in future to use these separated non-apoptotic sperm cells during ICSI for fertilization. From the above it is apparent that the spermatozoon has to play a vital role in the development of the embryo from fertilization to implantation and pregnancy. It is, however, important to note that besides the gametes, there are other critical factors which contribute to a successful in vitro fertilization (IVF) cycle, among these are the in vitro culture conditions. In this regard, this study compared two sequential embryo culture systems. It was found that the more complex medium resulted in better day three embryo quality and a better blastocyst formation rate and pregnancy rate. These findings highlight the importance of a holistic perspective towards the complexity of the factors involved in affecting embryo quality and pregnancy outcome. / AFRIKAANSE OPSOMMING: Manlike fertiliteit is vir baie jare gedefinieer as die in vitro vermoë van ‘n spermsel om ‘n eiersel te bevrug om sodoende embrios te verkry. Die spermsel se plasmamembraan bestaan uit ‘n hoë persentasie poli-onversadigde vetsure. As gevolg van die klein hoeveelhede sitoplasma van die spermsel het dit ‘n beperkte weerstand teen reaktiewe suurstof spesies (ROS) en is gevolglik baie sensitief vir oksidasie. Oksidasie lei tot DNS skade/fragmentasie. DNS fragmentasie kom in spermselle van alle ejakulate voor en is gewoonlik die gevolg van ROS produksie deur die leukosiete in die semen of vanaf die spermselle self. Onder sekere omstandighede kan DNS fragmentasie ook voorkom in die testis waar dit deel vorm van apoptose. Hierdie tipe DNS skade word gesien as laat tekens van geprogrammeerde seldood (apoptose). In oligo- en/of teratozoospermiese mans lei DNS fragmentasie tot verlaagde implantasie- en swangerskapssyfers. Die p-patroon normale sperm morfologie groep gee laer in vitro bevrugting en swangerskapsyfers as die g- en n-patrone. In hierdie studie doen ons verslag oor die statisties betekenisvolle korrelasie wat gevind is tussen die p-patroon normale sperm morfologie en DNS fragmentasie soos gemeet met die ‘terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling’ of te wel TUNEL toets. Hierdie bevinding is ‘n verdere verklaring vir die laer fertiliteits potensiaal van pasiënte wat voordoen met p-patroon sperm morfologie. ‘n Verdere doel van die studie was om die moontlike verband tussen ROS in die semen, spermatozoa DNS fragmentasie, apoptotiese status van die sperms en die motiliteits parameters van die spermatozoa te bepaal. ‘n Positiewe korrelasie is gevind tussen ROS en sperm apoptotiese status. Sperms met ongeframenteerde DNS is ook positief gekorreleer met sperm konsentrasie en motiliteit. Die resultate beklemtoon die belangrikheid van spermseleksie veral in pasiënte waar die keuse van behandeling intrasitoplasmiese sperm inspuiting (ICSI) is. ‘n Vroeë teken van apoptose is die eksternalisering van ‘phosphatidylserine’ (PS) vanaf die interne oppervlakte van die plasmamembraan na die eksterne oppervlak. PS het ‘n hoë affiniteit vir Annexin V. Apoptotiese sperms het die vermoë om ‘n oösiet te bevrug, maar kan lei tot die staking van embrio deling wanneer die vaderlike gene ‘n rol begin speel in embrio ontwikkeling. In hierdie studie het ons ‘n nuwe metode ontwikkel waarvolgens die spermatozoa in die ejakulaat op grond van hul apoptotiese status geskei kan word in apoptotiese en nie-apoptotiese fraksies. Die normale sperm morfologie van die nie-apoptotiese fraksie is betekenisvol beter as dié van die apoptotiese fraksie. Verdere verfyning van die tegniek kan daartoe lei dat dit in die toekoms toegepas kan word om vir nie-apoptotiese sperms te selekteer veral voor die uitvoering van ICSI. Uit die bogenoemde is dit duidelik dat die spermsel ‘n baie belangrike rol in die ontwikkeling van ‘n embrio, vanaf bevrugting tot implantasie en swangerskap, speel. Dit is egter ook belangrik om in gedagte te hou dat daar ander bydraende faktore tot ‘n suksesvolle in vitro swangerskap is, soos laboratorium toestande en embrio kultuursisteem. Om hierdie rede is daar ook twee kultuurmedia in hierdie studie vergelyk. Daar is bevind dat die meer komplekse medium beter kwaliteit embrios op dag drie lewer, asook meer blastosiste en ‘n hoër swangerskapsyfer. Dit is dus duidelik dat dit uiters belangrik is om ‘n holistiese perspektief te hê op die komplekse faktore wat ‘n invloed mag hê op bevrugting, embrio kwaliteit asook die swangerskapsyfer.

Thesis (D.Phil.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Chapter 1 provides literature based background information on the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the use of a sequential diagnostic schedule for couples in an assisted reproductive programme is emphasized. The author revisited the literature on chromatin packaging of spermatozoa and addresses this issue as an additional semen parameter providing information relating to DNA damaged spermatozoa. The chapter also includes evidence underlining the growing need for the implementation of the acrosome reaction as an important contribution to the assisted reproductive programme. Chapter 2 provides detailed descriptions of the material and methods used during the study. Chapter 3 is sub-divided into 5 sections, each of which represents a separate study that was prepared as a scientific paper. The study included 338 couples consulting for infertility treatment at various gynaecologists in Pretoria and Johannesburg. The diagnostic assisted reproductive laboratory support was provided by the Andrology laboratory of Drs du Buisson and partners from Pretoria. In the first study the role of chromatin packaging as an indicator of in vitro fertilization rates, the semen samples from 72 men were used to record their chromatin packaging quality as well as their sperm morphology classification. Significant different percentages CMA3staining (mean±SE) were recorded among the 2 morphology groups, namely 65.9%±3.5 and 44.5%±1.7 (p=0.001). Using cut off values for chromatin packaging established during the first study, the second study utilized semen from 140 men in the in vitro fertilization (IVF) and intracytoplasmic sperm injection programme (ICSI) to analyze for sperm concentration, motility, morphology and chromatin packaging (CMA3).IVF and ICSI data were stratified using 3 basic cut off values for CMA3staining, namely <44%, >44-60% and >60%. The study concluded that results on the chromatin packaging quality of spermatozoa could be used as an additional parameter of sperm quality since it could provide valuable information on decondensation status of a given sperm population. The third study aimed to establish zona pellucida induced acrosome reaction response (ZIAR) among 35 couples with normal and G-pattern sperm morphology and repeated poor fertilization results during assisted reproduction treatment. Interactive dot diagrams, divided patients into 2 groups i.e. ZIAR<15% and ZIAR>15% with mean fertilization rates of 49% and 79%, respectively. The study concluded that the ZIAR test has diagnostic potential, since it can assist the clinician to identify couples that will benefit from ICSI therapy. The forth study revisited the importance of micro-assay for acrosome reaction determinations in a diagnostic andrology laboratory. The micro-assay not only allows the use of a single zona pellucida, but also facilitates the future possibility of using recombinant zona pellucida proteins in a diagnostic test system. The final study in Chapter 3 includes results obtained from 49 couples (172 oocytes) and aimed to evaluate the role of chromatin packaging and sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies among 170 oocytes that failed in vitro fertilization (IVF). Odds ratio analyses indicated that being in the a group with elevated CMA3 staining i.e. >60%, the risk of decondensation failure increases 15.6 fold relative to normal CMA3 staining <44%. Chapter 4 underlines the validity of the sequential diagnostic approach and summarizes the results and value of a multistep diagnostic scheme. The chapter concludes with the recommendation that both chromatin packaging quality and zona pellucida mediation of the acrosome reaction should be part of the diagnostic tools in the assisted reproductive programme. / AFRIKAANSE OPSOMMING: Die literatuuroorsig in Hoofstuk 1 konsentreer in hoofsaak op die kliniese belang van sperm morfologie en die uitbreiding van die diagnostiese toetse en hantering van die egpaar in die reproduktiewe ondersteuningsprogram. Die kromatien pakkingskwaliteit van die spermsel word onderskryf as In belangrike toevoeging tot die diganostiese program, aangesien ONS skade dikwels saam met kromatiendefekte aangetref word. Die rol van die akrosoomreaksie word ook in detail literatuuroorsigtelik beklemtoon. Hoofstuk 2 bevat volledige inligting omtrent materiaal en metodes wat in die studie gebruik is. Hoofstuk 3 bevat die eksperimentele gegewens wat in 5 afsonderlike sub-afdelings as wetenskaplike publikasies aangebied word. Die studies bestaan uit data van 338 pasiënte, wat deur verskillende ginekoloë van Pretoria en Johannesburg gekonsulteer is waartydens drs. du Boisson en vennote van Pretoria die diagnostiese reproduktiewe laboratoriumdienste verskaf het. Die eerste studie stel dit ten doel om die belang en korrelasie van die spermsel kromatienpakkingskwaliteit van 72 mans te vergelyk met die morfologiese bou van sie sel. Aangesien morfologie reeds gevertig is as 'n kliniese voorspeller van bevrugting was dit nodig om hierdie parameter te vergelyk met die kromatienpakking van die sel. Twee afsnypunte word vir die normo-en teratozoospermiese mans identifiseer naamlik, 44.5%±1.7 en 65.9%±3.5 (p=O.001),respektiewelik. Die tweede studie gebruik die afsnypunte 44% en 66% om die in vitro bevrugting en intrasellulêre sperm inspuiting (ICSI) data te ontleed. Die resultate dui aan dat kromatienpakking In waardevolle bydrae tot die diagnostiek van die pasiënte lewer. Die derde studie stel dit ten doelom die waarde van die zona pellucida geinduseerde akrosoomreaksie (ZIAR) te bepaal. Die studie sluit die data van 35 egpare in wat almal normale of G-patroon morfologie het en verder onverklaarde swak bevrugtings resultate tydens in vitro bevrugtingsterapie. Interaktiewe punt diagram (interactive dot diagrams) verdeel die data in twee groepe naamlik, ZIAR<15% en ZIAR>15% met gemiddelde bevrugtingssyfers van 49% en 79%, respektiewelik. Die studie sluit af met die gedagte dat die ZIAR toets 'n groep pasiënte identifiseer met 'n besondere fisiologiese afwyking d.i. subnormale akrosoom respons op zona pellucida blootstelling. Die vierde afdeling van die hoofstuk onderstreep die belang van die mikro-tegniek vir die bepaling van die akrosoom reaksie, wat tydens die projek gebruik is Die vyfde afdeling van Hoofstuk 3 stel dit ten doelom 170 onbevrugde eierselle van 49 pasiënte te ontleed vir moontlike oorsake vir die mislukte bevrugting. Ondersoeke sluit in die kromatienpakking, sperm-zona binding, sperm dekondensasie en die teenwoordigheid van polêre liggaampies. Statisties blyk dit dat indien 'n kromtienpakking nie normaal is nie (>66%) het die spermsel 'n 15 keer groter kans om nie te dekondenseer nie. Hoofstuk 4 bespreek die noodsaaklikheid van die diagnostiese skedule by die hantering van die onvrugbare egpaar in.