Dissertation (PhD)--Stellenbosch University, 2007. / ENGLISH ABSTRACT: This research investigated human candidate genes for susceptibility to tuberculosis and the effect of various factors on time to sputum conversion in the admixed South African Coloured (SAC) population. Population stratification was formally tested and excluded. Population based casecontrol studies were the primary analysis method with a variety of genotyping methods. Candidate polymorphisms in RANTES, CCR5, CCR2 and SDF1, were not associated with tuberculosis susceptibility. Initially the RANTES polymorphism -403 was found to be associated with tuberculosis susceptibility but after the testing of additional samples the association was lost, illustrating the challenges with association studies. The C-type lectins DC-SIGN, encoded by the gene CD209, and L-SIGN are important pathogen-recognition receptors of the human innate immune response. Both lectins have been shown to interact with Mycobacterium tuberculosis. CD209 promoter polymorphisms, -336 and - 871, were both found to be associated with tuberculosis susceptibility. The haplotype containing CD209 -871G and -336A was strongly associated with the control group. The CD209 -336A allele has been found to be associated with increased DC-SIGN expression, which may be the underlying reason for an increased efficiency of host phagocytes. Susceptibility to tuberculosis in mice has recently been attributed to the Ipr1 gene. Eight polymorphisms in the human homologue, SP110, were investigated, including two novel polymorphisms. No significant associations were found with any of the polymorphisms investigated, including two polymorphisms that were previously found to be associated with tuberculosis susceptibility in West African populations. A cohort of 249 cases from a longitudinal study of first time pulmonary tuberculosis patients was available. The cohort was used to investigate if the vitamin D receptor gene (VDR) polymorphisms FokI, ApaI and TaqI were associated with tuberculosis susceptibility or time to sputum conversion, and to investigate other clinical and demographic factors affecting the rate of response to treatment. No association between the VDR genotype and tuberculosis was found in the case-control study. The cohort allowed for a reliable conversion time to be determined for smear (n=220) and culture (n=222). Analysis was carried out to determine which factors, including VDR FokI, ApaI, and TaqI genotypes, contribute to faster mycobacterial resolution in sputum. This was done by survival curves and Cox regression models. The results indicate that the extent of disease at diagnosis was predictive of both smear and culture conversion times in the final models. Smoking status and VDR genotype contributed independently to smear conversion time, with ApaI ‘AA’ and TaqI ‘T’ containing genotypes being predictive of a faster response to tuberculosis therapy. We can conclude that the time taken for an individual to convert to sputum negativity while on DOTS therapy, can be independently predicted by the VDR genotype. This may have implications for future immunomodulatory therapies. Identifying what contributes to susceptibility to tuberculosis will provide us with a better understanding of the human immune response to tuberculosis which may lead to the development of accurately targeted therapeutics and vaccines. / AFRIKAANSE OPSOMMING: Kandidaatgene vir die vatbaarheid vir tuberkulose en die effek van verskeie faktore op sputum oorgangstyd was in hierdie navorsingsstudie ondersoek in die Suid-Afrikaanse Kleurlingbevolking (SAC). Dié bevolking was ook getoets vir populasie-stratifikasie, waarvan daar geen bewyse gevind is nie. Populasiegebaseerde pasiënt-kontrole studies was die primêre metode van analise en verskeie genotipering metodes was gebruik. Polimorfismes in kandidaatgene soos RANTES, CCR5, CCR2 en SDF1 was nie met die vatbaarheid van tuberkulose geassosieer nie. Oorspronklik was daar ‘n assosiasie met die RANTES -403 polimorfisme, maar met die genotipering van addisionele individue het die assosiasie verdwyn. Resultate verkry vir die polimorfisme illustreer die uitdagings waaraan assosiasie studies onderworpe is. Die C-tipe lektiene DC-SIGN, wat gekodeer word deur CD209, en L-SIGN is belangrike patogeen herkenningsreseptore in die aangebore immuunreaksie. Interaksies tussen beide lektiene en Mycobacterium tuberculosis is voorheen gerapporteer. Die CD209 promoter polimorfismes, -336 en -871, was met die vatbaarheid van tuberkulose geassosieer. ‘n Haplotipe bestaande uit die CD209 -871G en -336A allele was sterk met die kontrole groep geassosieer. Die CD209 -336A alleel was geassosieer met ‘n toename in die DC-SIGN proteïen vlakke, wat moontlik ‘n onderliggende rede is vir die toename in die effektiwiteit van die gasheer se fagosiete. Vatbaarheid vir tuberkulose is onlangs toegeskryf aan die Ipr1 geen in muise. Agt polimorfismes, insluitend 2 voorheen onbekendes, was in die mens homoloog SP110 bestudeer. Geen positiewe beduidende assosiasie was met enige van die polimorfismes gevind nie ten spyte van die feit dat twee van hierdie polimorfismes voorheen met tuberkulose vatbaarheid geassosieer was in bevolkings van Wes-Afrika. ‘n Versameling van 249 TB pasiënte van ‘n longitudinale studie was beskikbaar. Dié groep was gebruik om polimorfismes FokI, ApaI and TaqI in die vitamien D reseptor geen (VDR) te bestudeer ten opsigte van vatbaarheid vir tuberkulose of sputum oorgangstyd sowel as ander kliniese en demografiese faktore wat die tempo van respons op behandeling kan affekteer. In hierdie studie was daar geen assosiasie gevind tussen die ontwikkeling van tuberkulose en die VDR genotipes nie. Die bepaling van ‘n betroubare oorgangstyd vir beide smeer en kultuur van die groep was moontlik. Analises was uitgevoer om te bepaal watter faktore bydrae tot vinniger resolusie van Mycobacteria in sputum. Resultate verkry het aangedui dat die aard van die siekte tydens diagnose voorspelbaar was van die oorgangstye van beide smeer en kultuur in die finale modelle. Die rookstatus van individue sowel as die VDR genotipes het onafhanklik bygedrae tot die oorgangstyd van die smeer, met ApaI ‘AA’ en TaqI ‘T’ bevattende genotipes wat ‘n vinniger reaksie op tuberkulose behandeling voorspel het. Ter opsomming, die tyd wat dit ‘n individu op DOTS terapie neem om na sputum negatief oor te gaan kan onafhanklik deur die VDR genotipe voorspel word. Dit kan moontlik implikasies hê vir ander immunomodulerende terapië in die toekoms. Die identifisering van faktore wat bydra tot die vatbaarheid van turberkulose sal ons in staat stel om ‘n beter begrip te hê van die immuunrespons teen tuberkulose wat moontlik kan lei tot die ontwikkeling van akkurate behandelings en inentings.

The accurate differentiation of a ventricular from a supraventricular origin of a wide QRS tachycardia (QRS > 120 milliseconds) is an important clinical problem. Misdiagnosis of this arrhythmia can lead to institution of inappropriate drug therapy acutely with potentially catastrophic consequences. Various diagnostic aids have been used to obtain electrocardiographic potentials to aid in the differentiation. This report assesses the clinical usefulness of oesophageal electrocardiography in the differentiation of wide complex tachycardias and describes a simple, safe technique to obtain oesophageal electrocardiograms. Eighteen consecutive patients between the ages of 27 and 71 years who were haemodynamically stable were selected for this study. The technique was performed in the following manner: A temporary pacing catheter was lubricated and passed nasally and advanced with the patient being instructed to swallow. Adjustments in catheter depth were made as necessary to obtain an optimal recording on a standard electrocardiograph recorder. Satisfactory placement with minimal patient discomfort was achieved within 6.5 minutes (average 4.5 minutes) in all cases. High quality tracings were obtained in every instance. In the 18 patients with tachyarrhythmia, AV dissociation consistent with ventricular tachycardia was demonstrated in 11 instances; in the remainder the diagnosis was supraventricular tachycardia. Of the 11 patients diagnosed as ventricular tachycardia, 9 were initially misdiagnosed as supraventricular tachycardia, whilst only 1 of 7 patients with supraventricular tachycardia was misdiagnosed. This study has demonstrated that oesophageal electrocardiography is useful in the differentiation of wide complex tachycardias. The technique outlined in this report is simple and offers the following advantages: the temporary pacing catheter is associated with minimal discomfort; the catheter allows easy manoeuverability within the oesophagus which allows proper depth to be easily obtained; the equipment used is routinely available. Therefore the technique offers a rapid, safe and simple method of obtaining an oesophageal electrocardiogram which is invaluable in the electrocardiographic differentiation of a wide complex tachycardia. / Thesis (M.Med.)-University of Natal, Durban, 1990.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: SEPARATION STUDY Traumatic experiences during childhood can have a negative impact on behaviour later in life. Kendier et al. (1992) found that the loss of a parent during childhood increased the risk to develop major anxiety disorders and could also lead to depressive-like behaviour (Furukawa et al., 1999). Methods: We subjected rat pups to maternal separation and determined the effects thereof on adult behaviour. We removed rat pups from their mothers for 3 hours daily from postnatal day 2 to 14. On day 60, the behaviours of the rats were tested using the elevated plus-maze and the open field test. Controls were reared normally. Behaviours: Amount of time spent and the number of entries into the arms of the maze were noted on the elevated plus-maze, while the total time spent in each zone (inner versus outer) and the number of zone crossings were noted for each rat on the open field arena. The latency to move from the initial placement in the outer zone to the inner zone as well as the number of quadrant crossings was also determined. Defecation, freezing, rearing and grooming behaviours were also noted. Neurotransmitter levels: Noradrenaline, serotonin and their metabolites were evaluated in maternally separated rats and compared to controls. Their concentrations at basal level, immediately after restraint stress and 15 minutes after restraint stress, were also determined. A HPLC method was followed in these determinations. ACTH Determinations: All rats were subjected to restraint stress for a lO-minute period. Trunk blood was collected for basal, as well as 15 and 60 minutes postrestraint stress for ACTH determinations. Results: Behaviours: The amount of entries was significantly reduced in the separated animals, indicating decreased locomotion. They spent significantly more time in the closed maze arms. A significant increase in defecation frequency and rearing behaviour was noted. These observations are typical of anxious behaviour. In the open field test, the behavioural results were less convincing. Only a significant increase in defecation frequency and a significant decrease in rearing behaviour in separated animals, were observed. Neurotransmitter levels: No significant differences were noted between separated animals and controls with respect to basal monoamine levels. However, noradrenaline levels were significantly decreased in the frontal cortex 15 minutes after restraint stress and immediately after restraint stress in the hypothalamus and hippocampus in separated animals. MHPG levels were significantly decreased in the frontal cortex immediately after restraint stress. No significant differences were found with respect to serotonin levels. However, significant increases were found in 5HIAA levels in the frontal cortex and hippocampus of separated rats, 15 minutes after restraint stress. The basal turnover ratios of serotonin (5HIAA/5HT) and noradrenaline (MHPGINA) did not yield significant results. However, immediately after restraint stress, a significant increase was found in serotonin turnover in the hypothalamus of separated rats when compared to controls. This turnover rate was also increased in separated rats, 15 minutes after restraint stress in the frontal cortex and hypothalamus. ACTH Determinations: Basal ACTH levels were significantly higher in separated animals. At 15 minutes post-restraint stress, the levels were significantly lower than controls, indicating a blunted stress response. Our results therefore showed that maternal separation could lead to anxious behaviours in adult life. These behavioural abnormalities were associated with alterations in the central nervous and neuroendocrinological systems, particularly in response to stressful situations. CRF STUDY The maternal separation study indicated that elevated CRF levels could possibly be causally related to abnormalities observed in the anxious animals. We therefore hypothesised that adverse development factors, such as maternal separation, predisposes individuals to develop psychopathologies later in life and that this process was driven by a presence of high CRF levels. Methods: Cannulas were implanted into the left lateral ventricles of normal rats, making use of stereotaxic procedures. CRF (3 flg/fll) was injected into the ventricles daily for 5 days. Saline controls were handled similarly, but only injected with saline for the same time period. Both groups of animals were then compared to naïve controls. Histology was performed to determine the correct placement of the cannulas. Behaviours: The Elevated Plus-maze was employed to determine whether their behaviours were anxious. The number of entries into the various arms of the maze as well as the amount of time spent in the open and closed arms was accumulated. Rearing, freezing, defecation and grooming were also noted. ACTH Determinations: The ACTH levels ofCRF-injected, saline-injected and naïve rats were determined 15 minutes after restraint stress. Results: Behaviours: A decrease in the number of entries into the closed arms of the maze was noted in the CRF-injected rats when compared to naïve controls. No significant differences were found between the groups with respect to the amount of time spent in the various arms and the behaviours noted during the experiment. ACTH Determinations: A decrease in ACTH levels was noted in CRF-injected rats 15 minutes after restraint stress when compared to naïve controls. Therefore, although the CRF injections did not alter the behaviour of the rat, they did exhibit a blunted stress response to the stressor. Conclusion: Our experiments led us to conclude that early adverse experiences, such as maternal separation, can lead to the development of psychopathologies later in life. CRF, however, is not pivotal in the development of these abnormalities; rather it seems that the neurochemical abnormalities (serotonin and noradrenaline) play a more important role in the development of these mental disturbances. Finally, we hypothesise that combination drug therapy that targets both the noradrenergic and serotonergic neurotransmitter systems could be preferred above those aimed at rectifying the individual neurotransmitter systems in the treatment of psychopathologies, such as anxiety disorders. / AFRIKAANSE OPSOMMING: MOEDERLIKE SKEIDINGS STUDIE Traumatiese gebeurtenisse wat gedurende kinderjare ervaar word, kan 'n negatiewe impak op die gedrag van dieselfde individue hê, as hulle volwassenheid bereik het. Kendier et al. (1992) het waargeneem dat die verlies van 'n ouer tydens die kinderjare, die risiko om angssteumisse te ontwikkel, dramaties verhoog en kan ook lei tot 'n depressiewe gemoedtoestand (Furukawa et al., 1999). Metodes: Ons het neonatale rotte aan moederlike skeiding blootgestel en die effekte daarvan op gedrag tydens hul volwasse lewe beoordeel. Ons het daagliks die moeders vir 3 ure van die kleintjies afweggeneem, vanafpostnatale dag 2 tot 14. Op dag 60, het ons die gedrag van die diere op die "elevated plus-maze" en die" open field test" getoets. Kontrole rotte het onder normale omstandighede opgegroei. Gedrag parameters: Die hoeveelheid tyd en aantal kere wat die rotte in die verskillende arms van die "elevated plus-maze" gespandeer het, was waargeneem. Die totale tyd in die "open field" toets se binneste ofbuitenste sones, die hoeveelheid kruisings tussen die twee sones, die tyd wat dit neem om beweging in die binneste sone te inisiëer, sowel as die hoeveelheid kwadrante wat gekruis was, is genotuleer. Defekasie, botstilstande, steiering, en versorgingsgedragte was ook waargeneem terwyl die rotte in die doolhowe was. Neurochemiese oordragstowwe: Die hippokampus, hipotalamus en frontale korteks van moerderlik-geskeide rotte en kontroles, was uit hul brein gedissekteer om die vlakke van noradrenalien, serotonien en hul metaboliete daarin te bepaal. Basale vlakke sowel as hul konsentrasies onmiddelik na stres en 15 minute na stres, was gedetermineer. 'n HPLC metode was gebruik vir hierdie bepalings. ACTH bepalings: Rotte, moederlik-geskei en kontroles, was onderwerp aan beperkingstres vir 'n tydsduur van 10 minute. Bloed was op die volgende tydsintervalle gekollekteer vir die bepaling van ACTH vlakke, naamlik basaal, 15 minute en 60 minute na die einde van stresperiode. Resultate: Gedrag: Op die "elevated plus-maze" was moederlik-geskeide rotte minder beweeglik omdat hul aanmerklik minder die arms van die doolhowe binne gegaan het. Hulle het ook baie meer tyd in die geslote arms gespandeer. Verder het die eksperimentele rotte meer defekasie bolusse uitgeskei en was die aantal steieringe uitgevoer, ook aanmerklik verhoog. Hierdie patroon van gedrag is tipies die van angstigheid. Neurochemiese oordragstowwe: Daar was geen betekenisvolle verskil tussen die basale neurotransmitter vlakke van moederlik-geskeide rotte en hul kontroles. Daarenteen was die vlakke van noradrenalien in die frontale korteks dramaties verhoog by die 15 minute tydsinterval na die stres, asook onmiddelik na die stres in die hipotalamus en hippokampus. MHPG vlakke was egter aanmerklik verlaag in die frontale korteks onmiddelik na die stres. Terwyl daar geen noemenswaardige verskil in serotonien vlakke waargeneem is nie, was die vlakke van 5HlAA betekenisvol verhoog in die frontale korteks en hippokampus van moederlik-geskeide rotte, 15 minute na die beperkingstres. Geen verskil in die omsettingsverhoudinge van basale serotonien (5HlAA/5HT) ofnoradrenalien (MHPGINA) vlakke is gevind nie. Daar was egter 'n betekenisvolle verhoging in die serotonien omset in die hipotalamus van moerdlik-geskeide rotte, onmiddelik na beperkingstres. Hierdie verskil het ook voorgekom 15 minute na die stresperiode in die hipotalamus, sowel as in die frontale korteks. ACTH bepalings: Rotte wat onderwerp was aan moederlike skeiding het verhoogde basale konsentrasies van ACTH getoon. Die ACTH vlakke was egter aanmerklik laer 15 minute na stres toe dit met kontrole groepe vergelyk is. Ons resultate toon dus dat moerderlike-skeiding wel tot angstige gedrag tydens die volwasse lewe kan lei. Hierdie afwyking in gedrag was geassosieër met abnormaliteite in die sentrale senuwee sisteem sowel as die neuroendokrienologiese sisteem van die dier, veralonder toestande van stres. Na gelang van ons bevindinge in die moerderlike skeidingstudie, het dit geblyk dat CRF 'n belangrike rol speel tot daarstelling van angstige gedrag. Daarom het ons in die tweede deel van ons studie gaan kyk ofverhoogde vlakke van CRF in die brein moontlik die gedrag van die rot kon verander. CRF STUDIE Metodes: Kannules was in die linker ventrikel van die breine van normale rotte geïmplanteer deur gebruik te maak van stereotaktiese prosedures. CRF (3 Ilg/IlI) was daagliks vir 5 dae aan die rotte toegedien. Rotte wat presies dieselfde gehanteer was het 'n fisiologiese soutoplossing ontvang. Hierdie rotte was met naïewe rotte vergelyk. Die korrekte plasing van kannules was met histologiese metodes bevestig. Gedrag: Die "elevated plus-maze" was gebruik om te bepaal of angstige gedragte by behandelde rotte ontlok was. Die aantal kere wat 'n rot die verskillende arms van die doolhofbinne gaan, sowel as die tyd wat die dier op elke arm deurbring was genotuleer. Die aantal steierings, botstilstande, defekasies en versorgingsbewegings was weereens waargeneem. ACTH bepalings: Die vlakke van ACTH was bepaal in al die rotgroepe, 15 minute nadat hulle aan 10 minute beperkingstres onderwerp was. Resultate: Gedrag: Rotte wat met CRF toegedien was, het op minder geleenthede die toe arms van die "elevated plus-maze" binne gegaan toe hulle met die naïewe groep rotte vergelyk was. Hierdie verskil was betekenisvol. Daar was geen ander noemenswaardige verskille ten opsigte van die ander gedragsparameter nie. ACTH bepalings: Daar was 'n afname in die ACTH vlakke, 15 minute na die stres toegedien was in rotte wat CRF ontvang het, in vergelyking tot die naïewe kontrole groep. Hierdie resultate dui daarop dat die toediening van CRF in die brein nie die rot se gedrag, maar wel die dier se respons op stres, beïnvloed het. Gevolgtrekking: In die lig van die voorafgaande resultate verky, blyk dit dat moederlike-skeiding tydens die vroeë kinderjare wel kan aanleiding gee tot angstige gedrag tydens volwassenheid. Ons studies dui ook aan dat CRF nie die primêre bron van hierdie gedrags afwykings is nie, maar dat abnormaliteite in die neurochemiese oordragstowwe (serotonien en noradrenalien) eerder die bepalende faktore is. Ten slotte, ons beveel aan dat geneesmiddels wat geskoei is om die serotonerge sowel as die noradrenerge sisteme aan te spreek, voordeel moet geniet in die behandeling van gedragstoomisse, soos angs.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is overwhelmingly prevalent in Botswana and South Africa and to date no interventions have been successful enough to curb the rapid spread of the virus. A number of HIV-1 vaccine strategies are being developed, however the breadth and efficacy of such candidate vaccines, many of which are based on the HIV-1 structural genes pol, gag and env, have mostly been found to be inadequate. The HIV-1 accessory genes are attractive components of HIV vaccines due to their role in viral pathogenesis, early expression and the high ratio of conserved CTl epitopes. Yet, because of undesirable properties questions regarding their safety as vaccine components are raised. In this study candidate tat, rev and nefmutants were assessed for efficient expression and inactivation of undesirable functionality. / Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat, Rev and Nef proteins were constructed. The coding sequences of the genes were codon-optimised for optimum protein expression and these synthetic genes were constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins were mutated at previously described sites by PCR-based site-directed mutagenesis to render them inactive for their respective functions. Corresponding wild-type Tat, Rev and Nef constructs were also made from viral isolates that were least dissimilar to the respective consensus amino acid sequences. tn vitro expression of the different constructs were assessed in 293 cells by Western blotting with polyclonal mouse sera, which were generated by DNA immunisation with one of the Tat, Rev and Nef constructs. The transactivation activity of Tat variants and Rev-mediated nuclear export activity of RRE-containing transcripts were studied in cotransfection experiments using reporter-gene-based assays while Nef functionality was assessed in a cotransfection assay with subsequent flow cytometric analysis of surface CD4 and MHC-I expression on 293 cells. Sequence analysis of the South African HIV-1 subtype C consensus sequences of Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence that was drawn up from a large number of viruses from southern Africa. These consensus sequences were also closer to individual viral isolate sequences than any individual sequences were, indicating that the use of a consensus sequence may serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not significantly higher than the wild-type constructs, however, the codon-optimised rev mutant exhibited markedly higher expression than the wild-type rev construct. Immunoreactivity of the protein with the mouse sera demonstrates expression and immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent CAT expression in 293T and Hela cells. Yet, this activity was significantly impaired using the single mutation, C3?, or the double mutation, C22C3? Compared to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4 downregulation was completely abrogated in one of the mutants, while both Nef mutants were entirely deficient for MHC-I downregulation. These data demonstrate the high expression levels and impaired functionality of sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens that may be used as single-standing vaccine components or form part of a multicomponent HIV-1 vaccine. / AFRIKAANSE OPSOMMING: Sedert die eerste gevalle van MIV in die vroeë 1980's beskryf is het die virus wêreldwyd versprei en 'n beraamde 28.1 miljoen mense in sub-Sahara Afrika was teen die einde van 2001 geïnfekteer. MIV-1 subtipe C kom verreweg die meeste voor in Botswana en Suid-Afrika en tans is daar geen suksesvolle tussenkoms wat die vinnige verspreiding van die virus kan stuit nie. 'n Aantal MIV-1 subtipe C entstofstrategieë word tans ontwikkel maar die spektrum en effektiwiteit van sulke entstowwe, waarvan baie op die MIV strukturele gene gag, pol en env gebaseer is, is tans onvoldoende. Die MIV-1 bykomstige gene is aantreklike entstofkomponente omdat hulle vroeg uitgedruk word, 'n belangrike rol in virale patogenese speel en omdat hulle 'n hoë verhouding van gekonserveerde sitotoksiese T-limfosiet (STL) epitope tot grootte besit. Vanweë hierdie gene se verskeie ongewenste eienskappe word vrae ten opsigte van hul veilige insluiting in enstofstrategieë geopper. Hierdie studie omskryf die evaluasie van kandidaat tat, reven nef mutante vir doeltreffende proteïenuitdrukking en funksionele onaktiwiteit. Plasmiedkonstrukte wat vir die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat, Rev en Nef proteïene kodeer is saamgestel. Die koderingsvolgordes van die gene is geoptimiseer vir optimale uitdrukking en die sintetiese gene is van oorvleuelende 50- mer oligonukleotiede vervaardig. Deur van PKR-gebaseerde site-directed mutagenese gebruik te maak is hierdie proteïene gemuteer op posisies wat voorheen geïdentifiseer is. Ooreenstemmende wilde-tipe Tat, Reven Nef konstrukte is gemaak vanaf virale isolate waarvan die aminosuurvolgordes die meeste ooreenstem met dié van die konsensusvolgorde. In vitro uitdrukking van die konstrukte in 293 selle is met behulp van immunoklad met poliklonale muissera bepaal. Die serum is gegenereer deur DNS immunisasie van muise met een elk van die Tat, Reven Nef konstrukte. Die transaktiverings-aktiwiteit van Tat variante en Rev bemiddelde uitvoer van RREbesittende transkripte uit die nukleus is in verklikkergeen kotransfeksie-eksperimente bestudeer. Nef se funksionaliteit is deur kotransfeksie en die daaropvolgende vloeisitometriese analise van 293 selle se oppervlak-CD4 en MHC-I uitdrukking bestudeer. Nukleotiedvolgorde-analise van die Suid-Afrikaanse MIV-1 subtipe C konsensus Tat, Reven Nef proteiëne toon 'n hoë vlak van ooreenkoms met 'n konsensusvolgorde wat afgelei is vanaf 'n groot aantal suider-Afrikaanse virusse. Hierdie konsensusvolgordes is ook meer soortgelyk aan individuele virale isolate as enige individuele volgordes. Vanuit hierdie data kan afgelei word dat die gebruik van so 'n konsensusvolgorde die genetiese diversiteit tussen 'n entstof en sirkuierende virusse kan verminder. Uitdrukkingsvlakke van die volgorde-geoptimiseerde tat en nef geenkonstrukte is nie merkbaar hoër as die van die wilde-tipe konstrukte nie. In teenstelling het die volgorde-geoptimiseerde rev mutant merkbaar hoër uitdrukkingsvlakke as die wildetipe getoon. Immunoreaktiwiteit van die proteïene met die muissera demonstreer dat die Tat, Reven Nef proteïene uitgedruk word en immunogenies in muise is. 'n Enkele C22 mutasie in Tat is nie genoeg om lTR-afhanklike CAT uitdrukking in 293T en Hela selle te inaktiveer nie. In teenstelling is hierdie aktiwiteit geïnhibeer vir Tat proteïene met die enkel mutasie C37 en die dubbel mutasie C22C37. In vergelyking met die funksionele aktiwiteit van die wilde-tipe Rev is dié van die Rev mutant M5M10 heeltemal geïnhibeer. Die wilde-tipe en geoptimiseerde, konsensus Nef proteïene het seloppervlak-CD4 en -MHC-I uitdrukking verlaag, maar hierdie effek van afregulering van CD4 uitdrukking was heeltemaal opgehef in een Nef mutant en van MHC-I uitdrukking in beide Nef mutante. Hierdie data demonstreer die hoë uitdrukkingsvlakke en geïnhibeerde funksionaliteit van volgorde-gemodifiseerde MIV-1 subtipe C konsensus Tat, Reven Nef DNS immunogene wat as enkelstaande enstof kan optree of deel kan uitmaak van 'n multi-komponent MIV-1 entstof.

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 (MD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Resistance to antituberculosis agents became evident soon after antituberculosis treatment was introduced for the first time. Combined drug therapy seemed to resolve this problem. Animal experimental studies, which showed that isoniazid (INH)-resistant strains of Mycobacterium tuberculosis were less infectious and pathogenic than drugsusceptible strains, gave further reassurance that drug resistance was not a major issue. Transmission of INH- and multiple-drug-resistant strains did, however, occur. Studies in children, who develop mainly primary drug resistant tuberculosis (TB), showed that drug resistance in adults was followed by a similar rise in drug-resistant (TB) in children, and that tuberculous infection rates in childhood contacts of INHresistant and drug-susceptible adult TB cases were the same. It was however, only after the significant rise in the incidence of TB and large outbreaks of multidrug-resistant (MDR) TB cases in developed countries (mainly because of the human immunodeficiency virus epidemic) in the early nineties that sufficient attention was again focussed on the problem of drug-resistant TB. Drugresistant tuberculosis, and more in particular MDR TB, posed a serious threat to global TB control programmes. Despite this renewed interest, childhood drug-resistant TB remained neglected. The incidence of drug-resistant TB among children, which could give a good indication of currently circulating strains in a community, is hardly known. The management of childhood contacts of adults with infectious MDR TB or children with MDR TB has also not been studied prospectively. All confirmed childhood TB cases from a specific geographic drainage area over a 3.5-year period were prospectively included in a drug resistance surveillance study. The incidence of drug resistance in children was comparable to the incidence of initial (primary plus undisclosed previous treatment) drug resistance documented in adults in the same area. The findings show that the incidence of drug-resistant TB in children in the Western Cape province is low, and probably reflects the level of primary drug resistance amongst organisms currently circulating in this community. The short- and long-term outcome of children <5 years of age in contact with infectious adult MDR TB cases was determined by prospective follow-up for 30 months. The initial evaluation showed an infection rate significantly higher in MDR TB contacts compared with contacts of drug susceptible cases, but the disease rate was lower. On follow-up, many more children became infected or developed disease. The finding that 90% of those who developed disease did so within the first 12 months, indicates that follow-up beyond 12 months is probably not cost-effective in resource poor countries. The results demonstrate that MDR TB is not less infectious than drug susceptible TB. Despite the fact that some children received chemoprophylaxis, 24% of the children eventually developed disease. This is not different from the expected prevalence of disease in childhood contacts <5 years of age of infectious drug-susceptible adult pulmonary TB cases. Restriction fragment length polymorphism analysis confirmed transmission from an adult source case to a child contact in 5 of 6 adult-child pairs in whom both isolates were available. If therefore an isolate of M tuberculosis for susceptibility testing cannot be obtained from a child in close contact with an infectious MDR TB case, the child should therefore be treated according to the drug susceptibility pattern of the source case's strain. Treatment of children with confirmed and probable MDR TB included 2 or 3 drugs to which the adult source case's isolate was susceptible in addition to pyrazinamide and high-dose INH. Duration of treatment ranged from 6 to 12 months depending on the severity of the disease. INH was included in the treatment regimen because low-level resistance to INH was present in about half the cases of primary INH resistance. The pharmacokinetics of INH in children confirmed that an adequate concentration and exposure time could be achieved for this purpose. Ethionamide often caused gastrointestinal adverse events, but these could be overcome in most cases by temporary dose adjustments. The fluoroquinolones, which are not generally recommended for use in children, possibly caused arthralgia in 1 of the17 children treated for ~6 months. This is in accordance with previous reports of the safety of these drugs in children for short- and medium-term treatment. TB disease occurred significantly less often in children who received appropriate chemoprophylaxis (according to the drug susceptibility pattern of the adult source case's isolate). Although this was not a randomised controlled trial, the group that received chemoprophylaxis was at higher risk for developing disease. This implies that prevention of TB in MDR contacts is possible. A prospective, randomised controlled study is necessary to evaluate the best drug combinations and the optimal duration of such chemoprophylactic regimens. / AFRIKAANSE OPSOMMING: Middelweerstandigheid het na vore gekom kort nadat antituberkulose behandeling vir die eerste keer in gebruik geneem is. Die gekombineerde gebruik van middels het klaarblyklik die probleem oorkom. Diere eksperimente wat getoon het dat isoniasied (INH)-weerstandige stamme van Mycobacterium tuberculosis minder infektief en patogenies IS as vatbare stamme, het verdere gerustelling gegee dat middelweerstandigheid nie 'n groot probleem is nie. Die oordrag van INH- en multi-middelweerstandige stamme het egter wel plaasgevind. Studies in kinders, wat hoofsaaklik primêre middelweerstandige tuberkulose (TB) ontwikkel, het getoon dat middelweerstandigheid in volwassenes gevolg is deur 'n soortgelyke toename in middelweerstandige TB in kinders en dat die voorkoms van tuberkuleuse infeksie in kinderkontakte van INH-weerstandige en middelvatbare volwasse TB gevalle dieselfde is. Dis egter eers toe daar 'n beduidende toename in die insidensie van TB en groot uitbrake van multimiddelweerstandige (MDR) TB gevalle in die ontwikkelde lande (hoofsaaklik as gevolg van die menslike immuungebrek virus epidemie) in die vroeë negentigerjare was dat daar opnuut aandag aan die probleem van weerstandige TB geskenk is. Middelweerstandige TB, en in besonder MDR TB, hou 'n ernstige bedreiging vir globale TB beheerprogramme in. Tenspyte van die nuwe belangstelling in middelweerstandige TB is die probleem in kinders steeds afgeskeep. Die insidensie van weerstandige TB in kinders is onbekend alhoewel dit 'n goeie weergawe van die huidig sirkuIerende stamme in 'n gemeenskap sou gee. Die hantering van kinderkontakte van volwassenes met infektiewe MDR TB of kinders met MDR TB is ook nog nie prospektiefbestudeer nie. Alle bevestigde kinder-TB gevalle van 'n spesifieke geografiese gebied is oor 'n 3.5 jaar tydperk prospektief in 'n middelweerstandige waarnemingstudie ingesluit. Die insidensie van middelweerstandigheid in kinders was vergelykbaar met die insidensie van inisiële (primêre weerstandigheid plus onbekende vonge behandeling) middelweerstandigheid in volwassenes van dieselfde gebied. Die bevindinge toon dat die insidensie van middelweerstandige TB in kinders in die Weskaap provinsie laag is. Dit weerspieël waarskynlik die vlak van primêre middelweerstandigheid in organismes wat tans in hierdie gemeenskap sirkuleer. Die kort- en langtermyn uitkoms van kinders <5 jaar oud wat in kontak met infektiewe volwasse MDR TB gevalle was, is prospektief tydens 'n 30-maande opvolg bepaal. Die aanvanklike evaluasie het 'n beduidend hoër infeksiekoers in die MDR TB kontakte in vergelyking met kontakte van middelvatbare gevalle getoon, maar die siektekoers was laer. Tydens die opvolgperiode het baie meer kinders infeksie of siekte ontwikkel. Aangesien 90% van dié wat siekte ontwikkel het, dit gekry het binne die eerste 12 maande, is opvolg ná 12 maande waarskynlik nie koste-effektief in hulpbronbeperkte lande nie. Die bevindinge toon dat MDR TB nie minder infektief is as middelvatbare TB nie. Tenspyte daarvan dat sommige kinders chemoprofilakse ontvang het, het 24% van die kinders uiteindelik siekte ontwikkel. Dit verskil nie van die verwagte siekte-insidensie van kinderkontakte <5 jaar oud wat in kontak met infektiewe volwasse middelvatbare pulmonale TB was nie. Restriksie fragment lengte polimorfisme analise het oordrag van volwasse brongeval na kinderkontak in 5 uit 6 volwasse-kind pare, van wie beide isolate beskikbaar was, bevestig. Indien daar dus nie 'n isolaat van M. tuberculosis vir vatbaarheidstoetse van 'n kind met nabye kontak met 'n infektiewe MDR TB geval beskikbaar is nie, behoort die kind volgens die middelvatbaarheidspatroon van die brongeval se stam behandel te word. Behandeling van kinders met bevestigde of waarskynlike MDR TB het 2 tot 3 middels waarvoor die volwasse brongeval se isolaat vatbaar was, ingesluit, tesame met pirasinamied en hoë-dosis INH. Die duur van behandeling het gewissel van 6 tot 12 maande op grond van die omvang van die siekte. INH is in die behandeling ingesluit omdat dit getoon is dat ongeveer die helfte van die gevalle met primêre INHweerstandigheid lae-vlak weerstandigheid het. Die farmakokinetika van INH in kinders het bevestig dat genoegsame vlakke en blootstellingstyd aan INH vir hierdie doel bereik kan word. Etionamied het dikwels gastrointestinale newe-effekte veroorsaak, maar dit kon in die meeste gevalle oorkom word. Die fluorokwinolone, wat nie oor die algemeen in kinders aanbeveel word nie, het moontlik artralgie veroorsaak in 1 uit 17 kinders wat vir ~6 maande behandel is, wat vorige verslae oor die veiligheid van hierdie middels in kort- en medium-termyn behandeling bevestig. TB-siekte het beduidend minder dikwels voorgekom in kinders wat toepaslike chemoprofilakse (volgens die middelvatbaarheidspatroon van die volwasse brongeval se isolaat) ontvang het. Alhoewel dit nie 'n ewekansig gekontroleerde studie was nie, het die groep wat chemoprofilakse ontvang het die hoogste risiko vir die ontwikkeling van siekte gehad. Dit dui daarop dat voorkoming van TB in MDR TB kontakte moonlik is. 'n Prospektiewe, ewekansig gekontrolleerde studie is nodig om die beste middel kombinasies en die optimale duur van so 'n chemoprofilaktiese behandeling te bepaal.

T -2 toxin is an immunosuppressive mycotoxin that has been conjoined with several symptoms and diseases as early as the turn of the century, but whose mechanisms of action are still being investigated. Accordingly, this study was an attempt to determine the cytotoxic effects of T -2 toxin on normal human lymphocytes in vitro, with particular emphasis on mitochondrial viability, cellular and nuclear morphology as well as the localisation of the subcellular sites of toxin interaction. The cytotoxicity of T -2 toxin was assessed with the use of a methylthiazol tetrazolium (MTT) assay. This assay targeted the succinate dehydrogenase activity of the lymphocytic mitochondria, over a range of concentrations of T-2 toxin at various incubation times. The morphology of treated lymphocytes was analysed with the use of transmission electron microscopy and the localisation of the toxin was accomplished via immunocytochemistry. DNA fragmentation studies formed an integral part of the analyses. The cytotoxicity assay indicated that not only was cell viability inversely proportional to both the dose and exposure time, but that the eftects of the different doses were only evident at prolonged incubation times (12-24 hours). The electron microscopy studies showed that T-2 toxin (1,56 ug/ml) induced apoptosis (cell suicide) in normal human lymphocytes. This was determined by the observation of chromatin condensation and nuclear disintegration within the toxin treated lymphocytes. Apoptosis seemed to occur independently of mitochondrial damage at 6 hours of exposure to T-2 toxin. The presence of polyribosomes within the treated lymphocytes indicated that protein synthesis was not inhibited. Anti-T-2 toxin conjugated gold label was present in all areas of damage, particularly within the nuclei of the T-2 toxin treated lymphocytes. The DNA fragmentation results showed that T-2 toxin induced fragmentation in lymphocytes, the extent of which was directly proportional to the exposure time. It appears that the early signs of T-2 toxin induced apoptosis in normal human lymphocytes can be determined by damage to the nucleus. / Thesis (M.Med.)-University of Natal, Durban, 1998.

Chemoprevention may be defined as the inhibition, delay or reversal of carcinogenesis by dietary compounds or their derivatives. "Imifino" is a collective name for many wild plants used predominantly by rural people as herbs in cooking. Many of these herbs possess medicinal properties. As the rural population is at higher risk of exposure to dietary carcinogens, such as mycotoxins, this pilot study was undertaken to determine whether the Amaranthus lividus plant held potential for use in chemopreventive strategies. The plant leaves were extracted to obtain individual solvent fractions. Cytotoxic profiling of the fractions using the SNO oesophageal adenocarcinoma cell line and normal human lymphocytes was achieved using the methylthiazol tetrazolium salt bioreduction assay. The SNO cell line, the A549 lung adenocarcinoma cell line and normal human lymphocytes were utilised for the evaluation of the anti-mycotoxigenic potential of the plant fractions in combination with two important dietary carcinogens, aflatoxin B1 and fumonisin B1. A specific biomarker assay (the induction of reduced glutathione) was employed using the SNO cell line. Flow cytometry was also conducted to determine the apoptotic properties of the acetone fraction on normal human lymphocytes. The results of the anti-mycotoxigenic study showed that certain fractions did have protective effects against both of the carcinogens tested. In addition, these effects were noted in the two cancerous cell lines, which were of different tissue origin. None of the fractions tested were toxic towards the normal human lymphocytes. The glutathione assay indicated that certain acetone fraction dilutions were inducive to reduced glutathione production. This plant is a promising candidate for further investigation concerning chemoprevention and the rural community could be educated on the possible benefits of this herb. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.

No abstract available. / Thesis (M.D.)- University of Natal, Durban, 1987.

Anthropometric measures, physiological variables and skills tests were performed on subjects selected from the provincial KwaZulu Natai Junior Hockey teams in South Africa. The main purpose of this study was to establish a data base of norms for boy and girls in the UB, U14, U16, UI8 and the U2I age groups. The tests were done at the beginning and at the end of season. The anthropometric measures included height, weight, percentage body fat and lean body mass; physiological variables included sit-ups, push-ups, sit-and-reach (flexibility), broad jump, winder and bleep tests , and the skills tests comprised a wide range of ball skill tests. As expected, anthropometric changes were observed across the age groups, due to growth. Amongst the older age groups the girls had reached height and weight values comparable to elite female players , but only the boys in the U2I had reached their adult height and were slightly taller than the elite male players. There was no significant difference in the profile between the attack and defence players in the boys, but amongst the girls the defence players tended to be heavier and taller than the attack players. In the physiological and skills tests there was no difference between positional players. In the comparison between pre and end season to determine the effectiveness of the training programmes, there was a change in the anthropometric characteristics because of growth. However, the physiological and skill tests revealed no consistent pattern of improvement in the test results from pre season to end season. This study provides the first set of norms for male and female junior hockey players in South Africa. Further studies are required to expand upon and update the data in the current study. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1997.