Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The risk of human immunodeficiency virus type-1 (HIV-1) infection and rate of progression towards development of the acquired immunodeficiency syndrome (AIDS) is determined by a combination of viral characteristics, immune function and host genetic variation. Although mutations of the chemokine and chemokine co-receptor genes and allelic variation of the major histocompatibility complex (MHC) have been studied extensively, variation in these host genetic factors does not explain the differences in HIV/AIDS susceptibility in all cases. This study represents the first analysis of new candidate genes implicated in iron metabolism and immune function in relation to HIV-1 disease in the African context. Both case-control association studies and genotype-phenotype correlations were performed to determine the potential functional significance of genetic variants that may be involved, either directly or indirectly, in susceptibility to HIV-1 disease in the South African population. Genotyping was performed to identify potentially important polymorphisms in the solute carrier family 11 member 1 (SLC11A 1), haemochromatosis (HFE) and protein-tyrosine phosphatase receptor-type C (PTPRC/CD45) genes in HIV-seropositive versus HIVseronegative individuals. This was followed by HLA-B27 genotyping in HIV-1 infected individuals with known disease status to determine the potential impact of combined genotypes for different mutations identified in the same study cohort. Preferential association with any of the mutations screened for in the CCR5, SLC 11A1, HFE or CD45 genes were not detected in HLA-B27 positive individuals identified. These findings were in accordance with the independent protective role of HLA-B27 in relation to disease progression in HIV-1 infected individuals. Although differences in allelic distribution were not significant between the study groups, an apparently African-specific mutation 32A~G, identified in an exonic splicing silencer element (ESS-1) of the CD45 gene, appeared to predominate in HIV-1 infected subjects with WHO Class I disease status and slow progression to AIDS. This mutation was present in 35.7% (5/14) of HIV-seropositive individuals with WHO Class I disease status, whilst absent in 22 HIV-seropositive patients with rapid disease progression. This finding may be related to differences in proportions of both CD4+ and CD8+ subsets observed following flow cytometry (FACs) analys.s in two HIV-seropositive individuals with mutation 32A~G, compared with an HIV-seropositive individual without this mutation. Analysis of the iron-related SLC11A1 and HFE genes did not reveal significant associations with modified risk of HIV-1 infection or progression to AIDS in our predominantly African study population. However, the effect of the virus on iron metabolism was demonstrated for the first time at the DNA level. Haemoglobin levels were significantly reduced in both HIV-seropositive (P=O.004) and HIV-seronegative (P=O.02) Black Africans with mutation IVS3-48c~g in the HFE gene, compared with mutationnegative individuals in both groups. Since this effect was more pronounced in HIV-infected individuals compared with controls, presence of the HFE mutation seems to result in an even stronger effect on haemoglobin levels, which may be related to the acute phase response following virus infection. This effect possibly results from genetic variation in a nearby gene involved in innate immunity, most likely in the HLA region on chromosome 6. It therefore seems possible that genetic variation in any of the host molecules involved in response to infection could contribute to clinical outcome. The significance of the multitude of host genetic factors investigated in this study, or previously implicated in susceptibility to HIV-1 infection and disease progression, revealed a complex interrelationship between the host and HIV-1. In some instances the disease process following HIV-1 infection depends on combined effects of different mutations occurring in the same individual, while independent effects of specific genes in conjunction with environmental influences may explain diverse clinical outcomes in others. / AFRIKAANSE OPSOMMING: Die risiko vir menslike immuniteitsgebrek virus tipe-1 (MIV-1) infeksie en die progressietempo vir ontwikkeling van die verworwe immuniteits gebrek sindroom (VIGS) word hoofsaaklik deur fn kombinasie van virale eienskappe, immuunfunksie en gasheer genetiese variasie bepaal. Alhoewel mutasies van die chemokien en chemokien koreseptor gene en alleliese variasie van die major weefsel-verenigbaarheidskompleks (MWVK) reeds omvattend bestudeer is, verklaar variasie van hierdie gasheer genetiese faktore nie noodwendig verskille in vatbaarheid vir MIVNIGS in alle gevalle nie. Hierdie studie verteenwoordig die eerste analise van nuwe kandidaatgene, geïmpliseer in yster metabolisme en immuunfunksie in die konteks van MIV-1 siekte in Swart bevolkingsgroepe. Beide gevalle-kontrole assosiasie-studies en genotipe-fenotipe korrelasies is uitgevoer om moontlik betekenisvolle verwantskappe met genetiese variante te bepaal, wat moontlik direk of indirek betrokke mag wees in vatbaarheid vir MIV-1 siekte in die Suid Afrikaanse populasie. Genotipering van die solute draer familie 11 lid 1 (SLC11A1), hemochromatose (HFE) en protein-tirosien fosfatase reseptor-tipe C (PTFRC/CD45) gene is uitgevoer in beide MIVseropositiewe en MIV-seronegatiewe individue. Daaropvolgend is genotipering van die menslike leukosien antigeen-B27 (MLA-B27) uitgevoer in MIV-1 geïnfekteerde individue met bekende siekte-status, om die potensiële impak van gekombineerde genotipes te bepaal vir verskillende mutasies wat in dieselfde studie populasie geïdentifiseer is. Voorkeur-assosiasie is nie waargeneem vir enige van die mutasies waarvoor geanaliseer is in die CCR5, SLC11A1, HFE of CD45 gene nie. Hierdie bevinding is in ooreenstemming met die onafhanklike rol van MLA-B27 in verwantskap met siekte progressie in MIV-1 geïnfekteerde individue. Alhoewel die alleelverspreiding van In Afrika-spesifieke mutasie 32A~GI wat in In eksoniese splytingsdemper-element (ESS-1) van die CD45 geen geïdentifiseer is, nie statisties betekenisvolle verskille getoon het tussen studiegroepe nie, is die mutasie oorheersend waargeneem in MIV-1 geïnfekteerde individue met WGO Klas I siektestatus en stadige progressie na VIGS. Hierdie mutasie was teenwoordig in 35.7% (5/14) van HIV-seropositiewe individue met WGO Klas I siekte-status, terwyl dit afwesig was in 22 HIV-seropositiewe pasiënte met vinnige siekteprogressie. Hierdie bevinding mag moontlik verband hou met verskille in verhoudings van beide die CD4+ en CD8+ substelle, soos waargeneem gedurende vloei sitometriese (VAS, FACs) analise in twee HIV-seropositiewe individue met mutasie 32A---+G, in vergelyking met en HIV-seropositiewe individu sonder hierdie mutasie. Analise van die yster-verwante SLC11A 1 en HFE gene het nie betekenisvolle assosiasies opgelewer met gemodifiseerde risiko vir MIV-1 siekte of progressie na VIGS in die hoofsaaklik Swart studie-populasie nie. Die effek van die virus op ystermetabolisme is wel vir die eerste keer op DNS vlak gedemonstreer. Hemoglobien vlakke was betekenisvol verlaag in beide MIV-seropositiewe (P=O.004) en MIV-seronegatiewe (P=O.02) Swart individue met die HFE geen IVS3-48C---+G mutasie, in vergelyking met mutasie-negatiewe individue in beide groepe. Aangesien hierdie effek meer uitgesproke was in MIVgeïnfekteerde individue as in kontroles, blyk dit dat die teenwoordigheid van die HFE mutasie die hemoglobienvlakke tot engroter mate beïnvloed weens die akute fase respons wat verband hou met die virusinfeksie. Hierdie effek kan moontlik toegeskryf word aan genetiese variasie in ennaasliggende geen wat in aangebore immuniteit betrokke is, heel moontlik in die MLA gebied van chromosoom 6. Dit wil dus voorkom asof genetiese variasie in enige van die gasheer molekules betrokke by respons op infeksie kan bydra tot die kliniese uitkoms. Die belangrike rol van die veelvuldige gasheer genetiese faktore wat in hierdie studie bestudeer is, of wat voorheen geïmpliseer is in vatbaarheid vir MIV-1 infeksie en siekte progressie, het enkomplekse inter-verwantskap tussen gasheer en MIV-1 geopenbaar. In sommige gevalle is die siekte-proses na MIV-1 infeksie afhanklik van gekombineerde effekte van verskillende mutasies in dieselfde individu, terwylonafhanklike effekte van spesifieke gene tesame met omgewings-invloede uiteenlopende kliniese uitkomste in ander mag verklaar.

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.

Dissertation (DSc)-- Stellenbosch University, 2008. / ENGLISH ABSTRACT: The overall aim of these studies was to investigate selected aspects of psychopathology, neurobiological abnormalities and treatment in schizophrenia. The following topics were researched: 1. Psychopathology: We explored the symptom structure of schizophrenia by means of principal components and factor analysis in two separate samples. a. The first study investigated the nature of symptoms in patients with a first-episode of schizophrenia, in a large cohort of patients who were participating in a multinational clinical trial. We compared our findings with similar analyses previously conducted in multi-episode schizophrenia patients. b. We then assessed the influence of culture on the symptom structure of schizophrenia by conducting a principal components and factor analysis of the symptom ratings in a large sample of South African Xhosa patients with schizophrenia, and comparing the results with those in other parts of the world. c. We investigated the occurrence of co-morbid depressive and anxiety symptoms, and their demographic and clinical correlates. The sample for this study comprised acutely psychotic patients who were participants in clinical drug trials conducted at our centre. d. To explore the relationships between obsessive-compulsive disorder and schizophrenia, we conducted a review of the relevant literature. 2. Neurobiological abnormalities: a. We performed a series of studies to investigate disorders of water homeostasis and vasopressin secretion in schizophrenia. To test the hypothesis that acutely psychotic patients have disordered regulation of water homeostasis, we applied a dynamic suppression test - a water loading test, with assessment of excretory capacity (including arginine vasopressin assay) in acutely psychotic patients. To evaluate whether a subset of patients with schizophrenia and co-morbid disordered water homeostasis sustained cerebral damage as a consequence of water intoxication we did the following experiment: We identified a cohort of subjects with schizophrenia and disordered water homeostasis and compared them with patients with schizophrenia without disordered water homeostasis in terms of cerebral ventricular size and cognitive function. To assess the prevalence of disordered water homeostasis in a long-term inpatient sample of psychiatric patients we conducted serum sodium screening tests. Those subjects with dilutional hyponatraemia were then further investigated for dysregulation of water homeostatic mechanisms. b. We studied neurological soft signs in a sample of subjects with first-episode schizophrenia followed up over a two year period. We investigated their occurrence, relationships to psychiatric symptoms and medication effects, their temporal stability and their outcome correlates. We also investigated their potential to predict outcome in schizophrenia 3. Treatment aspects A great deal of our work has focussed on the pharmacological treatment of schizophrenia. The following aspects of treatment are included in this thesis: a. Treatment effects on psychiatric symptoms: i. To assess the effects of ethnicity on treatment outcome in schizophrenia we compared the acute response to antipsychotic treatment in 3 ethnic groups, namely blacks, coloureds and whites. We included patients in this analysis who had participated in clinical trials in our department as well as the Department of Psychiatry in the University of the Free Sate. Patients had been treated under blinded conditions over a 6-week period. ii. After discussions with the late Dr David Horrobin, who had pioneered possible applications of the omega-3 fatty acids in the treatment of various psychiatric disorders, we became interested in further investigating the potential of this group of compounds as an affordable adjunct to treating schizophrenia. We assessed the antipsychotic potential of the omega-3 fatty acid, ethyl-eicosapentaenoic-acid (e-EPA) supplementation versus placebo supplementation in a small sample of subjects with schizophrenia who had been only partially responsive to antipsychotic treatment previously. We also conducted a review of the literature to evaluate the evidence for efficacy for the omega-3 fatty acids in schizophrenia according to published studies. b. Treatment effects on neurological abnormalities: i. In a single-blinded controlled study we compared a new generation antipsychotic to a conventional antipsychotic in the treatment of tardive dyskinesia (TD). This was a long-term (1 yr) study in patients with chronic schizophrenia and established tardive dyskinesia. ii. We also assessed the effect of omega-3 fatty acid (e-EPA) supplementation in treating TD. This was conducted in a larger sample (n=84) of patients with chronic schizophrenia and established TD. The blinded, placebo-controlled phase was 12 weeks. This was followed by an open-label extension for 40 weeks. c. Conventional versus new generation antipsychotic agents. Several evidence-based literature reviews of the efficacy and tolerability of the new generation of antipsychotics compared to the conventional agents were conducted. Some multinational, randomised, controlled clinical trials in which the author was principal investigator, are included in this thesis. Also, studies addressing patients with partial treatment refractoriness are included, as well as studies of the effects of antipsychotics on depressive symptoms, body mass and glycaemic control. Finally, we have included a pharmacoeconomic study comparing a conventional antipsychotic (haloperidol) with a new generation antipsychotic (quetiapine) in partially refractory patients in a South African setting. Findings and conclusions: 1. Psychopathology: Our studies demonstrated that the factor structure for the symptoms of schizophrenia is replicable across samples, and is not greatly influenced by ethnic and cultural factors. However, changes in the factor structures do occur over time. There are symptom domains that are present in first-episode schizophrenia but disappear as a distinct entity as the illness becomes chronic. Particularly, a motor component is evident in untreated patients, but disappears after initiation of treatment. We found that depression and anxiety are common co-morbid symptoms in schizophrenia, and have important clinical and outcome correlates. Depressive symptoms in the acute psychotic phase of schizophrenia are associated with a favourable prognosis and diminish as the symptoms of psychosis improve in response to antipsychotic treatment. However, persistent depressive symptoms are associated with a poorer prognosis, and require additional therapeutic intervention. 2. Neurobiological abnormalities: We investigated the occurrence of disordered water regulation in a population of psychiatric inpatients, and conducted further investigations on those identified, in order to establish mechanisms involved. Polydipsia and the syndrome of inappropriate antidiuretic hormone secretion (SIADH) were found to occur in a subset of patients with schizophrenia, and are associated with acute psychosis, as well as with some psychotropic medications. These patients are characterised by more severe cognitive impairment and evidence of cerebral atrophy. The condition can become life-threatening in the presence of other factors impeding water excretion, particularly thiazide diuretics. Neurological soft signs were investigated in a sample of patients with a first-episode of schizophrenia. These soft signs appear to be trait-like (present early in the illness, and stable over time), except for a motor sequencing factor. Patients performing poorly on this latter group of tests have a longer duration of untreated psychosis, and are at significant risk for developing TD. 3. Treatment aspects: Our studies suggest that there are important ethnic differences in antipsychotic treatment response, but that these differences could be explained by a number of environmental and biological factors. As was found with many studies worldwide, we found that the new generation antipsychotics have important efficacy and safety advantages over their predecessors. Risperidone was as effective as haloperidol in first-episode psychosis, but with a more favourable side-effect profile in terms of reduced extrapyramidal symptoms. Quetiapine treatment in partially refractory patients resulted in more responders compared to haloperidol, and fewer extrapyramidal symptoms. However, evidence of a different side-effect profile is emerging. Of particular concern is the finding that some of the new antispychotics cause weight gain, glucose intolerance and dyslipidaemias. We found that one novel antipsychotic, quetiapine, was not associated with significantly more weight gain or disordered glucose metabolism that a conventional agent, haloperidol. The omega-3 fatty acids, particularly EPA may have a role in the treatment of various psychiatric disorders. Our studies provided mixed results – the first found a significant beneficial effect on psychotic symptoms and dyskinesia scores for EPA supplementation, while the second failed to demonstrate a beneficial effect on TD or psychotic symptoms. We explored the early treatment response in first-episode psychosis and found, unlike that reported in multi-episode patients, some patients took a long time to respond. We also found that early treatment response was a significant predictor of later remission, as was duration of untreated psychosis, educational level and baseline excitement factor scores. Finally, our pharmacoeconomic study conducted for South African circumstances in patients with a partial response to conventional antipsychotic treatment showed cost-neutrality or cost-benefits for quetiapine compared with haloperidol treatment for direct costs. / AFRIKAANSE OPSOMMING: Die oorkoepelende doel van hierdie studies was om geselekteerde aspekte van psigopatologie, neurobiologiese abnormaliteite en behandeling in skisofrenie te ondersoek. Die volgende onderwerpe is nagevors: 4. Psigopatologie: Ons het die simptoomstruktuur van skisofrenie ondersoek deur middel van hoofkomponent- en faktoranalise in twee aparte steekproewe. a. Die eerste studie het die aard van simptome in pasiënte, met ʼn eerste-episode van skisofrenie, ondersoek in ʼn groot kohort van pasiënte wat deelgeneem het aan ʼn multi-nasionale kliniese proefneming. Ons het ons bevindinge vergelyk met soortgelyke analises wat voorheen gedoen is in multi-eposode skisofrenie pasiënte. b. Hierna het ons die invloed van kultuur op die simptoom struktuur van skisofrenie geassesseer deur ʼn hoofkomponent- en faktoranalise van die simptoomtellings uit te voer in ʼn groot steekproef van Suid-Afrikaanse Xhosa pasiënte met skisofrenie en die resultate te vergelyk met bevindinge in ander dele van die wêreld. c. Ons het die voorkoms van ko-morbiede depressiewe en angssimptome ondersoek, asook hul demografiese en kliniese korrelate. Die steekproef vir hierdie studie het bestaan uit akute psigotiese pasiënte wat deelnemers was in ʼn kliniese geneesmiddel proef wat uitgevoer is by ons sentrum. d. Om die verband tussen obsessief-kompulsiewe steurnis en skisofrenie te verken, het ons ʼn oorsig van die relevante literatuur gedoen. 5. Neurobiologiese abnormaliteite: a. Ons het ʼn reeks studies uitgevoer om steurnisse in water homeostase en vasopressien sekresie in skisofrenie te ondersoek. Om die hipotese dat akute psigotiese pasiënte versteurde regulering van water homeostase het te ondersoek, het ons ʼn dinamiese onderdrukkingstoets toegepas – ʼn water ladingstoets, met assessering van ekskresiekapasiteit (insluitend arginien vasopressien essai) in akute psigotiese pasiënte. Om te evalueer of ʼn onderafdeling van skisofrenie pasiënte met ko-morbiede versteurde water homeostase serebrale skade opgedoen het as gevolg van water intoksikasie, het ons die volgende eksperiment uitgevoer: Ons het ʼn kohort deelnemers met skisofrenie en versteurde water homeostase geïdentifiseer en hulle vergelyk met skisofrenie pasiënte sonder versteurde water homeostase in terme van serebrale ventrikulêre grootte en kognitiewe funksionering. Om die voorkoms van versteurde water homeostase in ʼn langtermyn binne-pasiënt steekproef van psigiatriese pasiënte te bepaal, het ons serum natrium siftingstoetse uitgevoer. Deelnemers met hiponatremie is hierna verder ondersoek vir disregulering van water homeostatiese meganismes. b. Ons het neurologiese sagte tekens in ʼn steekproef van deelnemers met eersteepisode skisofrenie bestudeer en opgevolg oor ʼn twee jaar tydperk. Ons het hulle voorkoms, verwantskappe met psigiatriese simptome en medikasie effekte, hulle temporale stabiliteit en hul uitkoms korrelate ondersoek. Ons het ook hulle potensiaal om die uitkoms in skisofrenie te voorspel, ondersoek. 6. Behandelings aspekte ʼn Groot meerderheid van ons werk het gefokus op die farmakologiese behandeling van skisofrenie. Die volgende aspekte van behandeling is ingesluit in hierdie tesis: a. Behandelingseffekte op psigiatriese simptome: i. Om die effek van etnisiteit op behandelingsuitkoms in skisofrenie te assesseer, het ons die akute respons op anti-psigotiese behandeling in 3 etniese groepe vergelyk, naamlik swart, gekleurd, en wit. Ons het pasiënte wat deelgeneem het aan kliniese proefnemings in ons departement sowel as die Departement Psigiatrie van die Universiteit van die Vrystaan ingesluit in hierdie analise. Pasiënte is behadel onder geblinde toestande oor ʼn tydperk van 6 weke. ii. Na besprekings met wyle Dr David Horrobin, wie die moontlike toepassings van omega-3 vetsure in die behandeling van verskeie psigiatreise steurnisse gepionier het, het ons begin belangstel in verdere ondersoek na die potensiaal van hierdie groep samestellings as ʼn bekostigbare toevoeging in die behandeling van skisofrenie. Ons het die anti-psigotiese potensiaal van die omega-3 vetsuur, etieleikosapentanoësuur (e-EPA) supplementasie versus plasebo supplementasie ondersoek in ʼn klein steekproef van deelnemers met skisofrenie wat slegs gedeeltelik responsief was op anti-psigotiese behandeling in die verlede. Ons het ook ʼn literatuuroorsig gedoen om die bewyse vir die effektiwiteit vir die omega-3 vetsure in skisofrenie te evalueer volgens gepubliseerde studies. b. Behandelingseffekte op neurologiese abnormaliteite: i. In ʼn enkelblinde kontrole studie het ons ʼn nuwe generasie anti-psigotiese medikasie vergelyk met ʼn konvensionele anti-psigotiese medikasie in die behandeling van tardiewe diskinesie (TD). Hierdie was ʼn langtermyn (1- jaar) studie in pasiënte met chroniese skisofrenie en vasgestelde TD. ii. Ons het ook die effek van omega-3 vetsuur (e-EPA) suplementasie geassesseer in die behandeling van TD. Dit was gedoen in ʼn groter steekproef (n=84) van pasiënte met chroniese skisofrenie en vasgestelde TD. Die blinde, placebo kontrole fase was 12 weke. Dit is gevolg deur ʼn nie-geblinde ekstensie vir 40 weke. c. Konvensionele versus nuwe generasie anti-psigotiese agente. Verskeie bewys-gebaseerde literatuuroorsigte oor die effektiwiteit en toleransie van die nuwe generasie anti-psigotiese agente in vergelyking met die konvensionele agente, is gedoen. Sommige multi-nasionale, ewekansige, kontole kliniese proefnemings waarin die outeur die hoofnavorser was, is ingesluit in hierdie tesis. Verder, studies wat die pasiënte met gedeeltelike behandelingsweerstandigheid aanspreek, is ingesluit, sowel as studies oor die effekte van anti-psigotiese agente op depressiewe simptome, liggaamsmassa en glisemiese kontrole. Laastens, het ons a farmakoekonomiese studie ingesluit wat die konvensionele anti-psigotiese behandeling (haloperidol) met ʼn nuwe generasie anti-psigotiese behandeling (quetiapien) in gedeeltelik weerstandige pasiënte in ʼn Suid-Afrikaanse ligging vergelyk. Bevindinge en gevolgtrekkings: 4. Psigopatologie: Ons studies het gedemonstreer dat die faktor struktuur vir die simptome van skisofrenie herhaalbaar is oor steekproewe, en dat dit nie grootliks beïnvloed word deur etnisiteit en kulturele faktore nie. Veranderinge vind egter in die faktor strukture wel plaas met verloop van tyd. Daar is simptoom domeine wat teenwoordig is in eerste-episode skisofrenie, maar verdwyn as ʼn afsonderlike entiteit soos wat die toestand chronies word. Spesifiek, ʼn motoriese komponent is duidelik in onbehandelde pasiënte, maar verdwyn na die aanvang van behandeling. Ons het gevind dat depressie en angs algemene ko-morbiede simptome in skisofrenie is en het belangrike kliniese en uitkoms korrelate. Depressiewe simptome in die akute psigotiese fase van skisofrenie word geassosieer met ʼn gunstige prognose en verminder soos wat die simptome van psigose verbeter in repons op anti-psigotiese behandeling. Egter, volgehoue depressiewe simptome word geassosieer met ʼn swakker prognose en benodig addisionele terepeutiese intervensie. 5. Neurobiologiese abnormaliteite: Ons het die voorkoms van versteurde water regulering ondersoek in ʼn populasie van psigiatriese binne-pasiënte en verdere ondersoek ingestel op dié wie geïdentifiseer is, om die betrokke meganismes vas te stel. Polidipsie en en die sindroom van onvoldoende antidiuretiese hormoon sekresie (SIADH) is gevind om voor te kom in ʼn onderafdeling van pasiënte met skisofrenie, en word geassosieer met akute psigose sowel as met somige psigotropiese medikasie. Hierdie pasiënte word gekenmerk deur meer ernstige kognitiewe beperking en bewyse van serebrale atrofie. Die toestand kan lewensbedreigend raak in die teenwoordigheid van ander faktore wat water ekskresie hinder, veral tiasied diuretikums. Neurologiese sagte tekens is ondersoek in ʼn steekproef van pasiënte met eerste-episode skisofrenie. Hierdie sagte tekens blyk om kenmerkend (teenwoordig vroeg in die siekte, en stabiel oor tyd) te wees, behalwe vir ʼn motoriese volgorde faktor. Pasiënte wat swak vaar op die laasgenoemde groep toetse, het ʼn langer durasie van onbehandelde psigose, en het ʼn beduidende risko om TD te ontwikkel. 6. Behandeling aspekte: Ons studies stel voor dat daar ʼn belangrigke etniese verskil is in anti-psigotiese behandelingsrespons, maar dat hierdie verskille verduidelik kan word deur ʼn aantal omgewings- en biologiese faktore. Soos wat gevind was vir verskeie studies wêreldwyd, het ons gevind dat die nuwe generasie anti-psigotiese agente belangrike effektiwiteit- en veiligheidsvoordele het bo hulle voorgangers. Risperidoon was net so effektief as haloperidol in eerste-episode psigose, maar met ʼn meer gunstige newe-effkte profiel in terme van verminderde ekstrapirimidale simptome. Quetiapien behandeling in veral refraktêre pasiënte het gelei tot meer respondeerders vergeleke met haloperidol, en minder ekstra pirimidale simptome. Alhoewel, bewyse van ʼn verskillende newe-effekte profiel is besig om na vore te kom. Van spesifieke belang is die bevinding dat sommige van die nuwe anti-psigotiese agente gewigstoename, glukose intoleransie en dyslipidemie veroorsaak. Ons het gevind dat een nuwe anti-psigotiese agent, quetiapien, nie geassosieer was met enige beduidende meer gewigstoename of versteurde glukose metabolisme as ʼn konvensionele agent, haloperidol, nie. Die omega-3 vetsure, spesifiek EPA mag moontlik ʼn rol in die behandeling van verskeie psigiatriese versteurings hê. Ons studies het gemengde resultate voorsien – die eerste het ʼn beduidende voordelige effek op psigotiese simptome en diskinesie tellings vir EPA supplementasie gevind, terwyl die tweede nie ʼn voordelige effek op TD of psigotiese simptome gevind het nie. Ons het die vroeë behandelingsrespons ondersoek in eersteepisode pasiënte en het gevind, in teenstelling met dit wat gerapporteer word in multi-episode pasiënte, dat sommige pasiënte ʼn lang tyd geneem het om te reaggeer. Ons het ook gevind dat vroeë behandelingsrespons ʼn beduidende voorspeller was van latere remissie, so ook die durasie van onbehandelde psigose, opvoedingspeil, en basisvlak opwindings-faktor tellings. Laastens het ons farma-ekonomiese studie, wat uitgevoer is vir Suid-Afrikaanse omstandighede in pasiënte met ʼn gedeeltelike repons op konvensionele anti-psigotiese behandeling, koste-neutraliteit of koste-voordele aangetoon vir quetiapien vergeleke met haloperidol behandeling vir direkte onkostes.

Dissertation (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Although schizophrenia is traditionally viewed as an illness with a very poor prognosis, research over the last few years indicates that early intervention may substantially improve the long-term outcome of this disorder. Several studies suggest that patients with first-episode psychosis (FEP) are more sensitive to, and require lower doses of antipsychotic medications than patients with more chronic forms of illness. However, the optimal dose of first-generation anti psychotics in patients with FEP has not been explored extensively and continues to be a controversial subject. This study evaluated the efficacy and safety of low-dose haloperidol in a South African cohort with FEP. The study was conducted in two phases: Phase 1 was an open-label, naturalistic study of 57 subjects with FEP who were commenced on 1mg of haloperidol for 4 weeks, after which gradual escalation of doses were allowed, if required. Subjects who failed to respond at haloperidol 10mg per day were switched to thioridazine. Failure to respond to thioridazine 600mg per day was interpreted to indicate treatment resistance. These subjects were then commenced on clozapine. The principal finding of this phase of the study was that the majority of subjects could be stabilized and maintained on very low doses of haloperidol (1.7 ± 1.0 mg/day at 12 months and 1.3 ±0.8 mg/day at 24 months). Ratings for extra-pyramidal side-effects did not increase significantly from baseline over the duration of the study, except in the case of tardive dyskinesia (TD), where a substantial number of subjects (12.3%) developed TD within 12 months of starting treatment. Phase 2 of the study was a double-blind, randomized controlled trial of low-dose (2mg/day) versus "standard dose" (8mg Iday) haloperidol. Forty subjects were included in this phase of the study; 20 in each treatment arm. The main finding was that there were no significant differences in treatment reponse between the two treatment groups. There were, however, significant differences between the two treatment groups in extrapyramidal side effects (EPSE), with the 8mg per day group exhibiting significantly higher levels of EPSE than the 2mg per day group. This was manifested by significant differences in scores on the Extrapyramidal Symptom Rating Scale (ESRS) and the Simpson-Angus Rating Scale. Furthermore, subjects in the 8mg haloperidol per day group required significantly higher doses of anticholinergic medication and had significantly higher mean levels of prolactin at the end of the study period. This study indicates that a majority of subjects with first-episode psychosis can be treated and maintained successfully with very low doses of haloperidol. It also shows that low-dose treatment is as effective as, and better tolerated than, "standard" doses. Despite the success with the low-dose treatment, however, there was still a much higher than expected incidence of tardive dyskinesia, a serious and potentially irreversible side-effect of neuroleptic treatment. / AFRIKAANSE OPSOMMING: Hoewel skisofrenie tradisioneel gesien is as 'n siekte met 'n uiters swak prognose, dui navorsing oor die afgelope jare daarop dat vroeë ingryping die langtermynuitkoms van hierdie toestand drasties mag verbeter. Resultate van verskeie studies dui daarop dat pasiënte met eerste-episode psigose (EEP) nie net meer sensitief is vir antipsigotiese middels nie, maar ook laer dosisse daarvan benodig tydens behandeling as pasiënte met meer kroniese vorms van psigotiese siekte. Desondanks is die kwessie van die korrekte dosis van eerste generasie antipsigotika in hierdie groep nog onvolledig nagevors en bly dit 'n omstrede onderwerp. Hierdie studie het ten doel gehad om die effektiwiteit en veiligheid van lae dosis haloperidol in 'n Suid-Afrikaanse populasie van pasiënte met EEP te evalueer. Die studie is uitgevoer in twee fases: Fase 1 was 'n oop, naturalistiese studie van 57 pasiënte met EEP wat aanvanklik behandel is met 1mg haloperidol per dag vir 4 weke, waarna geleidelike verhoging van dosisse toegelaat is, soos nodig. Diegene wat nie bevredigende respons getoon het op haloperidol 10mg per dag nie, is oorgeskakel na tioridasien. Ontoereikende respons teen 600mg/dag tioridasien is geïnterpreteer as 'n aanduiding van behandelingsweerstandigheid en behandeling met klosapien is begin. Die belangrikste bevinding van hierdie fase van die studie was dat die meerderheid pasiënte gestabiliseer en in stand gehou kom word op baie lae dosisse haloperidol (1.7 ± 1.0 mg/dag op 12 maande en 1.3 ±0.8 mg/dag op 24 maande). Metings van ekstra-piramidale newe-effekte (EPNE) het nie beduidend toegeneem oor die duur van die studie nie, behalwe in die geval van tardiewe diskinese (TO), waar 'n beduidende aantal pasiënte (12.3%) TO ontwikkel het binne 12 maande na aanvang van behandeling. Fase 2 van die studie was 'n dubbelblinde, ewekansig gerandomiseerde studie waarin behandeling met lae dosis haloperidol (2mg/dag) vergelyk is met "standaard" dosis haloperidol (8mg/dag). Veertig pasiënte is ingesluit in hierdie fase van die studie, 20 in elke behandelingsarm. Die hoofbevinding was dat daar geen beduidende verskille in respons op behandeling was tussen die twee groepe nie. Daar was egter beduidende verskille in EPNE, waar die 8mg/dag groep beduidend hoër vlakke van EPNE gehad het as die 2mg/dag groep. Hierdie verskil in EPNE is aangedui deur 'n statisties beduidende verskil in tellings op die Extrapyramidal Symptom Rating Scale (ESRS) en die Simpson- Angus Rating Scale. Verder het pasiënte in die 8mg/dag groep beduidend hoër dosisse antikolinerge medikasie benodig en ook hoër gemiddelde prolaktienvlakke gehad teen die einde van studie. Hierdie studie dui dus daarop dat die meerderheid van pasiënte met EEP suksesvol behandel en in stand gehou kan word met baie lae dosisse haloperidol. Die studie wys ook daarop dat behandeling met lae dosisse net so effektief is en beter verdra word as behandeling met "standaard" dosisse. Ten spyte van die suksesvolle gebruik van lae dosisse medikasie het die studie egter ook getoon dat daar "n baie hoër as verwagte insidensie was van TO, "n emstige en potensieelonomkeerbare newe-effek van neuroleptiese behandeling.

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2005. / Tuberculosis (TB) is one of the most serious infectious diseases known to mankind, with devastating outcomes in the poorest countries in the world. Isoniazid is the cornerstone of all first-line anti-TB regimens. Forty-eight percent of all drug resistant TB isolates in the Western Cape are Isoniazid mono-resistant, and the majority of these isolates belong to the Beijing/W strain family. Currently, the known molecular mechanisms which confer Isoniazid resistance in these isolates are attributed to mutations within the katG gene and account for up to 70% of all drug resistant TB isolates. Risk factors for the development of Isoniazid resistance can be attributed to either pathogen or host related factors and may partially account for the other 30% of Isoniazid resistant isolates. In this study, three aspects which may contribute to Isoniazid resistance were investigated: DNA repair in the bacterium, host response to anti-TB treatment and socioeconomic factors. A PCR based dot-blot strategy was used to screen for previously reported missense mutations in the mutT2, Rv3908 and ogt DNA repair genes of different strains of M. tuberculosis. All the Beijing isolates (drug resistant and susceptible), in contrast to the Atypical Beijing strains and other dominant strain families, exhibited missense mutations in all three base excision repair genes. It is therefore speculated that defects in the DNA repair genes (mutator phenotypes) of the Beijing isolates may contribute to the development of drug resistance and hence, may account for the large proportion of isolates that are Isoniazid mono-resistant. A novel method, based on primer extension, was initially developed to screen the NAT2 gene and then used to type individuals into fast, intermediate and slow acetylators of Isoniazid. The newly develop method, which is sensitive and accurate, improves the detection of Single Nucleotide Polymorphisms within the NAT2 gene, in contrast to the traditionally used methods. Utilising this method, it was found that the combination of fast and intermediate acetylators was significantly associated with Isoniazid resistance in the study community. This finding may have an important impact on TB control programmes, since it may allow for the administration of higher dosages of Isoniazid to fast/intermediate acetylators and a lower dose for slow acetylators. Clinical factors (compliance and retreatment after cure) and socio-economic factors (education, employment and income) were found to be significantly associated with the development of INH resistance. Diagnostic delay was also found to be a risk factor, since it may allow for transmission of TB during this period. The HIV prevalence in the study population is low and subsequently HIV status was not associated with the development of INH resistance. This study indicates that a combination of risk factors, both pathogen and host related, are involved in the development of Isoniazid resistance.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology)--Stellenbosch University, 2008. / For many centuries, scientists have engaged in a theoretical debate concerning the etiology of mood disorders, with very few ancient scholars speculating about the importance of genetic factors and affective temperaments as factors in the etiology of depression. Mood, emotion and cognition have been shown to be modulated by the serotonergic midbrain raphe system; implicated in the pathogenesis of psychiatric disorders like those of the affective spectrum. Evidence from neuroscience, genetics, and clinical investigation demonstrate that depression is a disorder of the brain. Brain imaging research is revealing that in depression, neural circuits responsible for moods, thinking, sleep, appetite, and behavior fail to function properly, and that the regulation of critical neurotransmitters is impaired. Genetics research, including studies of twins, indicates that genes play a role in depression. Vulnerability to depression appears to result from the influence of multiple genes acting together with environmental factors. Other research has shown that stressful life events, particularly in the form of loss such as the death of a close family member, may trigger major depression in susceptible individuals. Depression and anxiety have often been successfully treated by means of selective serotonin reuptake inhibitors. However, selective serotonin reuptake inhibitors do not solve all the problems inherent to the treatment of depression, for approximately 30 % of depressed patients do not respond to treatment and 20 % experience relapses whilst on treatment. Of consideration is the fact that the majority of drugs today are based on proteins, with 50 % of therapeutics on the market targeting cell membrane proteins. Up to this day the precise pathophysiology of mood disorders remains obscure, as does the neurobiology of normal mood regulation. Accordingly, there is a need for methods to identify the structural and/or signaling components which lead to changes in the brain, particularly the hippocampus, of subjects having mood disorders such as bipolar depressive disorder, chronic major depressive disorder and the like. Similarly, there is a need for the early detection, screening and diagnosis of individuals at risk for a mood disorder. As the serotonin tranpsorter is the primary target for therapeutic intervention in the treatment of numerous psychiatric disorders and considering the fact that at the structural level this protein’s function as transporter in membranes remains incompletely understood, investigating its function in psychiatric disorders are of importance . The objective of this study was to determine the role of the serotonin transporter in wild type and serotonin knockout rats, with regards to the hippocampus. Rat hippocampi were fractionated into cytosolic and membrane components, which were run and further separated in two dimensions. Firstly separation occurred by isoelectrical focusing (pI), follwed by gel iii electrophoresis (molecular weight). Gels were compared to see whether protein spots have changed between animals that have been differentially bred. Differentially expressed protein spots, as determined by PD Quest software, were excised, digested and analyzed by means of mass spectrometry. Our results indicated that metabolic, structural and cell signaling proteins were differentially expressed in both the ventral and dorsal hippocampus of the serotonin knockout rat. Futhermore, cellular stress proteins were found to be only differentially expressed in the ventral hippocampus. The majority of proteins identified in both hippocampal areas as well as both fractions, were assigned to energy metabolism. The cytosolic protein profile mirrored the pattern of the membrane protein profile. In conclusion, this proteomic study identified various protein groups that interacted with one another, thus establishing compensation for disrupted serotonin homeostasis.

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2007. / Connexins (Cx) are major proteins of gap junctions, dynamic pores mediating the relay of ions and metabolites between cells. Cxs 40, 43 and 45 are the predominant cardiac isoforms and their distinct distribution raises questions about their functional differences. Their cytoplasmic (C)-terminal domains are involved in protein-protein interactions. Furthermore, mutations in the myotonic dystrophy protein kinase (DMPK)-causative gene are associated with disruptions in cardiac conduction similar to that described for Cx knock-out mice. DMPK is a Cx43 ligand, raising the possibility that defects in Cx40 ligands may be involved in the development of cardiac conduction disturbances. We hypothesised that delineation of the protein ligands of the C-termini of Cx40 and of Cx45 (parallel study conducted by N Nxumalo) would help elucidate their functional roles. Yeast-two-hybrid methodology was used to identify putative Cx40 ligands. Primers were designed to amplify the C-terminus-encoding domain of the human Cx40 gene (Cx40), the DNA product was cloned into the pGBKT7 vector which was used to screen a cardiac cDNA library in Saccharomyces cerevisiae. Successive selection stages reduced the number of putative Cx40 ligand-containing colonies (preys) from 324 to 33. The DNA sequences of the 33 ligands were subjected to BLAST-searches and internet database literature searches to assign identity and function and to exclude false positive ligands based on subcellular location and function. Eleven plausible ligands were identified: cysteine-rich protein 2 (CRP2), beta-actin (ACTB), creatine kinase, muscle type (CKM), myosin, heavy polypeptide 7 (MYH7), mucolipin1 (MCOLN1), voltage-dependent anion channel 2 (VDAC2), aldehyde dehydrogenase 2 (ALDH2), DEAH box polypeptide 30 (DHX30), NADH dehydrogenase, 6, (NDUFA6), prosaposin (PSAP) and filamin A (FLNA). Cxs 40 and 45 showed differences in the classes of proteins with which they interacted; the majority of putative Cx40 interactors were cytoplasmic proteins, while Cx45 interactors were mitochondrial proteins. These results suggest that Cxs 40 and 45 are not only functionally different, but may also have different cellular distributions. Further analyses of these protein interactions will shed light on the independent roles of Cxs 40 and 45.

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2005. / The purpose of this study was to determine the physiological and kinanthropometric attributes, respiratory muscle strength, and core stability of successful endurance paddlers, and to investigate the associations of these characteristics with kayak endurance performance in the laboratory and the field. Twenty male competitive paddlers (age: 28 ± SD 7 years, height: 184 ± SD 7 cm and weight: 80 ± SD 7 kg) were categorised in two ability groups, Elite and Sub-Elite. Testing included kinanthropometric measurements, maximum aerobic capacity, pulmonary function, six core stability tests, a 30 min endurance performance test (EPT) on the K1 Ergo and a 10 km time trial (TT) on the water. Maximum inspiratory mouth pressure (MIP) was measured before and after the 30 min EPT on the K1 Ergo to assess respiratory muscle fatigue. The Elite paddlers demonstrated significantly greater values for sitting height (as a percentage of stature), relative VO2max, PPO, PPO/kg, MVV and MIP compared to the Sub-Elite paddlers (All P < 0.05). They also demonstrated a significantly greater average PO and average back stroke length during the 30 min K1 Ergo EPT (P < 0.05) and a significantly faster race time (44:10 ± 1:17 vs 47:34 ± 3:14 min:s) during the 10 km water TT (P < 0.05), compared to the Sub-Elite paddlers. The paddlers did not experience respiratory muscle fatigue (as determined by change in MIP) after the 30 min K1 Ergo EPT. Significant intraclass correlations coefficients of r = 0.81 for average PO (30 min K1 Ergo EPT), r = 0.76 for MIP, and r = 0.95 for 10 km performance time, revealed the high repeatability of these tests. Significant relationships were found between the two endurance performance tests (30 min K1 Ergo EPT and 10 km water TT, r = -0.64, P < 0.05) and between both tests and a number of kinanthropometric, physiological and respiratory muscle function parameters. Stepwise multiple regression analysis revealed that PPO and MVV predicted endurance performance (average PO) on the K1 Ergo (R2 = 0.75, SEE = 15 W), whereas relative VO2max and best MIP predicted 10 km performance time on the water (R2 = 0.64, SEE = 115 s). The results of this study suggest that superior maximum aerobic capacities and respiratory muscle function distinguish successful paddlers from less successful paddlers and may be used to predict kayak endurance performance in the laboratory as well as on the water. No respiratory muscle fatigue occurred during the 30 min K1 Ergo EPT, indicating that respiratory muscle fatigue may not be a limiting factor to 30 min kayak endurance performance. The core stability results demonstrated no relevance to kayak endurance performance.

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2006. / The ESAT-6 gene cluster regions are duplicated 5 times in the genome of Mycobacterium tuberculosis. ESAT-6 gene cluster region 1 is the most frequently studied region as it contains RD1 (region of difference 1). RD1 is a 9.5 Kb deletion region confirmed to be involved in mycobacterial virulence and pathogenesis, and is present in virulent M. bovis strains, yet absent in all attenuated M. bovis BCG vaccine strains. The antigens CFP-10 and ESAT-6, which both evoke strong T-cell responses in experimental animals and humans, are situated in the RD1 region, and are thought to be key antigens in mycobacterial virulence. The absence of this region from the genomes of all BCG vaccine strains, led to the conclusion that the mechanism of attenuation of M. bovis BCG was due to the loss of RD1. Studies have shown that this attenuation is attributed to the loss of cytolytic activity mediated by secreted ESAT-6 (and some of the genes responsible for its secretion), which in turn results in reduced tissue invasiveness. The potent T-cell antigens ESAT-6 and CFP-10 are secreted without ordinary sec-dependent secretion signals. A study of the potential functions of the proteins encoded by the ESAT-6 gene clusters shows that most of these proteins have a potential to function in a protein-dependent ATP-binding cassette active transport system. It has been shown that ESAT-6 gene cluster region 1 is responsible for the secretion of the ESAT-6 and CFP-10 genes contained in this region, explaining the absence of any ordinary sec-dependent secretion signals in the amino acid sequences of members of this family. In order to elucidate the regulation of expression of the ESAT-6 gene cluster region 1, shown to encode for a secretion system for ESAT-6 and CFP-10 and to be involved in virulence, an operon analysis and promoter identification experiments were carried out in this study. The analysis of the ESAT-6 gene cluster region 1 showed the existence of more than one operon in this region and three constitutively-expressed promoters driving the expression of the genes in the operons. These results provide insight into the functional relationship (regulatory and secretory mechanisms) between the genes contained within ESAT-6 gene cluster region 1.None of the other four ESAT-6 gene cluster regions have been proven to also encode secretion systems. Preliminary studies indicated that the ESAT-6 gene cluster region 3 is expressed in its entirety as one single operon and a strong promoter involved in the expression of this region was identified. Mtb9.9A (the ESAT-6 antigen of the ESAT-6 gene cluster region 5) have also been shown to evoke strong T cell responses and to be secreted without any ordinary secretion signal. During the present study, we thus aimed to investigate the secretion of Mtb9.9A in order to determine whether it is also secreted by a dedicated secretion system encoded by ESAT-6 gene cluster region 5. The fact that region 5 was shown to be the last of the four duplications is important, as a positive result with this region would indicate whether the other four gene clusters share a similar secretion function. ESAT-6 gene cluster regions 2, 4 and 5 were isolated in the present study to form part of subsequent ESAT-6 gene cluster region secretion studies. Mtb9.9A was cloned, expressed and purified for antibody-generation, Resulting antibodies were used in an antigen secretion analysis. The secretion analysis entailed the integration of the isolated ESAT-6 gene cluster region 5 into the genome of M. smegmatis and investigation of the influence of the genes (contained in region 5) on the secretion of a heterologously expressed Mtb9.9A-HA-tagged fusion protein. We therefore attempted to show whether the proteins encoded by the ESAT-6 gene cluster region 5 also function together as a mycobacterial membrane-bound complex involved in protein-dependent transport and if so, whether this transport system is responsible for the active secretion of the native ESAT-6 antigen (designated Mtb9.9A) of region 5. This study opens the way for the understanding of the regulation, transport- and secretion mechanisms of important T-cell antigens of the mycobacteria, thereby giving insight into and building onto our understanding of the pathogenicity of Mycobacterium tuberculosis. A better understanding of these mechanisms could lead to the development of efficient strategies to either terminate or enhance secretion of antigens, which in turn will have an impact on drug and vaccine design and development.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: The aim of this study was to investigate the effect of MBV and Coley’s Toxin on dendritic cells in vitro. The dendritic cell system of antigen presenting cells is the initiator and modulator of the immune response. The principle function of the dendritic cells is to present antigens to resting naïve T lymphocytes: these cells are the only APCs that prime naïve T cells and only mature DCs can carry out this function.Previous studies done on dendritic cells showed that bacterial peptides can induce the maturation of dendritic cells. With the results of these studies in mind we hypothesized that these two vaccines will also induce the maturation of dendritic cells. Chapter 1 is a literature review on the immune system explaining the organs and cells of the immune system. Chapter 2 includes a full description of DCs, the MBV and Coley’s toxin. Also included in this chapter is a short explanation of the principle of the technique being used for the identification and maturation of both mDCs and pDCs, namely the technique of flow cytometry. Chapter 3 describes the method for the phenotypic identification of DCs: the subsets are distinguished by their absence of expression of several lineage markers for lymphocytes, monocytes and NK cells and the expression of CD11c (in the case of myeloid DCs) and CD123 (in the case of plasmacytoid DCs). The inclusion of HLA-DR in addition to the previous described markers allows the discrimination of CD123+ DCs from basophils. The assay requires three tubes per sample which enables quick analysis of these rare subsets with a small sample volume. This assay was applied to peripheral blood samples obtained from healthy individuals and individuals with cancer, HIV and HIV and TB co-infected patients. Our results showed that the maturation status of DCs in HIV and lymphoma were low but those measured in the case of HIV + TB patients were even higher than in the control group. Chapter 4 and 5 describe the in vitro activation and maturation status of DCs following their incubation with bacterial-derived products. Interactions between DCs and microbial pathogens are fundamental to the generation of innate and adaptive immune responses and upon contact with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of human DCs to MBV and Coley’s Toxin. Previous studies showed DCs can be activated with killed Streptococcus pyogenes. With this study in mind it was hypothesized that the MBV and Coley’s Toxin used in this study might modulate DC maturation. The results of this study showed that the MBV and Coley’s toxin did induce the maturation of both pDCs and mDCs as measured by increased surface expression of costimulatory molecules such as CD80 and CD83. Chapter 6 presents the measurement of cytokines released after the PMBCs had been were incubated with Coley’s Toxin and Mixed Killed bacteria. The BD™ Cytometric Bead Array (CBA) flex set was used for the simultaneous detection of multiple soluble analytes. The results indicated that both Coley’s Toxin and the MBV activated the DCs and subsequently induced TH1 as well as a TH2 responses in the T cells present in the cell cultures. Finally, a general conclusion discussing the significance and implications of our results as well as possible future research required is discussed in Chapter 7. DCs are potent antigen presenting cells (APCs) which play a critical role in the regulation of the immune response. There is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections, immunodeficiency diseases and autoimmune diseases. / AFRIKAANSE OPSOMMING: Die doel van die studie was om die effek van ‘n gemengde bakteriële vaksiene en Coley se toksiene op dendritiese selle te toets in vitro. Die dendritiese sel sisteem speel ‘n belangrike rol in die modulering en reaksie van die immuun sisteem.Die hoof funksie van dendritiese selle is om antigene bloot te stel aan naïewe ongeaktiveerde T selle. Slegs volwasse dendritiese selle kan die T selle aktiveer. Vorige studies het bewys dat bakteriële peptiedes die veroudering van die dendritiese selle kan induseer. Met die resultate in gedagte het ons gehipotiseer dat die twee vaksienes ook die maturasie van dendritiese selle kan induseer. Hoofstuk 1 is ‘n literatuur studie wat handel oor die organe en selle van die immuun sisteem. Hoofstuk 2 gee n volle beskrywing van dendritiese selle, die gemengde bakteriële vaksiene en Coley se toksiene. Ingesluit in die hoofstuk is die beskrywing van die prinsiep van die tegniek, vloei sitometrie, wat gebruik word vir die identifikasie en veroudering status van die dendritiese selle. Hoofstuk 3 beskryf ‘n vloei sitometrie metode vir die fenotipiese identifikasie van dendritiese selle. Dendritiese sel tipes kan onderskei word deur die afwesigheid van sekere merkers vir limfosiete, monosiete en NK selle. Plasmasitoïede dendritiese selle druk CD123 uit en miloïede dendritiese selle druk CD11c uit. HLA DR is ook ingesluit saam met die bogenoemde merkers om die dendritiese selle te onderskei van basofiele. Vir elke toets word slegs drie buise geprosesseer en dus kan die subklasse vinning geanaliseer word. ʼn Klein volume bloed word benodig vir die toests. Perifêre bloed is gebruik vir die toets op bloed monsters van 10 gesonde individue en individue met kanker, HIV en HIV en TB. Die resultate van die studie het getoon dat die maturasie status van die dendritiese selle in HIV en limfoom was, maar in die geval van HIV en TB pasïente was die maturasie status selfs hoër as die van die kontrole groep. Hoofstuk 4+5 beskryf die aktivering en maturasie status van die dendritiese selle na inkubasie met die bakteriële produkte. Interaksie tussen dendritiese selle en patogene speel ‘n belangrike rol in die aktivering van die immuunstelsel. Wanneer dendritiese selle in aanraking kom met bakterieë of bakteriële komponente, matureer die dendritiese sel wat lei tot the uitdrukking van stimulerings molekules, HLA molekules end die uitskeiding van sitokiene. Die uitdrukking van die molekules lei tot limfosiet ontwikkeling en differensiasie. In die studie het ons gekyk na die reaksie van menslike dendritiese selle in die teenwoordigheid van die gemende bakteriële vaksiene en Coley se toksiene. Vorige studies het bewys dendritiese selle word geaktiveer deur Streptococcus pyogenes. Met die resultate in gedagte het ons gehipotetiseer dat die gemengde bakteriële vaksiene en Coley se toksiene ook die maturasie van dendritiese selle kan induseer. Die resultate van die studie het bewys dat die gemengde bakteriële vaksiene en Coley se toksiene die veroudering van beide pDCs en mDCs induseer. Die uitdrukking van verouderings merkers CD80 en CD83 is gemeet. Hoofstuk 6 beskryf ‘n vloei sitometrie metode om die sitokiene te meet wat afgeskei word nadat selle geinkubeer het in die teenwoordigheid van Coley se toksiene en die gemengde bakteriële vaksiene.Die BDTM CBA Flex set metode het dit moontlik gemaak om meer as een sitokiene te meet in net een buis Die resultate het getoon dat albei die vaksienes ‘n TH1 en TH2 reaksie veroorsaak. Laastens volg‘n algemene afleiding waar ons kyk na die toepassing en implikasies van die resultate asook toekomstige navorsings moontlikhede,word bespreek in Hoofstuk 7 Dendritiese selle speel ‘n kritiese rol in die regulering van die immuun reaksie. Verdere studies kan nou gedoen word om dendritiese selle terapeuties toe te pas vir die behandeling van kanker, autoimmuniteit, immuun onderdrukkende siektes en kroniese siektes.