Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Infertility is a very common problem worldwide. Recent data have shown that disorders of the male represent the most common single defined cause of infertility. This proposal examines the clinical significance and fundamental physiological aspects of human gamete interaction. These studies are focused on the assessment of the cellular-molecular mechanisms involved in human sperm binding to its homologous zona pellucida resulting in the physiologic induction of the acrosome reaction. We have developed and validated in vitro bioassays that assess specific steps of the fertilization process that are critical for early embryo development. The results of our translational research have already had a significant impact on the overall evaluation of male infertility and on the clinical management of the infertile man in the assisted reproduction arena. Furthermore, the unveiling of the basic mechanisms involved in human gamete interaction will ultimately allow for both (i) the development of new male reproductive diagnostic capabilities and (ii) the design of improved and safer therapies aiding conception in childless couples suffering from male infertility. / AFRIKAANSE OPSOMMING: Menslike onvrugbaarheid is 'n algemene wêreldwye probleem en onlangse data toon aan dat die manlike factor die grootste enkel bydraende factor tot hierdie toestand is. Die werk loods 'n intensiewe ondersoek na die kliniese betekenis en basiese fisiologiese aspekte wat 'n rol tydens spermsel en eisel interaksie speel. Hoofstuk 3 fokus op die sellulêre en molekulêre meganismes wat betrokke is tydens spermsel en eisel binding wat gevolglik lei tot akrosoomreaksie van die spermsel. Die werk verteenwoordig die resultate van 10 jaar se navorsing tussen die kandidaat en die promoter. Dit gee oorsprong aan 'n reeks bio-toetse wat die bevrugtingsproses koriografiese ontleed en verskaf dus 'n stap-vir-stap uiteenseting van menslike bevrugting en gevolglike embrio ontwikkeling. Die resultate in Hoostuk 4 bring vernuwing in die begrippe van die manlike faktor en die rol in die kinderlose huwelik. Die resulate soos in Hoofstuk 3 en 4 uiteengesit, vorm nie net die basis vir die moontlike ontwikkeling van nuwe diagnostiese benaderings tot die hantering van die man nies maar speel oojk 'n rol die daarstelling van verbeterde terapeutiese hantering van die kinderlose egpaar. Hoofstuk 5 gee kortliks riglyne en aanbevelings tot opsigte van die gebruik van die spermsel-zona pellucida bindingstoets en akrosomreaksie. Die kandidaat bevel aan dat die genoemde twee bio-toetse deel van die laboratorium ondersoeke van die man gebruik moet word.

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although the effect of inhaled corticosteroids (ICS) on the hypothalamic- pituitary-adrenal axis (HPA) has been regarded as a “benign physiological response”, a survey published in 2002 suggested that adrenal crisis is more common in asthmatic children on ICS than previously thought. Relying on clinical features to detect chronic adrenal insufficiency secondary to corticosteroids may not be wise, as these are non-specific and can therefore easily be missed. Accurate biochemical assessment of the whole axis to detect subclinical HPA suppression (HPAS) is thus desirable. A review of the literature indicates that basal adrenal function tests, including plasma cortisol profiles, do not identify which children can appropriately respond to stress. There is no evidence to suggest that the degree of the physiological adjustment of the HPA to ICS and/or nasal steroids (by reducing basal cortisol production), predicts HPAS. Cortisol profiles should therefore only be used to demonstrate differences in systemic activity of various ICS and delivery devices. Only two tests, considered as gold standard adrenal function tests [the insulin tolerance test (ITT) and the metyrapone test] can assess the integrity of the whole axis. / AFRIKAANSE OPSOMMING: Die outeurs van ´n opname wat in 2002 gepubliseer is stel voor dat ´n bynierkrisis meer algemeen by asmatiese kinders, wat inhalasie kortikosteroïede ontvang, voorkom as wat voorheen gedink is. Dit is strydig met die gevestigde opvatting dat die effek van IKS op die hipotalamiese-hipofise-bynier-as (HHB) ’n “goedaardige fisiologiese reaksie” is. Die kliniese kenmerke van kroniese bynierontoereikendheid sekondêr tot die gebruik van kortikosteroïede (KS) is nie-spesifiek en gevolglik onbetroubaar. ´n Akkurate biochemiese toets van subkliniese HBB onderdrukking (HHBO) sou gevolglik waardevol wees. ´n Literatuur oorsig toon dat toetse van basale bynierfunksie, insluitend plasma kortisol (K) profiele, nie kinders uitken wat toepaslik op stres sal reageer nie. Daar is geen bewyse dat die graad van fisiologiese aanpassing van die HHB, soos aangedui deur laer K-vlakke, na die gebruik van IKS en/of nasale steroïede (NS), HHBO voorspel nie. Serum K profiele is dus slegs van waarde om die sistemiese aktiwiteit van verskillende IKS en toedieningsstelsels te ondersoek. Slegs twee toetse, naamlik die insulien toleransie toets (ITT) en die metyrapone -(MTP)-toets (wat beide as die goue standaard van bynier funksie beskou word), kan die integriteit van die hele as meet. / Stellenbosch University / Medical Research Council / SA Thoracic Society / Harry Crossley Foundation / Red Cross Children’s Hospital.

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: The communicable disease tuberculosis (TB) is responsible for millions of deaths each year, on a global scale. At present the contribution of host genetics in TB is generally accepted and, together with environmental aspects (e.g. nutrition and crowding) and the causative bacterium, Mycobacterium tuberculosis (M. tuberculosis); it will possibly have a hand in the outcome of disease. Clearly, TB is a multifaceted disease and the repercussions for studying genetic susceptibility are that many genes will potentially be implicated. To date a variety of genes such as NRAMP1 and HLA have been implicated in influencing the host response to TB, albeit with varying effects in different populations. Some of the more recently implicated genes are the pattern recognition receptors, the Toll-like receptors (TLRs). Genetic variation in theses genes has been associated with a myriad of different diseases, including those of an infectious nature, such as TB. In the case of TB, TLR2 is the most prominent candidate with TLR8 and 9 more recently implicated. One of the more well known genes implicated with TB is the vitamin D receptor (VDR), as the antimicrobial gene cathelicidin (CAMP), one of the most important agents of mycobacterial killing, has a VDR response element in its promoter. TLR2, VDR and CAMP are all connected in a complex pathway essential for the host defence against M. tuberculosis. Nine single nucleotide polymorphisms (SNPs) in three TLR genes (TLR2,8 & 9) were investigated via a case-control approach to determine their potential role in human genetic susceptiblity to TB in the Coloured population of South Africa. The effect of the VDR polymorphism Cdx2 on the expression of cathelicidin mRNA and protein expression was also investigated. Three genes were found to contribute significantly to genetic host susceptibility in the Coloured population of South Africa. An allelic association (p = 0.031) was observed for the TLR8 (located on the X-chromosome) SNP rs3761624, with the A-allele being more prominent in females. Four haplotypes of TLR8 were found to be significantly linked to TB susceptibility with the three SNP haplotype rs3761624-rs3764879-rs3764880, specifically the allelic combination of G/C/A [p = 0.004, OR = 2.67(95% CI: 1.90-3.74)], showing a marked association (p = 0.001). The TLR9 introexon2 boundary SNP rs352139 was significantly associated with TB susceptiblity on a genotypic (P = 0.02) and allelic scale [p = 0.05, OR=0.70; (95% CI: 0.55–0.90)], with the T allele more frequent in controls. The TLR9 two SNP haplotype consisting of rs5743836 and rs352139 was linked (p = 0.037) to TB susceptibility, specifically the combination of the alleles A/T [p = 0.013, OR=0.71; (95% CI: 0.55–0.92)]. No gene-gene interaction between TLR2, TLR8 and TLR9 was observed. No significant conclusions could be drawn from the analysis of the mRNA and protein expression of CAMP in samples harbouring the different genotypes of the VDR polymorphism Cdx2. Genetic variations in the TLR8 and 9 genes were identified as potential factors that influence genetic host susceptibility to tuberculosis in the Coloured population of South Africa. / AFRIKAANSE OPSOMMING: Die oordragbare siekte tuberkulose (TB) is elke jaar verantwoordelik vir miljoene sterftes wêreldwyd. Die invloed van die gasheer genoom op TB vatbaarheid word huidiglik aanvaar, tesame met die invloed van omgewingsfaktore (dieet, oorbevolking ens.) en die bakterium Mycobacterium tuberculosis (M. tuberculosis). Dit is duidelik dat TB ‘n veelvlakkigesiekte is wat beïnvloed sal word deur ‘n menigte verskillende gene. ‘n Verskeidenheid gene is al betrek by TB genetiese vatbaarheid, onder andere NRAMP1 en HLA, hoewel hul effekte in uiteenlopende bevolkings verskil. Sommige van die onlangse gene wat betrek is in TB genetiese vatbaarheid is die Toll-like reseptore (TLRs). Genetiese variasie in hierdie gene is geassosieer met ‘n wye verskeidenheid van siektes, insluitend aansteeklik van aard, onder andere TB. In die geval van TB speel TLR2 ‘n prominente rol, terwyl TLR8 en TLR9 meer onlangs geïmpliseer is. Een van die meer bestudeerde TB vatbaarheidsgene is die vitamiene D reseptor geen (VDR). VDR is direk betrokke in die uitdrukking van die anti-mikrobiale geen cathelicidin (CAMP),’n integrale komponent in die vernietiging van mikobakterieë. Die CAMP geen het ‘n VDR respons-element in sy promotor. Die TLR2, VDR en CAMP gene word verbind deur ‘n komplekse netwerk wat integraal is tot die liggaam se vermoë om TB af te weer. Nege enkel nukleotied polimorfismes (ENPs) in drie gene (TLR2,8 & 9) is vir hierdie studie ondersoek, deur gebruik te maak van ‘n pasiënt-kontrole assosiasiestudies, om te bepaal watter rol hul speel in genetiese vatbaarheid vir TB in die Kleurling bevoling van Suid-Afrika, al dan nie. Die invloed van die VDR polimorfisme Cdx2 op die uitdrukking van die mRNS (boodskapper ribonukleïensuur) en proteïen van die geen CAMP is ook ondersoek. Ons het gevind dat drie gene beduidend bygedra het tot genetiese vatbaarheid vir TB in die Kleurling populasie. ‘n Alleel verwante assosiasie (p = 0.031) was gevind vir die TLR8 SNP rs3761624, waar die A-alleel meer algemeen was in vroue. Vier haplotipes vir TLR8 het beduidende assosiasies met TB vatbaarheid getoon. Die drie SNP haplotipe rs3761624-rs3764879- rs3764880, spesifiek die alleel kombinasie C/G/A [p = 0.004, OR = 2.67(95% CI: 1.90-3.74)] het sterk assosiasie (p = 0.001) met TB getoon. Die TLR9 intron-ekson2 grens SNP, rs352139 het beduidende assosiasie met TB getoon op ‘n genotipiese (p = 0.02) sowel as alleliese skaal [p = 0.05, OR=0.70; (95% CI: 0.55–0.90)], met die T alleel meer algemeen in kontroles. Die twee SNP haplotipe bestaande uit rs5743836 en rs352139 het TB vatbaarheid beïnvloed (p = 0.037), spesifiek die alleliese kombinasie van A/T [p = 0.013, OR=0.71; (95% CI: 0.55–0.92)]. Geen noemenswaardige interaksies tussen TLR2, 8 en 9 is gevind nie. So ook is geen beduidende resultate gevind vir die effek van die VDR SNP Cdx2 op die uitdrukking van CAMP mRNS en proteïen nie. Genetiese variasie in die TLR8 en 9 gene is geïdentifiseer as moontlike faktore wat gasheer genetiese vatbaarheid vir TB in die Kleurlingbevolking van Suid-Afrika beïnvloed. / WW Roome Trust

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: South Africa is ranked amongst the top tuberculosis (TB) burden countries in the world and the Western Cape has a particularly high incidence of the disease. Previous studies have showed that several genes may play crucial roles in susceptibility to TB. In this study, we investigated the role of three genes previously associated with susceptibility to TB and progression to disease. These genes were Surfactant protein D (SFTPD), Macrophage receptor with collagenous structure (MARCO) and CD14. The proteins from these genes bind M. tuberculosis and are involved in the uptake of the bacteria into macrophages. The study investigated the role of ten polymorphisms from SFTPD and MARCO within a South African Coloured (SAC) population, where tuberculosis is highly prevalent. A casecontrol study design was used and polymorphisms were genotyped with Taqman® genotyping assays and amplification refractory mutation system polymerase chain reaction (ARMS-PCR). The results were analysed for association with disease, linkage disequilibrium, haplotypes and gene-gene interactions. Allele and genotype frequencies were also determined which allowed for comparisons to other populations. Five SNPs were associated with TB: two in SFTPD (rs1923537; rs2255326) and three in MARCO (rs1318645; rs3943679; rs2119112). The associated SNPs were located in regions other than exons and the effects of polymorphisms in these regions are not well understood but studies in other genes have shown them to play a functional role. Gene-gene interaction analysis showed that polymorphisms interacted with each other within and between genes, illustrating the importance of epistasis and the complexity of the genetic influences on TB. In addition to the case-control association studies, the role of the rs2569190 promoter SNP in CD14 was assessed. Gene-expression analysis was conducted with qPCR and a reporter gene assay and results from both of these approaches showed that individuals with the TT genotype had a twofold greater expression level than individuals with the CC genotype. Previously, the TT genotype has been associated with stronger promoter activity and expression of soluble CD14 in serum. Since the TT genotype was present at a higher frequency in the control group, we speculate that greater expression of CD14 may contribute to a more TB resistant phenotype. The work presented in this study illustrates the importance of the host genetic component of TB. Genetic studies will eventually revolutionize the current treatment regime as the identification of vulnerable individuals and populations will aid in the development of personalised medicines. / AFRIKAANSE OPSOMMING: Suid-Afrika is een van die top tuberkulose (TB) lande in die wêreld en die Wes-Kaap het veral ‘n hoë insidensie van die siekte. Vorige studies het gewys dat verskeie gene bydra to die vatbaarheid vir tuberkulose. In hierdie studie het ons drie gene, wat voorheen vir vatbaarheid vir tuberkulose en progressie na die siekte ondersoek is, bestudeer. Hierdie gene is Surfactant protein D (SFTPD), Macrophage receptor with collagenous structure (MARCO) en CD14. Die proteïene van hierdie gene bind M. tuberculosis en is betrokke in die opname van die bakterieë in die makrofages. Hierdie studie het tien polimorfismes van SFTPD en MARCO in die Suid-Afrikaanse Kleurlingbevolking (SAK), wat ‘n hoë TB insidensie het, getoets. Pasiënt-kontrole assosiasie studies is gedoen en polimorfismes is gegenotipeer met Taqman® genotiperingsisteem en die amplifikasie refraktoriese mutasie sisteem polimerase ketting reaksie (ARMS-PCR). Die resultate is geanaliseer vir assosiasies met TB, koppelings disekwilibrium, haplotipes en geen-geen interaksies. Alleel en genotype frekwensies is ook bepaal en vergelyk met die van ander bevolkings. Vyf enkel nukleotied polimorfismes (ENPs) is met TB geassosieer: twee in SFTPD (rs1923537; rs2255326) en drie in MARCO (rs1318645; rs3943679; rs2119112). Die geassosieerde ENPs was nie in eksons nie. Die effek van polimorfismes in areas anders as eksons word nie goed verstaan nie, maar studies het bewys dat hulle wel ‘n funksionele rol kan hê. Geen-geen interaksie analise het gewys dat polimorfismes interaksies met mekaar binne sowel as tussen gene gehad het, wat die belangrikheid van epistase en die kompleksiteit van genetiese invloede op TB illustreer. Tesame met die pasiënt-kontrole assosiasie studies is die rol van die rs2569190 promoter ENP in CD14 ook ondersoek. Geenuitdrukkingsanalise is gedoen met qPKR en rapporteerder geen toetse. Die resultate van beide hierdie benaderings het gewys dat individue met die TT genotipe twee keer soveel uitdrukkingsvlakke gehad het as individue met die CC genotipe. Die TT genotipe is voorheen geassosieer met sterk promoter aktiwiteit en die uitdrukking van oplosbare CD14 in serum. Aangesien die TT genotipe meer in die kontrolegroep gevind is, spekuleer ons dat die hoër uitdrukking van CD14 kan bydra tot ‘n meer TB weerstandbiedende fenotipe. Hierdie werk illustreer die belangrikheid van die gasheer genetiese komponent in TB. Genetiese studies sal in die toekoms die huidige behandeling regime revolusioneer, aangesien die identifikasie van individue en bevolkings met ‘n hoë risiko om TB te ontwikkel sal bydra tot die ontwikkeling van persoonlike medisynes. / The National Research Foundation (NRF); the South African Medical Research Council (MRC); Stellenbosch University and the Harry Crossley Foundation.

Thesis (PhD)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Most individuals latently infected with Mycobacterium tuberculosis (Mtb) contain the infection by a balance of effector and regulatory immune responses. However, this balance can be influenced by steroid hormones such as glucocorticoids (GCs), which are known to increase the risk of reactivation of TB. The contraceptive medroxyprogesterone acetate (MPA), which also possesses selective glucocorticoid activity, is widely used in developing countries with approximately 60% of women on contraceptives using MPA in our study cohort. Therefore, our aim was to investigate the effect of this hormone on protective immune responses to BCG in HIV negative household contacts of active TB patients. When PBMCs of TB household contacts were stimulated with BCG in the presence of 10 μM MPA; this hormone displayed both glucocorticoid as well as progestogenic properties. Similarly to cortisol, MPA suppressed antigen specific expression of a range of cytokines including IL-1α, IL-1ra, IL- 17, TNFα, IL-5 and IFNγ. Dose response curves showed that MPA can also alter expression of some cytokines at lower contraceptive doses (in the nano molar range). To assess whether this effect of MPA in vitro also occurs in women using this hormone as contraceptive the PBMCs of MPA users and controls were stimulated with BCG and the levels of up to 29 different cytokines measured by luminex analysis. PBMCs of MPA users produced significantly lower levels of cytokines involved in immune responses against Mtb such as IL-12p40, IL-1α, IL-10, IL-13 and G-CSF, which corresponds with lower numbers of circulating monocytes observed in these women. These findings warrant further investigation and clinical trials should investigate the risk of progression from latent to active TB disease in women using this contraceptive. These trials, however, require a large number of participants and are prohibitively expensive; therefore it was decided to setup an Mtb/MPA mouse model to determine the effect of MPA on the disease outcome. BALB/c and C57BL/6 mice were injected with a weekly dose of one mg MPA or PBS and infected with 30 colony forming units of Mtb H37Rv one week after commencing the hormonal treatment. Both strains were included to establish which strain best represents the human model. Three and eight weeks post infection the MPA treated C57BL/6 mice had a significantly higher bacterial load in their lungs compared to untreated mice, whereas no difference was found in the bacterial loads of the BALB/c mice. MPA treated C57BL/6 mice had significantly lower serum levels of IL-10 and G-CSF and MPA treated BALB/c mice lower serum levels of IFNγ, when compared to untreated mice. Furthermore, cells isolated from the MLNs of MPA treated C57BL/6 mice, produced significantly less TNFα, significantly more IP-10 and less IL-10 in response to PPD, while MLN cells of MPA treated BALB/c mice produced significantly less IFNγ, IL-2, IL-17, GM-CSF and MCP-1. Data of the C57BL/6 mouse strain correlated with our human data and can it therefore be said that the C57BL/6 mouse strain, together with the serum concentration of MPA used in these experiments, is a good model to determine the effect of MPA in the context of a low dose Mtb infection. To conclude MPA use could therefore alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult of adolescent women in the future. / AFRIKAANSE OPSOMMING: Die meeste mense wat latent met Mycobacterium tuberculosis (Mtb) geïnfekteer is, hou die infeksie onder beheer deur ʼn balans te handhaaf tussen effektor en regulatoriese immuunresponse. Hierdie balans kan egter beïnvloed word deur steroïedhormone soos glukokortikoïede (GCs), wat bewys is om die risiko van die heraktivering van TB te verhoog. Die voorbehoedmiddel medroksiprogesteroon-asetaat (MPA), wat ook selektiewe glukokortikoïed-aktiwiteit toon, word wyd gebruik in ontwikkelende lande en omtrent 60% van die vrouens in ons studie-bevolking wat voorbehoedmiddels gebruik, gebruik MPA. Om dié rede wou ons die effek van hierdie hormoon op die beskermende immuun-response teenoor M.bovis Bacilli Calmette-Guérin (BCG) in HIV negatiewe huishoudelike kontakte (HHKe) van pasiënte met aktiewe TB ondersoek. Ons het gevind dat wanneer perifere bloed mononukleêre selle (PBMSe) met BCG gestimuleer word in die teenwoordigheid van 10 μM MPA, hierdie hormoon beide glukokortikoïede en progesterogeniese eienskappe toon. Soos kortisol het MPA die antigeenspesifieke-uitdrukking van ʼn reeks sitokiene, insluitend IL-1α, IL-1ra, IL-17, TNFα, IL-5 en IFNγ, onderdruk. Respons kurwes wat verskillende konsentrasies van hormoon insluit, het getoon dat MPA ook by laer (nano-molare) dosisse die uitdrukking van sommige sitokiene kon verander. Om te bepaal of hierdie in vitro effek van MPA ook in vrouens wat MPA as voorbehoedmiddel gebruik voorkom, het ons PBMSe van MPA-gebruikers and kontroles met BCG gestimuleer en die vlakke van tot 29 verskillende sitokiene met behulp van Luminexanalise gemeet. PBMSe van MPA-gebruikers produseer beduidende laer vlakke van IL-12p40, IL-1α, IL- 10, IL-13 en G-CSF, wat elk in imuunafweerreaksies teen Mtb betrokke is. Die afname in dié sitokiene het gepaard gegaan met laer hoeveelhede sirkulerende monosiete. Ons resultate regverdig verdere ondersoeke en kliniese proewe behoort die risiko van progressie vanaf latente tot aktiewe TB in vrouens wat hierdie voorbehoedmiddel gebruik te bepaal. Sulke proewe vereis egter groot getalle deelnemers en is skrikwekkend duur, om die rede het ons besluit om ʼn Mtb/MPA muis-model op te stel om sodoende die algehele effek van MPA op die uitkoms van die siekte te bepaal. BALB/c en C57BL/6 muise is met ʼn weeklikse dosis van een mg MPA of sout oplossing ingespuit en een week na die aanvang van die hormoon behandeling met 30 kolonie-vormende eenhede Mtb H37Rv geïnfekteer. Beide muis tipes was ingesluit om sodoende te bepaal watter tipe die mens data die beste verteenwoordig. Drie en agt weke na die infeksie het die MPA-behandelde C57BL/6 muise ‘n beduidende hoër bakteriële lading in hul longe gehad as die onbehandelde muise, maar was daar geen verskil in die bakteriële ladings in die longe van die BALB/c muise nie. MPA-behandelde C57BL/6 muise het beduidende laer serumvlakke van IL-10 en G-CSF gehad, terwyl MPA-behandelde BALB/c muise laer serumvlakke van IFNγ gehad het. Verder het ons gevind dat die geisoleerde limfosiete van MPA-behandelde C57BL/6 muise beduidend minder TNFα, beduidend meer IP-10 en minder IL-10 geproduseer het na stimulasie met PPD, terwyl die limfosiete van MPA-behandelde BALB/c muise beduidend minder IFNγ, IL-2, IL-17, GM-CSF en MCP-1 geproduseer het. Data van die C57BL/6 muise stem ooreen met die van ons mens studie en ons kan dus vermeld dat die C57BL/6 muise, tesame met die spesifieke serumkonsentrasie van MPA wat gebruik is, ʼn goeie model is om die effek van MPA in die konteks van ʼn lae-dosis Mtb-infeksie te bestudeer. MPA gebruik kan dus die vatbaarheid vir TB, asook die erns van die siekte verander en kan ook die effektiwiteit van nuwe BCG-gebaseerde entstowwe, veral prima-hupstoot enstowwe, wat moontlik in die nabye toekoms vir volwasse en adolessente vroue toegedien kan word, verander.

Thesis (PhD)--Stellenbosch University, 2013. / Bibliography / ENGLISH ABSTRACT: Introduction: Lipoatrophy is a common adverse effect of stavudine and this effect is strongly dose-dependent. Stavudine remains the most commonly used paediatric antiretroviral drug in sub-Saharan Africa, yet when the current study began in 2009, the prevalence and severity of lipoatrophy in children on antiretroviral therapy in sub-Saharan Africa had never been studied. The development of lipoatrophy may have serious and far-reaching consequences for patients and their families. The off-label stavudine dosing method, prescribed to children whose caregivers do not have access to a refrigerator, in which the contents of an adult capsule is mixed into tap water, has potential for over-dosing or under-dosing. In addition, children on stavudine continue to be exposed to a disproportionately high dose out of line with the reduced adult dose. Aims: 1. a) To investigate the prevalence and risk factors for lipoatrophy in HIV-infected children in Southern Africa b) To identify a simple anthropometric screening tool to detect early lipoatrophy in children 2. To validate the off-label stavudine dosing method prescribed to children whose caregivers do not have access to a refrigerator, with a view to reducing the recommended dose and thereby the side-effects. Methods: 1. a) We recruited pre-pubertal children on antiretroviral therapy from a family HIV clinic in our facility. Lipoatrophy was identified by two experienced paediatric HIV clinicians using a standardized grading scale. A dietician performed dietary assessment and anthropometric measurements. Previous antiretroviral exposures were recorded. A subset of recruits received Dual-Energy X-ray Absorbtiometry scanning. b) Anthropometric measurements in children with and without lipoatrophy were compared using multivariate linear regression adjusting for age and gender. The most discerning anthropometric variables underwent Receiver Operating Characteristic curve analysis to identify the most appropriate diagnostic cut-off. 2. a) Accuracy of the standard off-label stavudine dosing method was investigated using high-performance liquid chromatography to recover active drug from solutions made up using the prescribed method. This was compared to the stated drug content of the capsules. b) Bioavailability was investigated by performing a randomized crossover pharmacokinetic study wherein healthy HIV-seronegative adult volunteers received one of two generic stavudine capsule formulations, either intact or mixed in water using the prescribed method. Plasma stavudine concentrations were assayed by liquid chromatography tandem mass spectrometry. Results: 1. a) Prevalence of lipoatrophy was 36%, and incidence was 12% per person-year. Adjusted odds ratio for developing lipoatrophy was 1.9 (CI: 1.3–2.9) for each additional year of accumulated exposure to standard-dose stavudine. b) Baseline biceps skin-fold thickness correlated well with maximum lipoatrophy grading score at any site, giving a partial correlation coefficient of 0.33 (p=0.0006), and a receiver operating characteristic area-under-curve value of 0.75 (CI: 0.64 – 0.84). Biceps skin-fold thickness <5mm at baseline had a sensitivity of 89% (CI: 67–100%) and a negative predictive value of 97% (CI: 91–100%) for predicting which children would go on to develop lipoatrophy by 15 month follow-up. Specificity was 60% (CI: 46–75%) and positive predictive value was 32% (CI: 14–50%). 2. a) Recovery of active drug from solution was 97.1%, 97.4% and 93.8% for the proprietary and two generic formulations respectively. b) Pharmacokinetic parameters of the off-label dosing method were well within the target range of intact capsule dosing for both generics. Conclusions: 1. a) The prevalence and incidence of lipoatrophy in pre-pubertal children on antiretroviral therapy in South Africa is high. Cumulative exposure to standard-dose stavudine was the greatest risk factor for lipoatrophy. b) Biceps skin-fold thickness provided reasonable sensitivity and specificity to detect and predict lipoatrophy in pre-pubertal children on antiretroviral therapy. 2. The off-label dosing method for stavudine prescribed to children whose caregivers do not have access to a refrigerator is reasonably accurate and is bioequivalent to intact capsule administration. / AFRIKAANSE OPSOMMING: Inleiding: Lipoatrofie is 'n algemene nadelige uitwerking van stavudien en hierdie effek is sterk dosis-afhanklike. Stavudien bly die mees algemeen gebruikte paediatriese antiretrovirale medikasie in sub-Sahara Afrika, maar toe ons studie begin het, was lipoatrofie in kinders op antiretrovirale terapie in sub-Sahara Afrika nog nooit voorheen bestudeer nie. Die ontwikkeling van lipoatrofie kan ernstige en verreikende gevolge vir die pasiënt en hul familie hê. Die af-etiket stavudien dosering metode voorgeskryf aan kinders wie se versorgers nie toegang tot 'n yskas het nie het 'n aansienlike potensiäal vir oor-dosering of onder-dosering. Daarbenewens, is kinders op stavudien blootgestel aan 'n disproporsionele hoë dosis uit-pas met die verminderde volwasse dosis. Doelwitte: 1. a) Om ondersoek in te stel na die voorkoms en risiko faktore vir lipoatrofie in MIV-geïnfekteerde kinders in Suid Afrika b) Om 'n eenvoudige antropometriese instrument te identifiseer om vroeë lipoatrofie op te spoor in kinders op antiretrovirale medikasie 2. Om die af-etiket stavudien dosering metode wat voorgeskryf is aan kinders wie se versorgers nie toegang tot 'n yskas het nie te valideer, met 'n oog op die vermindering van die aanbevole dosis Metodes: 1. a) Ons het 'n groep van onder-puberteitsjarige kinders op antiretrovirale terapie gewerf uit 'n familie MIV kliniek in ons fasiliteit. Lipoatrofie is geïdentifiseer deur twee ervare MIV pediaters deur gebruik van 'n gestandaardiseerde gradering skaal. 'n Diëetkundige het diëet assessering en antropometriese metings uitgevoer. Vorige antiretrovirale blootstellings is aangeteken. In 'n subset was Dual-energie X-straal Absorbtiometry (DXA) skandering uitgevoer. b) Antropometriese metings in kinders met en sonder lipoatrofie is vergelyk met behulp van meerveranderlike lineêre regressie aangepas vir ouderdom en geslag. Die mees kieskeurige antropometriese veranderlikes het Receiver Operating Curve analise ondergaan om die mees geskikte diagnostiese afgesnypunt te identifiseer. 2. a) Akkuraatheid is ondersoek deur gebruik te maak van hoë werkverrigting vloeistofchromatografie om aktiewe medikasie vanuit oplossings te herstel, wat gemeng is soos aangedui deur die voorgeskrewe af-etiket dosering metode. b) Biobeskikbaarheid is ondersoek deur die uitvoering van 'n ewekansige oorgesteekde farmakokinetiese studie waarin gesonde MIV- negatiewe volwasse vrywilligers een van twee generiese stavudien kapsule formulerings ontvang het, óf heel of in water gemeng soos aangedui deur die voorgeskrewe af-etiket dosering metode. Plasma stavudien konsentrasies is gemeet deur vloeistofchromatografie tandem massaspektrometrie. Uitslae: 1. a) Voorkoms van lipoatrofie was 36%, en insidensie was 12% per persoon-jaar. Aangepaste Odds ratio vir die ontwikkeling van lipoatrofie was 1,9 (CI: 1,3-2,9) vir elke addisionele jaar van opgehoopte blootstelling aan standaard dosis stavudien. b) Biceps vel-vou dikte <5mm het 'n sensitiwiteit van 89% (CI: 83-96%) en 'n negatiewe voorspellende waarde van 90% (CI: 84-96%) vir die opsporing en voorspelling van lipoatrofie. 2. a) Herwinning van aktiewe medikasie uit oplossings was 97,1%, 97,4% en 93,8% vir die oorspronklike en twee generiese formulerings onderskeidelik. b) Farmakokinetiese parameters van die af-etiket dosering metode was wel binne die teikenband van ongeskonde kapsule dosering vir beide generiese formulerings. Gevolgtrekkings: 1. a) Die voorkoms van lipoatrofie in onder-puberteitsjarige kinders op antiretrovirale terapie in Suid-Afrika is hoog. Die bedrag stavudien waaraan kinders blootgestel is moet hersien word. Die standaard stavudien dosis vir kinders moet herge-evalueer word. b) Biceps vel-vou dikte het redelike goeie sensitiwiteit en spesifisiteit om lipoatrofie op te spoor en te voorspel. 2. Die af-etiket dosering metode vir stavudien voorgeskryf aan kinders wie se versorgers nie toegang tot 'n yskas het nie is redelik akkuraat en is bio-ekwivalent aan ongeskonde kapsule administrasie.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / Background and purpose. To limit ischaemic injury, rapid restoration of coronary blood flow is required, which will in turn reduce infarct size. However, reperfusion itself causes myocyte death – a phenomenon termed lethal reperfusion-induced injury, which limits protection of the ischaemic myocardium. Thus the reperfusion of irreversibly damaged myocytes may accelerate the process of cell necrosis. Additive protection of the ischaemic myocardium in the form of adjunct therapy remains a topic of intensive research. Levosimendan, a calcium sensitizing agent with positive inotropic effects has in several studies been found to alleviate the damaging effects of reperfusion injury. Levosimendan has been shown to be a KATP channel opener. These channels have been implicated to play an important role in ischaemic preconditioning (IPC). With this knowledge, the aim of this study was to determine whether levosimendan and IPC have certain cardioprotective mechanisms in common and whether protection with pharmacological preconditioning could be elicited with levosimendan. In this study, we investigated whether: 1) the isolated guinea pig heart could be protected by ischaemic preconditioning (IPC) and postconditioning (IPostC), 2) the heart could be pharmacologically pre- and postconditioned, using levosimendan (LPC & LPostC), 3) a combination of IPC & LPC had an additive protective effect on the heart, 4) the KATP (both mitochondrial and sarcolemmal) channels are involved in this protection and 5) the pro-survival kinases of the RISK (reperfusion injury salvage kinase) pathway are involved. Experimental approach. Isolated perfused guinea pig hearts were subjected to three different IPC protocols (1x5, 2x5 and 3x5 minutes of ischaemia) or levosimendan (0.1μM) preconditioning, before coronary artery occlusion (CAO – 40min@36.5ºC), followed by 30 minutes of reperfusion. Hearts were also subjected to a combination of IPC & LPC, to establish whether they had additive protective effects. In addition, hearts were pre-treated with levosimendan directly before induction of sustained ischaemia (without washout of the drug – levosimendan pre-treatment (LPT)) for 10min. With the postconditioning protocol, iii the hearts were subjected to 3x30second cycles of ischaemia/reperfusion or levosimendan/vehicle. In a separate series of experiments, hearts were treated with KATP channel blockers (for both sarcolemmal & mitochondrial), before LPC, LPT and LPostC. The endpoints that were measured were: cardiac reperfusion function, myocardial infarct size and RISK pathway expression and phosphorylation (PKB/Akt and extracellular signal-regulated kinase – ERK42/44). Results. IPC, IPostC, LPC & LPostC decreased myocardial infarct size significantly compared with their controls (21.9±2.2%, 21.4±2.2%, 20.6±3.1% and 20.6±1.8% respectively vs. 46.4±1.8% for controls, p<0.05). The combination of IPC & LPC had no additive protective effect. Pre-treating the hearts with levosimendan (without washout), before index ischaemia, proved to be the most effective method of cardioprotection (infarct size: 5.8±0.9% vs. 46.4±1.8% for controls, p<0.001). With LPT a significant increase (p < 0.05 vs. control) in phosphorylation of ER42/44 was also observed. An increase in the activity of one of the RISK pathway kinases, ERK42/44 seems to be one of the reasons for LPT’s efficacy. Treating the hearts with KATP channel blockers before subjecting them to LPC, LPT & LPostC abolished the protective effects induced by levosimendan, suggesting a role for the sarcolemmal and mitochondrial KATP channels in levosimendan-induced cardioprotection. Conclusions and implications. 1) Isolated guinea pig hearts could be pre- and postconditioned within the setting of ischaemia, 2) Hearts could be pharmacologically pre- and postconditioned with levosimendan, 3) levosimendan pre-treatment is the most effective way to reduce infarct size, possibly acting by increasing the phosphorylation of ERK42/44, 4) Myocardial protection was not increased by combining IPC & LPC (suggesting similar mechanisms of protection), 5) LPC, LPT and LPostC were abolished by both sarcolemmal and mitochondrial KATP channel blockers. .LPC and especially LPT, could be useful before elective cardiac surgery while LPostC may be considered after acute coronary artery events.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the most important constitutively expressed enzymatic source of nitric oxide (NO), a major cardiac signalling molecule. On the whole, NO is regarded as a cardioprotective molecule. The role of eNOS during ischaemia / hypoxia is controversial; however, it is generally accepted that ischaemia / hypoxia results in increased cardiac NO production. Most studies focus either on the whole heart or isolated cell models. As yet, no study has compared findings with regard to NO metabolism in these two distinct models, in a single study. We hypothesise that observations in a whole heart model with regard to increased NO production and eNOS involvement in ischaemia are the result of events on cellular level and that the increase in NO production observed during hypoxia in cardiomyocytes and endothelial cells is at least in part due to the increase in expression and / or activation of eNOS. Furthermore, we hypothesize that these effects are mediated via the PI3-K / PKB pathway. We aimed to measure and compare NO-production and eNOS expression and activation in the whole heart and isolated cardiac cells and measure PKB expression and activation in the cells under normoxic and ischaemic / hypoxic conditions. We also aimed to determine the effects of PI3-K / PKB pathway inhibition on NO production and eNOS expression and activation in isolated cardiac cells under normoxic and hypoxic conditions. Adult rat hearts were perfused and global ischaemia induced for 15 and 20 min. Tissue homogenates of perfused hearts were used for the measurement of nitrites and determination of expression and activation of eNOS. Expression of eNOS in the heart was also determined by immunohistochemical (IHC) analysis. Cardiomyocytes were isolated from adult rat hearts by collagenase-perfusion, and adult rat cardiac microvascular endothelial cells (CMEC) purchased commercially. In the cells, hypoxia was induced by covering cell pellets with mineral oil for 60 min. Cell viability was determined by trypan blue and propidium iodide (PI) staining and intracellular NO production measured by FACS analysis of the NO-specific probe, DAF-2/DA and by measurement of nitrite levels (Griess reagent). Results show that in ischaemic hearts, nitrite production increased by 12 % after 15 min ischaemia and 7 % after 20 min ischaemia. Total eNOS expression remained unchanged (Western Blot and IHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15 min ischaemia and decreased by 43% after 20 min ischaemia. In the cells, both viability techniques verified that the hypoxia-protocol induced significant damage. In isolated cardiomyocytes, NO-production increased 1.2-fold (by DAF-2/DA fluorescence), total eNOS expression increased 2-fold and activated eNOS increased 1.8-fold over control. In CMECs, NO-production increased 1.6-fold (by DAF-2/DA fluorescence), total eNOS increased by 1.8- fold and activated eNOS by 3-fold. With regards to our PI3-K / PKB investigations, results showed an increase of 84 % and 88 % in expression vii and activation of PKB (phospho Ser473) in hypoxic cardiomyocytes, respectively. In hypoxic CMECs, there was no change in PKB expression but there was a 69 % increase in phosphorylated PKB. NO production in wortmannin-treated hypoxic cardiomyocytes decreased by 12 % as compared to untreated hypoxic cells. In treated hypoxic CMECs, NO production decreased by 58 % as compared to untreated hypoxic cells. Treatment with wortmannin did not change the expression of eNOS protein in the cardiomyocytes, however, activated eNOS decreased by 41 % and 23 % under baseline and hypoxic conditions in treated cells respectively. There was a significant increase in NO production after exposure to O2 deficient conditions in all models investigated, a trend similar to what previous studies in literature found. However, the source of this NO is not fully understood although it has been discovered that NOS plays a role. Our data reveals similar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in the cells; however, the trends observed at 20 min ischaemia are in conflict with our cell data (i.e. decrease in activated eNOS). This may be due to the severity of the ischaemic insult in whole hearts and/or the presence of other cell types and paracrine factors in the whole heart. Hypoxia increased the activation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKB pathway reduced NO production and hypoxia-induced eNOS activation in cardiomyocytes. In conclusion, we have, for the first time, demonstrated that the increase in NO production during hypoxia is due (at least in part) to an increase in eNOS phosphorylation at Ser1177 and that this is mediated via the PI3-K / PKB pathway.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: It has recently been observed that the application of multiple short cycles of reperfusion and ischaemia, at the onset of reperfusion, elicits cardioprotection against injury due to prior sustained ischaemia. This phenomenon has been termed “postconditioning” (postC) and is of special interest due to its clinical applicability. Although much work has been done to delineate the mechanism of protection, there is still controversy regarding the precise algorithm of postC, the importance of the reperfusion injury salvage kinases (RISK), as well as uncertainty about the possible role of p38 MAPK and the protein phosphatases in postC cardioprotection. The aims of this study were therefore: I. To develop and characterise a cardioprotective postC protocol in the ex vivo rat heart, using both the retrogradely perfused and working heart models. II. To characterise the profiles of PKB/Akt, ERK p42/p44 and p38 MAPK associated with the postC intervention. III. To investigate the possible role of the serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A) in the mechanism of postC. Hearts from male Wistar rats were perfused in both the retrograde Langendorff (at a perfusion pressure of 100 cmH2O and diastolic pressure set between 1 and 10 mmHg) and working heart models (preload: 15 cmH20 and afterload: 100 cmH20). Several different postC protocols were tested for their cardioprotective effect, as analysed by infarct size (IFS; determined by triphenyltetrazolium chloride (TTC) staining) and functional recovery. Experimental parameters tested were the number of cycles (3,4 or 6), the duration of the cycles (10, 15, 20 or 30 seconds), the method of application (regional or global) and temperature during the intervention (36.5 or 37 °C). Different sustained ischaemic insults were also utilised: 35 minutes regional (RI) or 20, 25, 30 and 35 minutes global ischaemia (GI). Hearts treated with a cardioprotective postC intervention or standard reperfusion after sustained ischaemia, were freeze-clamped at 10 and 30 minutes reperfusion in both perfusion models. Tissue samples were then analyzed using Western blotting, probing for total and phosphorylated PKB/Akt, ERK p42/p44 and p38 MAPK. The contribution of PKB/Akt and ERK p42/p44 activation to cardioprotection was also investigated by administration of inhibitors (A6730 and PD098059 respectively) in the final 5 minutes of ischaemia and the first 10 minutes of reperfusion, in the presence and absence of the postC intervention. The effect of these inhibitors were analyzed in terms of IFS and kinase profiles. The possible role of the phosphatases in postC was investigated by observing the effect of cantharidin (a PP1 and PP2A inhibitor) treatment directly before sustained ischaemia (PreCanth) or in reperfusion (PostCanth), in the presence and absence of postC, on IFS and kinase profiles. A postC protocol of 6x10 seconds global reperfusion / ischaemia, at 37°C, was found to give the best and most consistent reduction in infarct size in both the Langendorff (IFS in NonPostC: 47.99±3.31% vs postC: 27.81±2.49%; p<0.0001) and working heart (IFS in NonPostC: 35.81±3.67% vs postC: 17.74±2.73%, p<0.001) models. It could however only improve functional recovery in the Langendorff model (after 30 minutes GI: rate pressure product (RPP) recovery: NonPostC = 12.27±2.63% vs postC = 24.61±2.53%, p<0.05; and after 35 minutes GI: left ventricular developed pressure (LVDP) recovery: NonPostC = 28.40±7.02% vs postC = 48.49±3.14%, p<0.05). This protection was associated with increased PKB/Akt (NonPostC: 0.88±0.26 AU (arbitrary unit) vs postC: 1.65±0.06 AU; p<0.05) and ERK p42 (NonPostC: 2.03±0.2 AU vs postC: 3.13±0.19 AU; p<0.05) phosphorylation. Inhibition of PKB/Akt activation with A6730 (2.5 μM) abrogated the infarct sparing effect of postC. Administration of cantharidin, either before of after ischaemia, in the absence of postC, conferred an infarct sparing effect (IFS in PreCanth: 15.42±1.80%, PostCanth: 21.60±2.79%; p<0.05) associated with an increase in the phosphorylation of MAPK p38 (administration before ischaemia: NonCanth: 1.52±0.26 AU vs PreCanth: 2.49±0.17 AU, p<0.05; and administration after ischaemia: NonCanth: 5.64±1.17 AU vs PostCanth: 10.69±1.29 AU, p<0.05) and ERK p42 (when administered in reperfusion; NonCanth: 2.24±0.21 AU vs PostCanth: 3.34±0.37 AU; p<0.05). Cantharidin treatment combined with the postC intervention did not elicit an additive infarct sparing effect (postC: 17.74±2.72%, PreCanth-postC: 13.30±3.46% and PostCanth-postC: 15.39±2.67%). In conclusion: a postC protocol of 6x10 seconds global ischaemia / reperfusion, at 37°C, confers the best infarct sparing effect in both the Langendorff and working rat heart models. This protection is associated with ERK p42 and PKB/Akt phosphorylation, although only PKB/Akt is necessary for cardioprotection. We could not find evidence for PP1 and PP2A involvement in postC, although inhibition of these phosphatases per se does elicit an infarct sparing effect. The latter observation suggests that phosphatase activation during ischaemia / reperfusion is potentially harmful. / AFRIKAANSE OPSOMMING: Dit is onlangs waargeneem dat toediening van meervoudige siklusse herperfusie / iskemie, met die aanvang van herperfusie, die hart teen iskemie / herperfusie beskadiging beskerm. Hierdie verskynsel, bekend as postkondisionering (postC), geniet tans baie aandag vanweë die kliniese toepaslikheid van die ingreep. Ten spyte van intensiewe navorsing om die betrokke meganisme van beskerming vas te stel, is daar steeds kontroversie oor die presiese algoritme van die ingreep, asook die betrokkenheid van die sogenaamde iskemie herperfusie oorlewings kinases (RISK). Daar bestaan ook onsekerheid oor die rol van die stres-kinase, p38 MAPK, asook die proteïen fosfatases in die meganisme van beskerming teen iskemiese besering. Hierdie studie het dus drie doelstellings gehad: I. Ontwikkeling van ‘n postC protokol wat beskerming ontlok in die rothart ex vivo, deur gebruik te maak van beide die retrograad geperfuseerde ballon model, asook die werkhart model. II. Analiese van die profiele van die kinases PKB/Akt, ERK p42/p44 en p38 MAPK tydens herperfusie van postC en kontrole (NonPostC) harte. III. Ondersoek na die moontlike rol van die serien / treonien proteïen fosfatases tipe 1 en tipe 2A (PP1 en PP2A) in die meganisme van postC beskerming. Harte van manlike Wistar rotte is geperfuseer in beide die retrograad geperfuseerde ballon (d.i. die Langendorff) model (teen ‘n konstante perfusie druk van 100 cmH20 en ‘n diastoliese druk gestel tussen 1 en 10 mmHg), asook die werkhart model (teen ‘n voorbelading van 15 cmH20 en ‘n nabelading van 100 cmH20). Verskeie moontlike postC protokolle is getoets vir hul vermoë om kardiobeskerming te ontlok, in terme van funksionele herstel en infarktgrootte (IFS), soos bepaal deur trifenieltetrazolium chloried (TTC) kleuring. Die eksperimentele veranderlikes tydens die postC protokol wat ondersoek is, sluit in: die aantal siklusse (3, 4 of 6), die duur van die siklusse (10, 15, 20 of 30 sekondes), die wyse van postC toediening (streeks of globaal) en laastens die temperatuur tydens die ingreep (36.5 of 37 °C). Daar is ook gebruik gemaak van verskillende periodes iskemie: 35 minute streeks iskemie (RI), asook 20, 25, 30 en 35 minute globale iskemie (GI). Na 10 of 30 minute herperfusie is harte wat blootgestel is aan ‘n kardiobeskermende postC ingreep of gewone standaard herperfusie na iskemie, in beide perfusie modelle, gevriesklamp. Die weefsel proteïen-inhoud is verder geanaliseer deur van die Western blot tegniek gebruik te maak vir bepaling van die totale en fosforileerde vlakke van PKB/Akt, ERK p42/p44 en p38 MAPK. Die funksionele belang van PKB/Akt en ERK p42/p44 is verder ondersoek deur die effek van ‘n geskikte inhibitor (onderskeidelik A6730 en PD098059, toegedien tydens die laaste 5 minute van iskemie en die eerste 10 minute van herperfusie), in die teenwoordigheid en afwesigheid van die postC ingreep, op infarktgrootte en kinase aktiwiteit te monitor. Die moontlike rol van proteïen fosfatases in postC is ondersoek deur die effek van cantharidin (‘n PP1 en PP2A inhibitor) op infarktgrootte en kinase profiele te ondersoek. Cantharidin is óf onmiddelik voor iskemie óf tydens herperfusie toegedien, in die aan – en afwesigheid van die postC ingreep. Daar is bevind dat ‘n postC protokol van 6x10 sekondes globale iskemie / herperfusie, teen 37°C, die mees effektiewe en konstante verlaging in infarktgrootte teweeg gebring het in beide die ballon model (IFS in NonPostC: 47.99±3.31% vs postC: 27.81±2.49%; p<0.0001), asook die werkhart (IFS in NonPostC: 35.81±3.67% vs postC: 17.74±2.73%, p<0.001). Funksionele herstel kon egter slegs ontlok word in die ballon model (na 30 minute GI: tempo druk produk (RPP) herstel: NonPostC = 12.27±2.63% vs postC = 24.61±2.53%, p<0.05; en na 35 minute GI: linker ventrikulêre ontwikkelde druk (LVDP) herstel: NonPostC = 28.40±7.02% vs postC = 48.49±3.14%, p<0.05). Die infarkt-besparende effek van postC was geassosieer met ‘n toename in die fosforilasie van beide PKB/Akt (NonPostC: 0.88±0.26 AU (arbitrêre eenhede) vs postC: 1.65±0.06 AU; p<0.05) en ERK p42 (NonPostC: 2.03±0.2 AU vs postC: 3.13±0.19 AU; p<0.05). Inhibisie van PKB/Akt met A6730 (2.5 μM) het die infarkt-besparende effek van postC opgehef. Inhibisie van PP1 en PP2A opsigself, deur toediening van cantharidin óf voor óf na iskemie (in die afwesigheid van postC), het ‘n infarkt-besparende effek ontlok (IFS in PreCanth: 15.42±1.80%, PostCanth: 21.60±2.79%; p<0.05). Hierdie kardiobeskerming was geassosieer met ‘n toename in die fosforilasie van beide p38 MAPK (met toediening voor iskemie: NonCanth: 1.52±0.26 AU vs PreCanth: 2.49±0.17 AU, p<0.05; en toediening na iskemie: NonCanth: 5.64±1.17 AU vs PostCanth: 10.69±1.29 AU, p<0.05), asook ERK p42, indien cantharidin toegedien is tydens herperfusie (NonCanth: 2.24±0.21 AU vs PostCanth: 3.34±0.37 AU; p<0.05). Kombinasie van cantharidin behandeling met postC toediening kon egter nie ‘n kumulatiewe infarkt-besparende effek uitlok nie (postC: 17.74±2.72%, PreCanth-postC: 13.30±3.46% en PostCanth-postC: 15.39±2.67%). In samevatting: ‘n PostC protokol van 6x10 sekondes globale iskemie / herperfusie, teen 37°C, ontlok die mees effektiewe infarkt-besparende effek in beide die ballon, sowel as die werkhart modelle. Alhoewel hierdie beskerming geassosieer is met ‘n toename in die fosforilasie van beide PKB/Akt en ERK p42/p44 tydens herperfusie, is dit slegs PKB/Akt wat van funksionele belang is in die meganisme van kardiobeskerming. Ons kon geen bewyse vind vir die betrokkenheid van PP1 en PP2A in postC beskerming nie, alhoewel inhibisie van hierdie fosfatases opsigself infarkt-besparend is. Laasgenoemde waarneming toon dat fosfatase aktivering tydens iskemie / herperfusie skadelike gevolge mag hê.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings especially clinical trails. It is a powerful tool for the measuring of chemical (the fluorochrome we add) and physical (size and complexity) characteristics of individual cells. As these instruments became major diagnostic and prognostic tools, the need for more advanced quality control, standardized procedures and proficiency testing programs increased as these instrumentations and their methodology evolve. Minor instrument settings can affect the reliability, reproducibility and sensitivity of the cytometer and should be monitored and documented in order to ensure identical conditions of measurement on a daily basis. This can be accomplished by following an Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program. Currently there are no such programs available in South Africa and poorer Africa countries. HIV is a global concern and the laboratories and clinics in these places are in need of such IQA programs to ensure quality of their instrumentation and accurate patient results. Quality assurance programs such as CD Chex® and UK Nequas are available but due to bad sample transport, leave the receiving laboratories with nightmares. It would be best if there was a laboratory in South Africa that could provide the surrounding laboratories with stabilized whole blood samples that can be utilized as IQA. The transport of these samples can be more efficient due to shorter distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general terminology and concepts of immunology. Chapter two describes in detail the impact stabilized whole blood had on clinical immunology concerning Quality Control and Quality Assurance. The objective of this study is to stabilize whole blood with a shelf life of greater than 30 days to serve as reference control material for South African Immunophenotyping. It is further an objective to use these in-house stabilized control samples for poorer African countries as Internal Quality Assurance reference material. It is a still further objective to stimulate various lymphocyte subsets to express activation antigens and then stabilize these cells for more specialized immunological test and can serve as a QC for those required samples. Study design: In Chapter three, the method currently used to stabilize whole blood was modified. The stability of different concentrations of a first stabilizing agent (Chromium Chloride hexahydrate) was investigated. Incubation periods and concentrations of paraformaldehyde as second stabilizing agent were investigated. Blood samples from healthy individuals (n=10) were stabilized and monitored for the routine HIV phenotypic surface antigens over a period of 40 days. These samples (n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™ versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and monitored to identify phenotypic cell surface molecules for as long as possible. They were quantified on both flow cytrometric instruments. In addition, these stabilized samples (n=3) were investigated as control blood for calibration purposes on the BD FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40 Ligand on the T helper cell surfaces. These activated antigens were analyzed on the BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future. Results: In Chapter three, the ten individual stabilized samples had non-significant P values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™, resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic markers until day 84. The cells populations were easily identifiable and could be quantified on both BD FACSCalibur™ and BD FACSCount™ instruments. In Chapter four; for the activation study purposes, activated T helper lymphocytes expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface expression, 6 hour incubation was optimum. The stimulus of choice in this case was 4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25 expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69, CD25 and HLA-DR were stabilized in the same manner and stability could be achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA) designed to simulate whole blood having defined properties in clinical laboratory situations. In future kits can be developed with a low, medium and high control sample for the various immunological phenotypic determinants. Another kit can be compiled where various activation markers can be identified, quantified with a “zero”, low and high control. These whole blood IQA kits and “activation IQA kits” can be implemented for training of newly qualified staff, competency testing of staff, method development, software testing, panel settings and instrument setting testing. Control samples ideally must have a number of properties in order to be effective. For instance stability during storage times, preferably lasting more than a few weeks, reproducibility and ease of handling. These will provide the information on day-to-day variation of the technique or equipment which will enhance accuracy and improve patient care. / AFRIKAANSE OPSOMMING: Inleiding: Vloeisitometrie tegnologie het verskeie tradisionele laboratorium toetse vervang as gevolg van beter akuraadheid, sensitiwiteit en vinniger beskikbaarheid van resultate in ‘n kliniese omgewing, veral kliniese proewe. Vloeisitometrie is ‘n kragtige tegniek om chemiese (fluorokroom byvoeging) en fisiese (sel grote en kompleksiteit) karakter eienskappe van individuele selle te meet. Met die toename in gebruik en gewildheid van hiedie instrumente, neem die behoefde toe vir gevorderde kwaliteit kontroles, gestandardiseerde prosedures, met profesionele toets programme tesame met metode ontwikkeling. Klein verstellings aan instrument parameters beinvloed die betroubaarheid, herhaalbaarheid en sensitiwiteit van ‘n sitometer en moet gemonitor (en dokumenteer) word om identiese kondisies van leesings op ‘n daaglikse basis te verseker. Dit kan bereik word deur in te skakel met ‘n interne kwaliteits versekerings program [IQA: “Internal Quality Control”] en/of ‘n eksterne kwaliteits versekerings program [EQA: “External Quality Control”] te volg. Op die oomblik is daar geen sulke kwaliteits versekerings programme in Suid Afrika en/of in die verarmende Afrika lande beskikbaar nie. MIV is ‘n wêreldwye bekommernis en laboratoriums en klinieke in hierdie gedeeltes van die land verlang ‘n dringende behoefdte vir sulke “IQA” programme om kwaliteit van instrumentasie en akkurate pasiënt resultate te verseker wat tot beter behandeling van pasiënte lei. Kwaliteit versekerings programme soos “CD Chex®” en “UK Nequas” is beskikbaar, maar baie probleme met verwysing na monster integriteit as gevolg van tydsame vervoer en aflewering kondisies word hiermee geassosieër. Die behoefte het ontstaan vir ‘n laboratorium in Suid Afrika wat direk die omliggende laboratoriums, hospitale en klinieke kan voorsien met gestabiliseerde blood monsters wat gebruik kan word as “IQA”. Die vervoer en aflewerings kondisies van hierdie monsters sal aansienlik verbeter as gevolg van die korter aflewerings afstand wat direk die beperkte temperatuur wisseling beinvloed. Doel van studie: Die doelwit van hoofstuk een is om vir die leser ‘n inleiding te gee tot terminologie en konsepte van immunologie en die immune sisteem. Hoofstuk twee beskyf die impak wat gestabiliseerde heelbloed het op die kliniese immunologie met betrekking tot kwaliteit beheer en kwaliteit versekering. Die doelwit van hierdie studie is om heelbloed te stabiliseer sodat die rakleeftyd meer as 30 dae is en sodoende as verwysings-materiaal kontroles vir Suid Afrikaanse immunofenotipering kan dien. Dit is ‘n verdere doelwit om hierdie tuis-gestabiliseerde kontrole monsters te gebruik as “IQA” verwysings materiaal in verarmende Afrika lande. Die doelwit van hoofstuk vier is om limfosiete te stimuleer om verskeie aktiverings merkers uit te druk op hul selmembrane en dan te stabiliseer en dié te gebruik as Kwaliteits Kontroles vir die meer gespesialiseerde immunologiese toetse. Studie ontwerp: Hoofstuk drie beskryf ‘n aangepaste en verbeterde metode van heel bloed stabiliseering. Stabiliteit word ondersoek in ‘n verskyndenheid konsentrasies van ‘n primêre stabiliseerings agent (chromium chloried heksahidraat) en inkubasie periodes met paraformaldehied as tweede stabiliseerings agent word deeglik gedokumenteer. Bloedmonsters van gesonde indiwidië (n=10) was gestabiliseer en gemonitor vir roetine MIV membraanoppervlak antigene oor ‘n periode van 40 dae. Hierdie monsters (n=10) was gelees en geanaliseer op ‘n BD FACSCalibur™ en vergelyk met ‘n BD FACSCount™ vloeisitometer instrument. Drie gestabiliseerde heelbloed monsters (n=3) was gemonitor vir ‘n periode vir so lank moontlik die fenotipiese selmembraan molekules identifiseerbaar was en die kwantiteit bepaalbaar was. Hierdie drie monsters was gemeet op beide instrumente. As ‘n addisionele doelwit, was hierdie drie gestabiliseerde monsters ondersoek om as moontlike kalibrasie materiaal (verteenwoordig ‘n normale bloedmonster) te dien vir die BD FACSCount™ instrument in die oggende voor pasiënt monsters gelees kan word. In hoofstuk vier was limfosiete geϊsoleer en geaktiveer met ‘n verskyndenheid stimulante om optimale aktiveerings-antigene uit te druk op T helper selmembrane (byvoorbeeld CD25, CD69, HLA-DR en CD40 Ligand). Hierdie geaktiveerde monsters was geanaliseer op die BD FACSCalibur™ en daarna gestabiliseer. Na stabilisasie van die geaktiveerde limfosiet monsters was dit gemonitor oor ‘n tydperk so lank moontlik data plotte leesbaar en selpopulasies identifiseerbaar was. Hierdie monsters kan dien as ‘n moontlike “IQA” toets stel vir ‘n meer gespesialiseerde immunologiese aktiveerings kontrole doeleindes. Resultate: In hoofstuk drie; tien individiële gestabiliseerde heelbloed monsters het gedui op geen-beduidende P waardes (P > 0.05) vir CD3, CD4 en CD8 persentasies en absolute waardes; gemeet vanaf DAG 3 vergelykbaar tot-en-met DAG 40. Met korrelasie statistiek en vergelyking van die BD FACSCalibur™ met die FACSCount™ instrumente, is die volgende opgemerk; R2 = 0.9848 vir die CD4 absolute waardes en ‘n R2 = 0.9636 vir die CD8 absolute waardes. Drie gestabiliseerde monsters (n=3) was gemonitor vir MIV roetine fenotipeering tot en met DAG 84. Die selpopulasies was duidelik identifiseerbaar en die kwantitatief meetbaar op albei instrumente (BD FACSCalibur™ en BD FACSCount™). Hoofstuk vier: geaktiveerde T helper lymphosiete het 25 – 35% membraan CD40 Ligand uitgedruk op hul selmembrane. Die stimulant van keuse was ionomysien teen ‘n optimale konsentrasie van 4μM. Die optimale inkubasie tydperk was vier ure by 37°C in 5% CO2 kondisie. Ses uur inkubasie in 4μM ionomysien by 37°C in ‘n 5% CO2 omgewing was optimal vir die CD69 selmembraan uitdrukking en het 84.21% opgelewer. Vir CD25 selmembraan uitdrukking was die selle vir ses ure met phietoheamagglutinin (PHA) gestimuleer by 37°C in 5% CO2 kondisie en het 43% CD25 selmembraan uitdrukking opgelewer. HLA-DR selmembraan uitdrukking: selle was vir ses ure saam met PHA by 37°C in 5% CO2 kondisie inkubeer en het 43.32% opgelewer. CD40 Ligand aktivering/gestabiliseerde limfosiete het tot en met dag 23 stabiliteit getoon. Die ligand was duidelik identifiseerbaar en kwantifiseerbaar. Geaktiveerde lymphosiete wat CD69, CD25 en HLA-DR selmembraan merkers uitdruk het na die stabiliseerings proses stabiliteit getoon tot-en-met dag 16. Gevolgtrekking: Die doel van hierdie studie was om verwysingskontroles voor te berei sodat dit vars heelbloed naboots met uitkenbare eienskappe vir kliniese situasies. ‘n Toets kontrolestel met verwysings materiaal vir drie vlakke (byvoorbeeld ‘n lae, medium en hoë kontrole) absolute selwaardes en persentasies kan voorberei word vir roetine immunologiese fenotiperings merkers (CD3/CD4/CD8/CD45). Meer gespesialiseerde kontrolestelle vir meer spesifieke doeleindes kan opgemaak word wat ‘n verskydenheid van limfosiet aktiveringsmerkers bevat met byvoorbeeld ‘n “nul”, lae en hoë verwysings kontrole daarin. Hierdie heelbloed kan dien as “aktiveerde interne kwaliteits verwysings materiaal” en kan gebruik word om nuut aangestelde laboratorium werkers en nuut gekwalifiseerde studente op te lei. Hierdie verwysings materiaal / kontroles kan aangewend word vir bevoegdheids doeleindes (byvoorbeeld vir SANAS akkreditasie doeleindes), vir metode ontwikkeling, vir sagteware toetsing, vir paneel opstelling en instrument verstellings doeleindes. Die kontroles moet ‘n verskydenheid eienskappe bevat om effektief te wees. Byvoorbeeld, stabiliteit tydens storing, gewenslik meer as ‘n paar weke, herhaalbaar en maklik handteerbaar. Hierdie kontroles sal inligting voorsien op ‘n daaglikse basis tydens wisseling van tegnieke of instrumentasie wat akuraatheid beinvloed en op die ou-end direk pasiënt versorging bevoordeel.