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

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

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

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

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

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

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

Bibliography / Thesis (PhD ( Interdisciplinary Health))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Introduction: It is increasingly being recognized that how intensive care services are delivered may have a greater impact on patient outcome than the individual therapies. Uncertainty regarding the optimal physiotherapy service provision model in a surgical intensive care unit (ICU) exists. Methodology: The aims of this study were to 1) develop an evidence-based physiotherapy protocol; 2) validate the content of the protocol; and 3) conduct an explorative intervention trial to compare usual care to the estimated effects of providing a physiotherapy service guided by an evidence-based physiotherapy protocol by a dedicated physiotherapist. A systematic review process was used to synthesize the evidence in eight subject areas. The GRADE system was used to formulate best practice recommendations and algorithm statements. Forty-two experts from a variety of disciplines were invited to participate in a Delphi process. Finally, the evidence-based physiotherapy protocol was implemented in a surgical ICU over four three-week intervention periods by a group of research therapists. The outcomes measured included ventilator time, ventilation proportions, failed extubation proportions, length of ICU and hospital stay, mortality, functional capacity, functional ability and cost (using nursing workload as proxy). Results: Fifty-three research reports in eight subject areas were identified, 23 draft best-practice recommendations and 198 algorithm statements were formulated. The draft protocol consisted of five clinical management algorithms. Fifteen international research experts and twelve national academics in the field of critical care agreed to participate in the Delphi process. Consensus was reached on the formulation of 87% (20/23) recommendations and the rating of 66% (130/198) statements. The risk of an adverse event during the protocol care intervention period was 6:1000 treatment sessions (p=0.34). Patients admitted to the unit during the protocol care intervention period were less likely to be intubated (RR 0.16 95%CI 0.07 – 0.71; RRR 0.84 NNT 5.02; p=0.005) or fail extubation (RR 0.23 95%CI 0.05 – 0.98; RRR =0.77 NNT 6.95; p=0.04). The mean difference in the daily unit TISS-28 score between the two condition periods was 1.99 95%CI 0.65 – 3.35 (p=0.04). Patients managed by the protocol tended to remain in the hospital for a shorter time after unit discharge (p=0.05). There was no difference in the time spent on the ventilator (p=0.50), mortality (p=0.52) or in the six minute walk distance (p=0.65). In addition there was no difference in the proportion of patients who reached independence in any of the Barthel Index activities measured within 48 hours of discharge from the unit. Conclusions: The use of an evidence-based physiotherapy protocol for the comprehensive physiotherapeutic management of patients in a surgical ICU was feasible and safe. The preliminary results of this study suggest that a physiotherapy service, which is guided by an evidence-based protocol and offered by a dedicated unit therapist, has the potential to lower the cost of ICU care and facilitate the functional recovery of patients after unit discharge. This information can now be considered by administrators to optimize the physiotherapy service provided in ICU. / AFRIKAANSE OPSOMMING: Inleiding: Daar word toenemend erken dat die wyse waarop dienste gelewer word, ‘n groter impak mag hê op die uitkoms van pasiënte as die spesifieke modaliteite in gebruik. Onsekerheid heers tans oor die optimale fisioterapie diens model om te volg in ‘n chirurgiese intensiewe sorg eenheid (ISE). Metodologie: The doel van hierdie projek was om 1) ‘n bewysgesteunde protokol te ontwikkel; 2) die geldigheid van die protokol te bevestig; en 3) om deur middel van ‘n eksploratiewe studie die uitkoms van pasiënte te vergelyk wanneer die fisioterapie diens gelewer word aan die hand van die bewysgesteunde protokol deur ‘n toegewyde fisioterapeut, teenoor wanneer die gewone fisioterapie diens gelewer word. Die empiriese bewyse in agt onderwerp areas is gesintetiseer na afloop van ‘n sistematiese literatuur oorsig proses. Die GRADE sisteem is gebruik om beste praktyk aanbevelings en algoritme stellings te formuleer. Twee en veertig kundige persone van verskeie disiplines is genooi om deel te neem aan die Delphi proses om die geldigheid van die protokol te bevestig. Uiteindelik is die geldige bewysgesteunde protokol oor ‘n tydperk van vier drie weke intervensie periodes deur ‘n groep navorsings terapeute in ‘n chirurgiese ISE geïmplementeer. Die tyd wat pasiënte geventileer is, die proporsie pasiënte wat geïntubeer en geherintubeer is in die tydperk, die lengte van ISE en hospitaal verblyf, mortaliteit, funksionele kapasiteit asook funksionele vaardigheid en koste (deur die verpleeg werkslading te gebruik as ‘n indikasie van koste) is gemeet. Resultate: Drie en vyftig navorsings verslae in agt onderwerp areas is geïdentifiseer, 23 konsep aanbevelings en 198 algoritme stellings is geformuleer. Die konsep protokol het uit vyf algoritmes bestaan. Vyftien internasionale en twaalf nasionale kundiges het die uitnodiging aanvaar om aan die delphi proses deel te neem. Konsensus is bereik vir die formulering van 87% (20/23) van die aanbevelings en die gradering van 66% (130/198) van die algoritme stellings. Die risiko vir ‘n ongunstige episode tydens die protokol intervensie periode was 6:1000 sessies (p=0.34). Pasiënte wat tydens die protokol intervensie periode tot die eenheid toegelaat is was minder geneig om geïntubeer te word (RR 0.16 95%CI 0.07 – 0.71; RRR 0.84 NNT 5.02; p=0.005) of om ‘n ekstubasie te faal (RR 0.23 95%CI 0.05 – 0.98; RRR =0.77 NNT 6.95; p=0.04). Die gemiddelde verskil in die daaglikse eenheid TISS-28 telling tussen die twee intervensie periodes was 1.99 95%CI 0.65 – 3.35 (p=0.04). Patiente wat tydens die protokol intervensie periode behandel is was geneig om vinniger uit die hospitaal ontslaan te word nadat hul uit die eenheid ontslaan is (p=0.05). Daar was geen verskil in die ventilasie tyd, (p=0.50) die mortaliteit (p=0.52) of die afstand wat pasiente in ses minute kon aflê binne 48 uur na ontslag uit die eenheid (p=0.65) nie. Daar was ook geen verskil in die proporsie pasiente wat onafhanklikheid bereik het in enige van die kategorieë van die Barthell Index instrument nie. Gevolgtrekking: Die gebruik van die protokol vir die omvattende hantering van pasiënte in ‘n chirurgiese eenheid is haalbaar en veilig. Die voorlopige resultate van hierdie studie dui daarop dat wanneer ‘n fisioterapie diens in ‘n chirurgiese ISE gelewer word aan die hand van ‘n bewysgesteunde protokol deur ‘n toegewyde fisioterapeut dit die potensiaal het om ISE koste te verminder en die funksionele herstel van pasiente na ontslag uit die eenheid te fasiliteer. Hierdie inligting kan nou deur administrateurs oorweeg word om ‘n optimale fisioterapie diens in ‘n chirurgiese ISE te verseker.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Background to the study OLA can give rise to certain problems: 1. A significant decrease in lung volume is reported to occur in the dependent lung during OLA in the LDP. This decrease in lung volume can result in an acute increase in opposition to RV ejection. The potential problem is that the right ventricle is a thin walled structure that can generate considerably less work than the thicker walled LV. It possesses little reserve to deal with an acute rise in afterload as may occur during acute lung injury or after lung resection. Therefore, this increase in afterload during OLA may potentially impair RV-PA coupling. Albeit this potential problem exists, the changes in RV afterload and how the right ventricle performs during OLA have not been well studied. 2. Arterial hypoxemia, due mainly to venous blood being shunted via the non-ventilated lung, may present a clinical problem during one lung ventilation. a. The relative resistances of the pulmonary vascular beds of the dependent ventilated and nondependent non-ventilated lungs are an important factor governing shunting and thus arterial oxygenation during one lung anesthesia. A high non-ventilated lung PVR and low ventilated lung PVR will facilitate good arterial oxygenation during OLA. An increase in non-ventilated lung PVR is governed predominantly by hypoxic pulmonary vasoconstriction. A low opposition to pulmonary blood flow in the dependent lung is facilitated predominantly by a high alveolar oxygen tension and normal lung volume, albeit other factors also play a role in this regard. b. The saturation and oxygen content of mixed venous blood will contribute significantly to the arterial oxygenation in the presence of a large shunt as occurs during OLA. i. On the one hand, venous desaturation as a cause of hypoxemia during one lung anesthesia has not as yet been systematically addressed in the literature. ii. On the other hand, if RV afterload increases to such a degree that it leads poor RV performance, this may cause impairment of global circulatory efficiency and lead to mixed venous desaturation. The question that has been raised is whether inotrope infusions could improve RV and LV performance, cardiac output, and thereby the efficiency of the circulation. Increases in the efficiency of the circulation will result in an improvement in mixed venous and arterial oxygenation in the presence of a large shunt. Nonetheless, the administration of inotrope infusions in the presence of a shunt and during OLA has been reported to aggravate hypoxemia. Thus at the time of conducting the study, conflicting reports of whether increasing cardiac output and thereby mixed venous oxygenation would increase or decrease arterial oxygenation during OLA In the light of the above, the researcher thus investigated RV afterload, RV performance and coupling to its load during OLA. The study also addressed the question whether different levels of inotrope infusion or PEEP hadbeneficial or deleterious effects on RV afterload, RV performance and coupling to its load during OLA. Furthermore, if cardiac output increased during OLA secondary to the infusion of inotropes, would this improve the efficiency of the circulation, mixed venous oxygenation and thus the arterial oxygenation during OLA, or would it worsen shunt and arterial oxygenation during OLA? Control group: OLA and the opposition to pulmonary flow Pulmonary arterial elastance increased by between 18 to 36% during OLA and mean PAP rose by 32% after initiation of OLA This increase in mean PAP on initiation of OLA is greater than that observed by certain investigators but similar to that seen previously in patients with damaged lungs. The question arose as to why pulmonary artery pressure rises during OLA? From consideration of Ohm’s law, pressure may be regarded as the product of flow and resistance (Mark, Slaughter et al. 2000). The increase in mean PAP during OLA is due to two reasons. 1. Firstly, the pressure versus flow curve is likely to be steeper during OLA. This is because pulmonary vascular recruitment and dilatation (pulmonary vascular reserve) is more limited in scope in these patients than is usual and most likely accounts for the increase in pulmonary artery pressure during OLA. The reasons for the limited pulmonary vascular reserve in the DL during OLA include: a. The pulmonary vascular bed of patients subjected to OLA is frequently abnormal because of its underlying pathology, b. During OLA in the lateral decubitus position, lung volume decreases to a greater degree than during two-lung anesthesia (Klingstedt, Hedenstierna et al. 1990). c. This decrease in lung volume will be further aggravated by DLT malpositions, secretions and blood, and absorption atelectasis due to the use of high concentrations of oxygen (Hedenstierna 1998; Krucylak, Naunheim et al. 1996). d. Excessive amounts of extrinsic or intrinsic PEEP during OLA can compress the intra-alveolar capillaries and deleteriously affect the pulmonary vascular resistance (Ducros, Moutafis et al. 1999; Inomata, Nishikawa et al. 1997; Bardoczky, Yernault et al. 1996; Yokota, Toriumi et al. 1996). 2. Secondly, there is greater flow through this vascular bed that possesses a higher resistance. It is noteworthy that the increase in mean PAP did not exceed a value of 25 mm Hg during OLA, even though cardiac output increased by 30%. However, in studies conducted in patients with “damaged lungs”, greater increases in PA pressure (accompanied by a decrease in RVEF) have been reported to occur on PA ligation. A question arises as to why differences exist between PA clamping and OLA? The answer may well be that the observed plateau in the rise of PA pressure during OLA is as a result of progressive diversion of flow to the NDL as PA pressure rises. Support for such a suggestion comes from the observation that concomitant with increases in PA pressure during OLA, HPV is progressively inhibited and shunt fraction progressively rises. This increase in shunt fraction that has been observed to occur as PA pressure rises, reflects an increase in diversion of pulmonary blood flow to the NDL. The impact of diversion of this blood to the NDL is that it possibly acts as a safety mechanism limiting increases in PA pressure and other indices of opposition to pulmonary flow during OLA. This “blow-off effect” will protect the RV until PA clamping occurs.Control group: OLA and RV function The current study represented the opportunity to investigate the significance of the abovementioned increases in PA pressures and elastance on RV performance during OLA. The current study indicates that at the moderate (30%) increases in PAP that accompanied the initiation of OLA, RV performance, as judged by stroke volume, cardiac index, RVEF and RVSWI, did not deteriorate compared to the baseline awake status. In fact, cardiac output increased following surgical incision: this was probably due to sympathetic nervous system stimulation. This observation also fits in with other studies in which RV performance usually only begins to deteriorate when indices of opposition to RV ejection reach 200 to 250% of baseline. Furthermore, a constant preload, as indicated by unchanged central venous and pulmonary artery wedge pressures, and right ventricular end-diastolic volumes were observed throughout the study period. In other words, this increase in RV afyterlad did not cuse the RV to dilate durign OLA. The relationship between stroke work and afterload will vary, depending on the contractile reserve of the ventricle. In this regard, it could be concluded that under the conditions operative in the current study, the RV was operating on the upslope of the RVSWI versus Ea relationship. This supports the observation that RV function is well preserved during OLA. In conclusion, regarding the indices of opposition to pulmonary flow and RV performance during OLA, it can be concluded that: 1. Opposition to RV ejection increases. This is evidenced by a 30% rise in mean PAP and 18 to 36% increase in pulmonary arterial elastance. 2. Right ventricular performance as indicated by RVSWI, RVEF and stroke volume does not decrease during OLA compared with when the patients awake or subjected to two-lung anesthesia. 3. Furthermore, coupling between the RV and its load is well preserved during OLA. This would imply that the RV operates at close to maximal efficiency during OLA and that RV stroke work reserve is present during OLA. It is likely that the RV, which continues operating as a flow pump as it does in normal life, easily copes with the small increases in RV afterload during OLA. Dobutamine during OLA: opposition to pulmonary flow and RV performance The effects of dobutamine infusions on RV performance during OLA can be summarised as follows: 1. Low rates of dobutamine infusion (3 ug.kg-1.min-1) increased cardiac output, stroke volume, and RVSWI. The administration of dobutamine 3 ug.kg-1.min-1 was not accompanied by increases in RV afterload. Therefore, low infusion rates of dobutamine did benefit RV-PA coupling during OLA. 2. However, administration of higher dosages of dobutamine (5 and 7 ug.kg-1.min-1) during OLA was associated with increases in certain indices of opposition to pulmonary blood flow. For example, PA elastance, mean PA pressure, and PVR increased by 30% to 40% compared to both when the patients were awake and when both lungs were being ventilated. Furthermore, PA compliance decreased by up to 61% when dobutamine 5 and 7 ug.kg-1.min-1 were infused compared to the OLA step when dobutaminewas not administered. The increases in mean PAP and PVR are considered to be of limited clinical significance. However, the decrease in PA compliance during the infusion of the highest dosage of dobutamine is clinically significant. PA compliance represents one of the factors determining vascular impedance in the Windkessel model of the circulation. The increases in opposition to pulmonary flow and lack of progressive increase in indices of RV performance are in contrast to what is expected to occur on administration of increasing dosages of the inotrope and pulmonary vasodilator, dobutamine. The reasons for the increase in opposition to pulmonary flow include exhaustion of the pulmonary vascular reserve during OLA at the high cardiac indices of 5 to 5.5 l.min-1.m-2. This aspect overshadowed the expected pulmonary vasodilator effects of dobutamine. Moreover, it is probable that the increase in RV afterload was significant enough to prevent right ventricular performance increasing as would be expected with the administration of progressively higher dosages of inotrope. While dobutamine was being administered during OLA, mean PAP increased to a maximum of 24.9 ± 6.2 mm Hg at a cardiac index of 5.5 ± 1.2 l.min-1.m-2. However during OLA, in the control group, mean PAP was 24.0 ± 7.7 mm Hg at the maximum cardiac index of 4.4 ± 1.1 l.min-1.m-2. This represented a relatively limited rise in PA pressure compared with administration of dobutamine alone. The most likely reason why there may have been a limited increase in mean PAP while dobutamine was being administered is that the “blow off” effect of the NDL vasculature limited the rise in PA pressure. Oxygenation during OLA With regard to oxygen flux, venous and arterial oxygenation during OLA in the control group, the following was observed: 1. Induction of anesthesia and the approximately 1O Celsius decrease in temperature induced an approximately 40% decrease in VO2 that continued during OLA. 2. Initiation of OLA resulted in an increase in cardiac output compared to baseline OLA and awake states. 3. The consequence was an increase in S􀀀��������O2 from 75% and P􀀀��������O2 from 5.4 kPa when the patients were awake to a P􀀀��������O2 of 9.0 ± 1.7 kPa and S􀀀��������O2 of 90.6 ± 4.7% during one-lung anesthesia. 4. During OLA, the significant increase in venous oxygenation resulted in an increase in arterial oxygenation compared to the awake state in spite of the approximately 37% shunt occuring during OLA. 5. Under conditions in the present study, dobutamine administration during OLA did not improve, but maintained the already high venous and arterial oxygenation compared with OLA alone. Therefore, the study hypothesis, that dobutamine would induce improvement in RVF and the increase in cardiac output during OLA would improve arterial oxygenation, does not hold in the current study. The hypothesis that dobutamine administration and improving cardiac output during OLA would increase arterial oxygenation was therefore rejected. However, the rejection of the hypothesis means that the findings of the current study are in contrast to the findings of Mathru et al, and Nomoto and Kawamura. These authors demonstrated that inotrope administration resulted in an increase in arterial oxygenation. Nonetheless, the different results are not at odds with each other. In fact, these differences help to clarify the effect of increases in cardiac output on arterial oxygenation in the presence of asignificant shunt. The differences between the studies can be explained in the following way. Conditions in the current study resulted in a favourable DO2/VO2 ratio and a high starting P􀀀��������O2 even before dobutamine administration was commenced. Therefore the venous saturations were on the flat part of the oxygen dissociation curve and also on the flat part of the relationship between cardiac output and arterial oxygen content originally described by Kelman, Nunn and colleagues. Further increases in cardiac output and the DO2/VO2 ratio would not be expected to, and did not, increase P􀀀��������O2, S􀀀��������O2, or C􀀀��������O2. Thus, arterial oxygenation content and saturation did not change subsequent to the increase in cardiac output associated with the administration of dobutamine in the current study. In contrast, in the Mathru study, the low starting venous saturations and tensions were improved by increases in the DO2/VO2 ratio. As the starting venous saturation was “low,” significant benefit in arterial oxygenation was obtained on increasing cardiac output in that study. One significant concern for the clinician regarding the administration of the inotrope dobutamine during OLA is that it may increase shunt fraction (Qs/Qt) and thereby decrease arterial oxygenation during one lung ventilation. The influence of dobutamine on arterial oxygenation during OLA may theoretically be related to the balance of the following divergent effects: 1. By improving the relationship between oxygen delivery and consumption, dobutamine increases P􀀀��������O2. This increase will benefit arterial oxygenation in the presence of a large shunt, 2. The above has to be weighed against possible increases in VO2 induced by dobutamine, the consequence of which will be a decrease in P􀀀��������O2. Such increases in VO2 were not seen on administration of dobutamine in the current study, 3. An increase in PA pressure accompanying the increased cardiac output will oppose HPV and increase shunt in both the dependent and non-dependent lungs, 4. Direct inhibition of HPV by dobutamine and, 5. The influence of P􀀀��������O2 on HPV (i.e. high levels of venous oxygenation will inhibit whereas low levels will potentiate HPV). Nonetheless, in spite of the concerns (risk) of hypoxemia on administering dobutamine during OLA, dobutamine administration did not decrease PaO2 or arterial oxygen saturation, and neither did it increase the cost of oxygenation compared to when OLA was conducted in the absence of dobutamine infusions. In addition, the findings of studies conducted by Mathru and colleagues, Nomoto and Kawamura and the current study indicate that under usual clinical conditions present during OLA in the LDP, the administration of low dosages of dobutamine do not increase shunt fraction. In fact, the beneficial effect of the increase in cardiac output on venous oxygenation resulted in an increase in arterial oxygenation in the study by Mathru and colleagues; similar mechanisms were most likely operative in the study conducted by Nomoto and Kawamura. Therefore, there is currently no evidence that the administration of dobutamine in dosages of up to 7 ug.kg-1.min-1 increases shunt and worsens arterial oxygenation in humans subjected to OLA in the LDP. It is apparent that the vasodilatory effects of dobutamine resulting in a possible increase in shunt fraction (Qs/Qt) is therefore not the only factor to consider when studying its effects on arterial oxygenation. What is also of great relevance whenconsidering the effects of an inotrope on arterial oxygenation is the effect of inotropic drugs on the venous oxygen content. It is possible that Qs/Qt could be increased by the administration of inotrope. Nonetheless, if venous oxygenation is favourably affected by the administration of dobutamine, then a depressant effect on arterial oxygenation by an increase in the amount of blood passing via the shunt may be negated. If the increase in venous oxygenation is very significant, there may even be benefits in terms of arterial oxygenation, as was the case in the current study. This approach to how the quality of the blood passing via the shunt affects arterial oxygenation shifts the emphasis on prevention and treatment of hypoxemia during OLA from the lung to the efficacy of the circulation. In other words, the emphasis is shifted from what predominantly happens to the non-ventilated lung (HPV) to primarily the efficacy of oxygen flux during OLA. Extrinsic and intrinsic PEEP and OLA The effects of PEEP on hemodynamics and oxygenation observed during OLA in the current study may be summarised as follows. When PEEP5 was applied to the DL during OLA in the current study: 1. Neither right ventricular function, hemodynamics, oxygen flux nor arterial oxygenation was affected by the application of PEEP5 compared to the step when no external PEEP was applied. 2. Significant amounts of intrinsic PEEP were present during OLA in the control group patients. The degree of intrinsic PEEP was weakly related to the degree of obstructive airways disease present on preoperative LFT’s. 3. The most likely reason why PEEP5 did not make a difference to oxygenation or hemodynamics was the existence of similar amounts of intrinsic PEEP during OLA. These findings confirm Myles’s contention that low levels of intrinsic PEEP may have salutary effects on oxygenation during OLA. When PEEP10 was applied to the DL during OLA in the current study, it led to a decrease in stroke volume. This decrease is predominantly due to a decrease in preload, as PVR does not increase to levels that are known to impair RV performance. The decrease in the DO2/VO2 ratio that was induced by PEEP10 predictably decreases P􀀀��������O2 and can potentially lead to impairment of arterial oxygenation. It can therefore be concluded that greater (excessive) amounts of PEEP under more unfavourable circulatory conditions than were observed in the current study, may have deleterious cardio-respiratory effects. In summary, optimising DL volume plays an important role in determining arterial oxygenation. However, the therapeutic index for PEEP is narrow and the anesthesiologist needs to know firstly when the lung volume of the DL approaches FRC and secondly, how to avoid dynamic hyperinflation of that lung. One significant problem is that the best method of monitoring FRC during OLA is not clear at present. / AFRIKAANSE OPSOMMING: Agtergrond tot die studie Eenlongnarkose mag tot sekere probleme aanleiding gee. ’n Betekenisvolle afname in volume van die onderlong vind in die laterale decubitus posisie tydens eenlongnarkose plaas. Hierdie afname in longvolume mag egter ’n akute verhoging in regter ventrikulêre nalading tot stand bring. Die probleem is egter dat die regter ventrikel ’n dunwandige struktuur is wat potensieel baie minder werk as die dikwandige linker ventrikel kan genereer. Die regter ventrikel het min reserwe om ’n akute verhoging in nalading te weerstaan soos wat gebeur met akute longbesering of na longreseksie. Dus die verhoging in nalading wat gepaard gaan met eenlongnarkose mag die koppeling tussen die regter ventrikel en die pulmonale arterie belemmer. Alhoewel hierdie potensiële probleem bestaan, is die verandering albei in regter ventrikulêre nalading en hoe die regter ventrikel funksioneer tydens eenlongnarkose nog nie goed bestudeer nie. 1. Arteriële hipoksemie, hoofsaaklik te wyte aan die groot aftakking via die long wat nie geventileer word nie, mag kliniese probleme tydens eenlongnarkose teweegbring. 2. Die weerstand wat die pulmonale vaskulêre beddens van die geventileerde en nie-geventileerde longe bied teen bloedvloei is belangrike faktore wat aftakking en dus arteriële oksigenasie tydens eenlongnarkose beheer. ’n Hoë weerstand van die nie-geventileerde long en ’n lae weerstand van die geventileerde long se pulmonale vaskulêre beddens sal bevredigende arteriële oksigenasie tydens eenlongnarkose fasiliteer. ’n Verhoging in die pulmonale vaskulêre weerstand van die nie-geventileerde long is hoofsaaklik te wyte aan hipoksiese pulmonale vasokonstriksie. ’n Lae pulmonale vaskulêre weerstand in die geventileerde onderlong is hoofsaaklik gefasiliteer deur ’n hoë alveolêre suurstofspanning en ’n normale long volume, alhoewel alle faktore ook ’n rol in hierdie verband speel. 3. In die teenwoordigheid van die groot aftakking wat bestaan tydens eenlongnarkose, sal die saturasie en suurstof inhoud van gemeng veneuse bloed ’n betekenisvolle bydrae aan arteriële oksigenasie maak. a. Veneuse saturasie as ’n oorsaak van hipoksemie tydens eenlongnarkose, is nog nie sistematies in die literatuur ondersoek nie. b. Indien regter ventrikulêre nalading tot so ’n mate verhoog dat dit tot swak ventrikulêre uitwerp lei, mag dit ’n oorsaak wees van ontoereikendheid van die globale bloedsomloop en tot gemeng veneuse desaturasie lei. Die vraag is dus of verhoging van die kardiale omset deur inotrope ondersteuning die toereikendheid van die sirkulasie kan verbeter. Verbeterde sirkulasie toereikendheid sal tot ’n verhoging in gemeng veneuse en arteriële oksigenasie lei in die teenwoordigheid van ’n groot aftakking. Nietemin, die toediening van inotrope in die teenwoordigheid van ’n groot aftakking tydens eenlongnarkose gerapporteer om hipoksemie te vererger tydens eenlongnarkose. Dus ten tye van die uitvoer van dié studie, is daar uitdrukking gegee tot teenstrydige opinies in die literatuur oftewel verhoging in kardiale omset arteriële oksigenasie sal verbeter of versleg tydens eenlongnarkose.In die lig van die agtergrond hierbo, het die navorser dus regter ventrikulêre nalading, regter ventrikulêre funksie en koppeling van die regter ventrikel met sy lading tydens eenlongnarkose ondersoek. Die studie het ook die vraag benader of inotroop infusies of PEEP goeie of slegte gevolge sou hê op regter ventrikulêre nalading, regter ventrikulêre funksie en koppeling van die regter ventrikel aan sy lading tydens eenlongnarkose. Sou die kardiale omset en die toereikendheid van die sirkulasie sou verbeter sekondêr tot die toediening van inotrope tydens eenlongnarkose, gemeng veneuse oksigenasie en dus arteriële oksigenasie tydens eenlongnarkose verbeter, of sou dit aftakking en arteriële oksigenasie versleg tydens eenlongnarkose? Kontrole groep Pulmonêre elastansie het tussen 18 en 36% verhoog en gemene pulmonale arterie druk het met 32% tydens eenlongnarkose vermeerder. Die verhoging in gemene pulmonale arterie druk met die aanvang van eenlongnarkose is groter as die waardes gesien deur sekere navorsers maar gelyk met waardes gevind in pasiënte met beskadigde longe. Die vraag ontstaan dan hoekom styg pulmonale arterie druk tydens eenlongnarkose? volgens Ohm se Wet, mag druk as die veelvoud van vloei en weerstand beskou word. Die verhoging in gemene pulmonale arterie druk tydens eenlongnarkose is daarvolgens hoofsaaklik te wyte aan twee redes. 1. Eerstens, die kurwe van druk teenoor vloei is waarskynlik styler tydens eenlongnarkose. Hierdie is omdat pulmonale vaskulêre werwing en verwyding (pulmonale vaskulêre reserwe) is meer beperk as nornaal in pasiënte met longsiekte. Hierdie is die waarskynlikste rede hoekom pulmonale arterie druk tydens eenlongnarkose verhoog. Die redes hoekom die pulmonale vaskulêre reserwe in die onderste long tydens eenlongnarkose beperk is sluit in die volgende: 1.1 Die pulmonale vaskulêre bed van pasiënte onderwerp aan eenlongnarkose mag abnormaal wees weens die onderliggende long patologie, 1.2 Tydens eenlongnarkose in die laterale decubitus posisie, is long volume in hoë mate verminder as tydens tweelongnarkose, 1.3 Die voorafgenoemde vermindering in longvolume sal verder verminder word deur wanposisies van die dubbellumenbuis, sekresies en bloed, en absorpsie atelektase. 1.4 Te hoë vlakke van PEEP, oftewel intrinsiek of ekstrensiek van oorsprong, sal die intraalveolêre vate toedruk en so die pulmonale vaskulêre weerstand verhoog. 2. Tweedens, is daar groter vloei deur hierdie vaskulêre bed wat ‘n hoër weerstand bevat. Dit is opmerkingswaardig dat die verhoging in gemene pulmonale arterie druk ‘n waarde van 25 mmHg nie oorskry het nie tydens eenlongnarkose, alhoewel kardiale omset met 30% verhoog het. In pasiënte met beskadigde longe, het vorige studies egter bewys dat groter verhoging in PA druk gebeur tydens afbinding van die pulmonale arterie. Die vraag ontstaan dus hoekom daar verskille bestaan tussen wat gebeur tydens afbind van die pulmonale arterie en eenlongnarkose? Die antwoord mag wees dat die beperking in die styging in PA druk tydens eenlongnarkose as gevolg van ‘n progressiewe afleiding van bloedvloei na die nie-geventileerde long gebeur sodra pulmonale arterie druk styg tydens eenlongnarkose. Die implikasie van die afleiding van bloed na die nie geventileerde long is dat dit as ‘n veiligheids meganisme optree en verdere styging in pulmonale arterie druk beperk tydens eenlongnarkose. Hierdie afblaas meganisme sal die regter ventrikel beskerm tot en met PA afbind.Kontrole groep: eenlongnarkose en regter ventrikulêre funksie Die huidige studie bied die geleentheid om die betekenis van die voorafgenoemde verhoging in PA drukke en elastansie op regter ventrikulêre funksie tydens eenlongnarkose te ondersoek. Die huidige studie dui aan dat die 30% verhoging in pulmonale arterie druk wat met die aanvang van eenlongnarkose plaasvind, glad nie regter ventrikulêre funksie belemmer nie indien dit vergelyk word met die basislyn wakker staat. In teendeel, kardiale omset het verhoog na chirurgiese insnyding: hierdie verhoging is waarskynlik te wyte aan simpatiese senuwee stimulasie na die chirurgiese insnyding. Hierdie waarnemings pas in ook met ander studies waartydens regter ventrikulêre ejeksie alleenlik begin om af te neem indien die indekse van opposisie tot regter ventrikulêre ejeksie 200 tot 250% van basislyn bereik. Verder, die induksie van voorlading, naamlik sentrale veneuse druk, pulmonale arterie wigdruk en regter ventrikulêre einddiastoliese volumes is onveranderd tydens die huidige studie; dit beteken die ventrikel het nie gedilateer het nie tydens die verhoging in regter ventrikulêre nalading. Die verband tussen slagwerk en nalading sal varieer, afhanklik van die kontraktiele status van die ventrikel. In hierdie opsig, kon ons aflei dat die regter ventrikel, onder omstandighede wat tydens diė studie plaasgevind het, gefunksioneer het op die stygende been van die verband tussen regter ventrikulêre slagwerk en pulmonale arterie elastansie. Hierdie waarneming ondersteun die argument in die vorige paragraaf dat die regter ventrikel funksie behoue is tydens eenlongnarkose. Ter opsomming omtrent die indekse van opposisie tot pulmonale vloei en regter ventrikulêre funksie tydens eenlongnarkose: 1. Opposisie tot regter ventrikulêre uitwerp verhoog. Die bewys hiervoor is ’n 30% verhoging in gemene pulmonale arterie druk en ’n 36% verhoging in pulmonale arterie elastansie. 2. Ten spyte van die verhoging in weerstand teen RV uitwerping, het regter ventrikulêre funksie (soos bepaal deur regter ventrikulêre slagwerk indeks, regter ventrikulêre ejeksie fraksie en slag volume), nie verminder tydens eenlongnarkose in vergelyking met die waardes verkry wanneer die pasiënte wakker is of aan tweelongnarkose onderwerp is. 3. Ons kon ook aflei dat die koppeling tussen die regter ventrikel en sy lading goed behoue is tydens eenlongnarkose. Die implikasie hiervan is dat regter ventrikulêre slagwerk reserwe teenwoordig is tydens eenlongnarkose. Tydens eenlongnarkose funksioneer die regter ventrikel as ’n vloeipomp, net soos in normale lewe; dit beteken dat en die klein verhoging in regter ventrikulêre nalading wat ondervind word tydens eenlongnarkose maklik getolereer word. Dobutamien tydens eenlongnarkose: opposisie tot pulmonale vloei en regter ventrikulêre funksie Die uitwerking van dobutamien op regter ventrikulêre funksie tydens eenlongnarkose kan as volg opgesom word: 1. Lae dosisse dobutamien (3 μg.kg-1.min-1) verhoog kardiale omset, slagvolume en regter ventrikulêre slagwerkindeks. Die toediening van dobutamien 3 μg.kg-1.min-1 het nie saamgegaan met ‘n verhoging in regter ventrikulêre nalading nie. Dus, lae dosisse van dobutamien het wel die koppeling tussen die regter ventrikel en die pulmonale vaskulatuur tydens eenlongnarkose verbeter.2. Nietemin, albei die hoër dosisse van dobutamien (5 en 7 μg.kg-1.min-1) tydens eenlongnarkose het verhogings in die opposisie tot pulmonale bloedvloei teweeggebring. Byvoorbeeld, PA elastansie, gemene PA druk en pulmonale vaskulêre weerstand het met 30 tot 40% verhoog in vergelyking met die waardes gekry toe die pasiënte wakker was en toe albei longe geventileer is. ’n Belangrike opmerking in hierdie opsig is dat pulmonale arterie vervormbaarheid tydens eenlongnarkose met 61% verminder het tydens albei dobutamien 5 en 7 μg.kg-1.min-1. Die verhogings in gemene pulmonale arterie druk en pulmonale vaskulêre weerstand is, volgens mening, nie van kliniese of statistiese betekenis nie, alhoewel die vermindering in PA vervormbaarheid tydens die dobutamien 7 μg.kg-1.min-1 infusie wel van kliniese betekenis is. PA vervormbaarheid weerspieël een van die faktore wat vaskulêre impedansie in die 3- element Windkessel model van sirkulasie het. Die verhoging in opposisie tot pulmonale vloei en die afwesigheid van progressiewe verhogings in indekse van regter ventrikulêre funksie is nie wat verwag word indien die dosisse van die inotroop en pulmonale vasodilator dobutamien, progressief verhoog word. Die redes hoekom die opposisie tot pulmonale vloei verhoog tydens die toediening van dobutamien sluit in die uitwissing van die pulmonale vaskulêre reserwe tydens eenlongnarkose. Tydens die hoë kardiale indekse van 5 tot 5.5 μg.kg-1.min-1. is die pulmonale vaskulêre reserwe uitgeput en die meganisme het die verwagte pulmonale vaskulêre vasodilatasie van dobutamien oorskadu. Bowendien is dit waarskynlik dat die verhoging in regter ventrikulêre nalading betekenisvol genoeg was om te verhoed dat regter ventrikulêre funksie progressief verhoog soos sou verwag word met die administrasie van hoër dosisse inotroop. Die administrasie van dobutamien tydens eenlongnarkose het gemene pulmonale arterie druk verhoog tot ’n maksimum van 24,9 ± 6.2 mm Hg teen ’n kardiale indeks van 5.5 ± 1.2 l.min-1.m2. Nietemin is gemene pulmonale arterie druk 24.0 ± 7.7 mm Hg teen die maksimum kardiale indeks in die kontrole groep van 4.4 ± 1.1 l.min-1.m-2 tydens eenlongnarkose in die kontrole groep. Hierdie weerspieël dus ’n relatief beperkte verhoging in pulmonale arterie druk in vergelyking met die verhoging in pulmonale arterie druk wat gebeur het tydens die administrasie van dobutamien. Die waarskynlikste rede hoekom daar ’n beperkte verhoging in pulmonale arterie druk sou gewees het tydens die infusie van dobutamien is die afblaas effek van die nie-geventileerde long wat die verhoging in PA druk beperk het. Oksigenasie tydens eenlongnarkose Die volgende waarnemings is gemaak in verband met suurstof vloed, veneuse en arteriële oksigenasie tydens eenlongnarkose in die kontrole groep: 1. Die kombinasie van Induksie van narkose en die 1ºC vermindering in temperatuur het saamgegaan met ’n 40% vermindering in suurstof verbruik tydens twee long narkose. Hierdie vermindering in suurstof verbruik het voortgegaan tydens eenlongnarkose. 2. Die aanvang van eenlongnarkose is geassosieerd met ’n verhoging in kardiale omset in vergelyking met albei die basislyn eenlongnarkose en wakker state. 3. Die gevolge van punte 1 en 2 hierbo is dat die gemengde veneuse suurstof saturasie vanaf 75% en die gemeng veneuse suurstof spanning vanaf 5.4 kPa (toe die pasiënte wakker was) gestyg het tydens4. Tydens eenlongnarkose het die betekenisvolle verhoging in veneuse oksigenasie veroorsaak dat daar ’n verhoging in arteriële oksigenasie was in vergelyking met wanneer die pasiënte wakker was. Hierdie styging in arteriele oksigenasie was ten spyte van die 37% aftakking wat teenwoordig was tydens eenlongnarkose. 5. Onder toestande in die huidige studie, het dobutamien tydens eenlongnarkose nog arteriële nog veneuse oksigenasie verbeter nie, maar die arteriele oksigenasie het konstant gebly. ’n Belangrike observasie wat daarmee saamgaan is dat dobutamien toediening nie met ’n daling in arteriële suurstof spanning geassosieer is nie. Vervolgens, die hipotese dat die verhoging in kardiale omset geassosieer met dobutamien toediening tydens eenlongnarkose ’n verhoging in arteriële oksigenasie beweeg bring, is dus verwerp. Die verwerping van die hipotese van die deel van die studie beteken dat die bevindinge die teenoorgestelde is van die studies gepubliseer deur Mathru en sy kollegas en Nomoto en Kawamura. Hierdie outeurs het gedemonstreer dat die toediening van inotrope ’n verhoging in arteriële oksigenasie teweeg gebring het. Nietemin is die teenoorgestelde gevolgtrekkinge nie teenstrydig met mekaar nie. Inteendeel hierdie verskille help ons om die effek van ’n verhoging in kardiale omset of arteriële oksigenasie in die teenwoordigheid van ’n betekenisvolle aftakking duidelik te maak. Die verskille tussen die studies kan op die volgende manier verduidelik word. Toestande wat in die huidige studie teenwoordig was het veroorsaak dat die verband tussen suurstof lewering en verbruik baie hoog was en dat die gemeng veneuse suurstof spanning baie hoog was om mee te begin alvorens dobutamien geinfuseer is. Dus is die veneuse saturasies op die plat deel van albei die suurstof dissosiasie kurwe en ook van die verband tussen kardiale omset en arteriële suurstof inhoud oorspronklik deur Kelman, Nunn en kollegas beskryf. Verdere verhogings in kardiale omset sou dus nie verwag word, en het nie, verhogings in gemeng veneuse suurstof spanning, gemeng veneuse suurstof saturasie of gemeng veneuse suurstof inhoud teweeg gebring. Dus, arteriële suurstof inhoud en saturasie het nie verander na die verhoging in kardiale omset wat teweeg gebring is deur die toediening van dobutamien in die huidige studie. Inteendeel, in die studie deur Mathru en kollegas, is die lae aanvanklike veneuse saturasie en spanning verbeter deur verhogings in die verband tussen suurstoflewering en suurstofverbruik. Omdat die veneuse saturasie aan die begin van die Mathru studie laag was, is betekenisvolle voordeel in arterieël oksigenasie teweeg gebring deur om die kardiale omset te verhoog. ’n Groot bekommernis vir die klinikus is dat die aftakking mag verhoog met die toediening van die inotroop dobutamien tydens eenlongnarkose en, op die manier, arteriële oksigenasie mag verminder. Die invloed van dobutamien op arteriële oksigenasie tydens eenlongnarkose mag teoreties te wyte wees aan die balans van die volgende uiteenlopende faktore: 1. Deur om die verband tussen suurstof lewering en verbruik te verbeter, sal dobutamien gemeng veneuse suurstof spanning verhoog. Hierdie verhoging sal arteriële oksigenasie verbeter in die teenwoordigheid van ’n groot aftakking, 2. Die bogenoemde moet teenoor potensiële verhogings in suurstofverbruik deur dobutamien oorweeg word. Die gevolge hiervan sou potensieel ’n vermindering in gemeng veneuse suurstof spanning wees. Sulke verhogings in suurstof verbruik is nie tydens die huidige studie gesien nie,3. ’n Verhoging in pulmonale arterie druk wat saamgaan met die verhoogde kardiale omset sal hipoksiese pulmonale vasokonstriksie teenwerk wat die aftakking in albei die geventileerde en nie geventileerde longe sal verhoog, 4. Direkte inhibisie van hipoksiese pulmonale vasokonstriksie deur dobutamien en, 5. Die invloed van gemeng veneuse suurstof spanning op hipoksiese pulmonale vasokonstriksie moet ook oorweeg word (d.i. hoe gemeng veneuse suurstof parsiele druk sal hipoksiese pulmonale vasokonstriksie inhibeer). Nietemin, ten spyte van die bekommernisse rondom hipoksemie tydens die toediening van dobutamien tydens eenlongnarkose, het dobutamien toediening nie ’n verlaging in arteriële suurstof spanning teweeg gebring nie, en ook het dit nie die koste van oksigenasie verhoog nie. Verder, die bevindinge van studies tydens eenlongnarkose in die laterale decubitus posisie deur Mathru en sy kollegas, Nomota en Kawamura en ook die huidige studie, dui aan dat die toediening van lae dosisse van dobutamien nie toe ’n verhoging in aftakking lei nie. Inteendeel, die voordelige effekte van die verhoging in kardiale omset op veneuse saturasie het veroorsaak dat daar ’n verhoging in arteriële saturasie is in die studie deur Mathru en sy kollegas soortgelyke meganismes is waarskynlik ook van toepassing in die studie wat gedoen is deur Nomoto en Kawamura. Dus, dwars deur die literatuur, is daar geen huidiglike bewys dat die toediening van dobutamien tot en met dosisse van 7μg.kg-1.min-1 aftakking verhoog of arteriële oksigenasie versleg in mense onderworpe aan eenlongnarkose in die laterale decubitus posisie. Dit is duidelik dat die vasodilatoriese effekte van dobutamien wat moontlik ’n verhoging in aftakking fraksie teweeg kan bring, nie die enigste faktore is om te oorweeg wanneer die middel se invloed op arteriële oksigenasie bestudeer word nie. Dit is ook van kliniese belang om die invloed van inotrope middels op veneuse suurstof inhoud te oorweeg. Dit is moontlik dat ’n aftakking verhoog kan word deur die toediening van ’n inotroop. Nietemin, mag die negatiewe effek wat die toediening van ’n inotroop sal inhou op arteriële oksigenasie deur middel van sy verhoging in aftakking, negeer word indien veneuse oksigenasie voordelig beïnvloed is. Verder, indien die verhoging in veneuse oksigenasie wat teweeggebring word deur die toediening van inotrope baie betekenisvol is, mag die gevolg hiervan wees dat arteriële oksigenasie voordelig beïnvloed word soos die geval in die huidige studie was. Die huidige benadering waar die kwaliteit van die bloed wat deur die aftakking vloei die arteriële oksigenasie beïnvloed, skuif die klem van voorkoming en behandeling van hipoksemie tydens eenlongnarkose van die long na die toereikendheid van die sirkulasie. Met ander woorde, die klem is geskuif van wat gebeur in die nie-geventileerde long (hipoksie pulmonale vasokonstriksie) tot primêr die toereikendheid van suurstof flux tydens eenlongnarkose. Ekstrinsieke en intrinsieke PEEP tydens eenlongnarkose Die invloed van PEEP op hemodinamika en oksigenasie tydens eenlongnarkose in die huidige studie mag as volg opgesom word. Toe PEEP5 tydens eenlongnarkose toegedien is: 1. Nie regter ventrikulêre funksie, hemodinamika, suurstof flux nog arteriële oksigenasie is beïnvloed deur die toediening van PEEP5 in vergelyking met die stap wanneer geen eksterne PEEP toegedien is nie. 2. Betekenisvolle hoeveelhede intrinsieke PEEP is teenwoordig tydens eenlongnarkose in die kontrole groep.Die hoeveelheid intrinsieke PEEP wat teenwoordig was, is swak maar betekenisvol verwant aan die graad obstruktiewe lugwegsiekte wat teenwoordig was gemeet deur pre-operatiewe longfunksie toetse. 3. Die waarskynlikste rede hoekom PEEP5 nie ’n verskil gemaak het aan oksigenasie of hemodinamika nie is die teenwoordigheid van soortgelyke hoeveelhede intrinsieke PEEP tydens eenlongnarkose. Hierdie bevinding bevestig Myle’s se beweringe dat lae vlakke intrinsieke PEEP voordelige effekte op oksigenasie tydens eenlongnarkose mag hê. PEEP10 toediening aan die onderlong tydens eenlongnarkose in die huidige studie het tot ’n vermindering in slagvolume gelei. Hierdie vermindering is primêr veroorsaak deur ’n vermindering in voorlading en nie die gevolg van ’n verhoging in pulmonale vaskulêre weerstand nie. Die gevolgtrekking is gemaak omdat regerventrikulere enddiastoliese volume verlaag het maar pulmonale vaskulêre weerstand het nie verhoog tot vlakke wat bekend is om regter ventrikulêre funksie te belemmer nie. Die vermindering in die verhouding tussen suurstof lewering en suurstof verbruik wat geïnduseer is deur PEEP10 het (voorspelbaar) gemeng veneuse suurstof spanning verminder en kon potensieël gelei het tot belemmering in arteriële oksigenasie. Indien minder voordelige sirkulatoriese toestande geheers het tydens die huidige studie, sou groter (oorbodige) hoeveelhede PEEP slegter kardiorespiratoriese gevolge tot gevolg gehad het. Ter opsomming, optimalisering van die volume van die onderlong tydens eenlongnarkose speel ’n belangrike rol in die bepaling van arteriële oksigenasie. Nietemin, die terapeutiese indeks vir PEEP is nou en die narkotiseur het die behoefte om te weet wanneer die volume van die onderlong optimaal is. In die opsig, is ’n betekenisvolle probleem tydens eenlongnarkose dat meting van funksionele residuele kapasiteit nie huidiglik maklik is nie

This indoor environment study formed part of the South Durban Health Study (SDHS) that investigated the health effects of exposure to ambient air pollution. Homes of children from seven communities corresponding schools were recruited to participate. This study was designed to determine characteristics in the homes that are associated with higher or lower levels of allergens and fungal aerosols. Homes were inspected using a field tested walkthrough checklist to collect data on home characteristics associated to adverse health effects. The characteristics include dampness, visible mould, type of flooring, type of bedding, type of heating systems, and building type and age. Dust samples for allergen analysis were collected from the bedding and the floor of the sleep area used by the children. Air samples from all rooms in the house were collected on malt extract agar, the media used for identifying and quantifying airborne fungal aerosols. More than 70% of the homes were single units standing on their own, 20% were attached houses (flats or apartments) and the rest (10%) were informal houses. Construction material of the homes comprised of bricks (93%), wood (5%) and other material (2%) such as corrugated iron of which 94% were formally constructed. Dampness signs were observed in 51% of the homes and visible mould growth 13% of them. In all them, at least one characteristic that is hypothetically associated to elevated house dust mite allergens was found. Levels of mould (Asp f 1) allergen and house dust mite (Der p 1 and Der f 1) allergen were comparable to levels found in other parts of the world. Asp f 1 allergen levels ranged between 0.32-1.379g/g and Der p 1 and Der f 1 allergen levels ranged from undetectable to 49.61 and from undetectable to 39.319g/g of dust respectively. Some home characteristics from walkthrough checklist were associated with Asp f 1, Der p1 and Der f 1 allergen levels when simple regression analysis was performed. Asp f 1 was significantly associated with single family home [OR= 0.004 (95%CI 0.004–0.35)] and polyester filled pillows [OR= 0.07 (95%CI 0.01– 0.61)] in logistic regression models. Der p 1 allergen was associated with observed extent of roof dampness [OR= 0.33 (95%CI 0.13–0.81)]. Fungal aerosol mixture consisted of Cladosporium spp. as the predominant genus together with other genera such as Aspergillus, Penicillium and Fusarium were, to a lesser extent, identified in the samples from the homes. Mean concentration of total indoor fungal aerosol of indoor and outdoor were 1108 CFU/m3 and 1298 CFU/m3 respectively. Individual genera of fungi in the childrens sleep area had mean levels of 783 CFU/ m3, 30CFU/ m3, 64CFU/ m3, 48CFU/ m3 and 43 CFU m3 for Cladosporium spp., Aspergillus spp., Penicillium, spp., Fusarium spp. and Rhizopus spp. respectively. Simple regression showed some conditions in the homes to be predictors of higher levels of total fungal aerosols. In a linear regression models, total outdoor fungal levels were a protective effect on total indoor fungal levels [C= 0.542 (95%CI 0.437–0.647)] whilst homes with hard floors had about 25 CFU/m3 [C= 5.235 (95%CI 0.557–9.913)] in the homes were significantly associated. This study showed the need to adapt observational instrument/ checklist/ questionnaire to suit the environment or the study area of interest. As other studies and findings indicated, the best way to assess exposure to biological pollutants indoors needs a combination of two or more methods, i.e. direct and indirect methods. / Thesis (MMedSc-Occupational and Environmental Health)-University of KwaZulu-Natal, 2007.