Der Transport von Sauerstoff im Organismus ist die Hauptaufgabe von Eisen im Säugetier und somit auch im Pferd. Unter bestimmten Einflussfaktoren, wie z.B. akuten Infektionen, kann es zu einer Umverteilung von Eisen im Gewebe und im Blut bei Pferden kommen. Ziel dieser Arbeit ist es, verschiedene Eisenparameter wie Serum-Eisen, Ferritin, ungesättigte Eisenbindungskapazität (UIBC), totale Eisenbindungskapazität (TIBC) und die Eisensättigung beim adulten gesunden und kranken Pferd zu bestimmen. Dafür wurde eine adulte Pferdepopulation anhand von bestimmten Einschlusskriterien, wie dem Serum-Amyloid A (SAA) Wert, der Anzahl an Blutleukozyten und weiteren klinischen Symptomen, wie einer erhöhten Körperinnentemperatur, die auf eine akute systemische Entzündung hindeuten können, eingeteilt. Sowohl bei den gesunden (n = 71) als auch bei den kranken Pferden (n = 65) wurde einmalig eine Blutprobe zur Bestimmung der hämatologischen, der klinisch-chemischen sowie der Eisenstoffwechselparameter entnommen. Durch die Ergebnisse der SAA-Gehalte im Blut bestätigte sich die Diagnose der akuten systemischen Entzündung bei den kranken Pferden, da die Pferde SAA-Werte > 7 μg/ml (Referenzwert für gesunde adulte Pferde, LABOKLIN GMBH & CO.KG) zeigten und sich ihre medianen Gehalte bei 412 μg/ml befanden. Die Eisenparameter im Serum, wie der Ferritin-Gehalt, der Eisen-Gehalt, die UIBC, die TIBC und die Eisensättigung zeigten hochsignifikante Unterschiede zwischen den gesunden und den kranken Pferden. Es ließ sich ein Abfall der Konzentrationen des Eisens, der Eisensättigung und ein Anstieg der Ferritin-Konzentration und der UIBC und TIBC im Serum bei den kranken Pferden darstellen. Diese Veränderungen der Eisenstoffwechselparameter lassen vermutlich auf eine Umverteilung von Eisen im Organismus bei einer akuten systemischen Entzündung schließen und könnten möglicherweise im Zusammenhang mit anderen Akute-Phase-Proteinen als Marker von Entzündungen genutzt werden.:Inhalt 1. Einleitung ......................................................................................................................... 1 2. Literaturübersicht ............................................................................................................. 2 2.1 Chemische Grundlagen, Verteilung und Vorkommen ................................................ 2 2.2 Eisenmetabolismus .................................................................................................... 2 2.2.1 Aufnahme und Resorption von Eisen beim Säugetier ......................................... 2 2.2.2 Transport und Speicherung von Eisen beim Säugetier ........................................ 3 2.2.3 Verluste und Wiederverwertung von Eisen beim Säugetier ................................. 4 2.3 Physiologische Funktionen von Eisen beim Säugetier ............................................... 4 2.3.1 Funktionseisen ..................................................................................................... 4 2.3.2 Speichereisen ...................................................................................................... 6 2.4 Rolle des Eisens in Entzündungsprozessen beim Pferd .......................................... 10 2.4.1 Rolle von Eisen bei Infektionserregern .............................................................. 10 2.4.2 Rolle von Eisen bei Entzündungsprozessen ...................................................... 11 2.5 Eisen in der Fütterung .............................................................................................. 13 2.5.1 Bedarf von Pferden ............................................................................................ 13 2.5.2 Eisengehalte in Futtermitteln ............................................................................. 13 2.5.3 Supplementierung von Eisen ............................................................................. 14 2.6 Eisenmangel beim Pferd .......................................................................................... 14 2.7 Toxizität von Eisen ................................................................................................... 15 3. Publikation ..................................................................................................................... 17 3.1 Untersuchung zum Eisenstoffwechsel im Blut bei gesunden adulten Pferden und bei Pferden mit einer akut entzündlichen Erkrankung .......................................................... 17 4. Diskussion ..................................................................................................................... 29 5. Zusammenfassung ........................................................................................................ 35 6. Summary ....................................................................................................................... 37 7. Literaturverzeichnis ........................................................................................................ 39 8. Danksagung ................................................................................................................... 44

Biochemical markers of bone turnover allow inference of the events occurring at the bone tissue level and may detect changes in bone cell activity earlier than densitometric technologies. Serum concentrations of receptor activator for nuclear factor kappa-beta ligand (RANKL), osteoprotegerin (OPG), osteocalcin, and N-telopeptide (NTx) were measured in women aged 20 + 1.5 years (mean + SD) who underwent 32 weeks of unilateral isokinetic concentric or eccentric muscular strength training. Changes in serum biomarkers were compared with changes in arm and leg flexor and extensor muscle strength. Dual X-ray absorptiometry (DXA) measures of bone mineral density (BMD) and bone mineral content (BMC) of the total forearm, total tibia, and total body also were assessed. The mean serum OPG concentration increased from 4.6 + 1.9 pmol/L to 5.2 + 2.1 pmol/L (â 14.9 %, mean + SD; p = 0.05, n = 20) following long-term isokinetic exercise training that also increased elbow extensor and knee flexor muscular strength (p < 0.05) and total forearm BMD (p = 0.04). The ratio of OPG/RANKL also increased over the course of the study (p = 0.045). Serum concentrations of other measured bone biomarkers did not change during training. Serum concentrations of OPG, a suppressor of osteoclastogenesis, increased with high-load muscular strength training that led to local increases in muscle strength and BMD. These adaptations may represent an exercise-mediated suppression of osteoclast differentiation and activity. The central role of the RANKL-OPG cytokine system in the regulation of bone cell biology is well established. Further research is needed to confirm the efficacy of using serum OPG and RANKL as biomarkers of bone cell metabolism in healthy populations undergoing long-term exercise interventions. / Master of Science

No / Biosimilar pharmaceuticals are complex biological molecules that have similar physicochemical properties to the originator therapeutic protein. They are produced by complex multi-stage processes and are not truly equivalent. Therefore, for a biosimilar to be approved for market it is important to demonstrate that the biological product is highly similar to a reference product. This includes its primary and higher order structures and its aggregation behaviour. Representative lots of both the proposed biosimilar and the reference product are analysed to understand the lot-to-lot variability of both drug substances in the manufacturing processes. Whilst it is not easy to characterise every variation of a protein structure at present additional analytical technologies need to be utilised to ensure the safety and efficacy of any potential biosimilar product. We have explored the use of Taylor dispersion analysis (TDA) to analyse such batch to batch variations in the model protein, bovine serum albumin (BSA) and compared the results to that obtained by conventional dynamic light scattering analysis (DLS). Inter and intra batch differences were evident in all grades of BSA analysed. However, the reproducibility of the TDA measurements, enabled the stability and reversibility of BSA aggregates to be more readily monitored. This demonstrates that Taylor dispersion analysis is a very sensitive technique to study higher order protein states and aggregation. The results, here, also indicate a correlation between protein purity and the physical behaviour of the samples after heat shocking. Here, the protein with the highest quoted purity resulted in a reduced increase in the measured hydrodynamic radius after heat stressing, indicating that less unfolding/aggregation had occurred. Whilst DLS was also able to observe the presence of aggregates, its bias towards larger aggregates indicated a much larger increase in hydrodynamic radii and is less sensitive to small changes in hydrodynamic radii. TDA was also able to identify low levels of larger aggregates that were not observed by DLS. Therefore, given the potential for immunogenicity effects that may result from such aggregates it is suggested that TDA may be suitable in the evaluating detailed batch to batch variability and process induced physical changes of biopharmaceuticals and biosimilars.

Alzheimer's disease (AD) was first described more than 100 years ago and is today the most common cause of dementia. It is one of the progressive neurodegenerative diseases that affect 47 million people around the world between the ages of 60 and 90. One of the contributing factors to AD is extracellular amyloid – β (Aβ) plaques that form as a result of protein aggregation. These Aβ proteins are neurotoxic, leading to degeneration of brain neurons and loss of cognitive abilities. Because AD largely affects society, researchers are constantly working to find a cure, which currently does not exist. The purpose of this study was to use Drosophila melanogaster as a living organism model for the expression of two types of Aβ proteins related to AD, Arctic (Glu22Gly) and TandemAβ, and to study the survival of these AD flies when Bovine serum albumin (BSA) was added to the fly food. The hypothesis was that BSA would be effective in slowing down and/or preventing formation of toxic Aβ-aggregates. The focus was therefore to investigate whether the AD flies would live longer if they were allowed to eat Bovine serum albumin and whether the soluble/insoluble Aβ levels in these flies would decrease in comparison to the control AD flies that were not allowed to eat BSA. The effect of BSA on toxicity was evaluated using survival assay on male flies and the levels of soluble/insoluble Aβ were evaluated using Meso Scale Discovery (MSD) on female flies. In both experiments, the following six groups of flies were examined: myow1118 ± BSA; myoArctic ± BSA; myoTandemAβ ± BSA. Conclusions from the studies are that the survival of AD flies could not be extended by adding 0.61 mM BSA to the food, rather the data showed a weak but significant toxic effect in the presence of BSA in the AD flies. However, MSD data showed a reduction of insoluble Aβ aggregates and an equilibrium shift from insoluble Aβ aggregates to soluble Aβ aggregates in the presence of BSA in the AD flies. Equilibrium shifts were particularly detectable in Myo-TandemAβ flies fed with BSA. In Myo-Arctic flies fed with BSA only reduction of insoluble Aβ could be detected. This shows that it is not the amount of Aβ aggregates that is decisive for toxicity, but rather the presence of specific aggregates that have toxic properties. If BSA shows good results in further studies, it could be used in the future to improve AD symptoms in patients.

Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Several alternatively-spliced mRNA transcripts of the follicle stimulating hormone receptor (FSHR) have been identified in sheep, including FSHR-1 (G protein-coupled form), FSHR-2 (dominant negative form), and FSHR-3 (growth factor type-1 form). Coupling of the FSHR to signaling pathways which activate different downstream effectors leads to speculation that specific splice variants may be transcribed under differing physiological conditions. This is the first study to correlate expression patterns of FSHR-1, FSHR-2, and FSHR-3 and development of follicles in the mature sheep ovary. In Experiment 1, 8 Suffolk-cross ewes were allowed to come into estrus naturally and were euthanized 24 (n=3), 36 (n=3), and 48 (n=2) hours after the onset of estrus. In Experiment 2, 7 Suffolk-cross ewes received CIDRs for 14 days. At CIDR removal, PMSG (500IU) was administered to treatment ewes (n=3), while controls (n=4) received no PMSG. Ewes were euthanized 24 (n=4; 2 CIDR only, 2 PMSG) or 36 (n=3; 2 CIDR only, 1 PMSG) hours later. All visible follicles were aspirated and pooled according to follicular diameter: small (≤ 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm), and preovulatory (≥ 6.1 mm). Granulosa cells were separated from follicular fluid by centrifugation. Total RNA was extracted from granulosa cells (GC) and reversed transcribed. The resulting cDNA was subjected to qPCR, using primer sets designed to amplify each variant specifically. For Experiment 1, regardless of time after onset of estrus, relative expression of FSHR-3 exceeded that of both FSHR-1 and FSHR-2 in medium follicles (p < 0.01), and tended to be higher in small follicles (p=0.09). For Experiment 2, treatment with PMSG did not significantly alter expression patterns of FSHR variants (p=0.18). The FSHR-3 was expressed higher than FSHR-2 in all follicle sizes (p < 0.01) and was numerically more highly expressed than FSHR-1, although this difference was not significant (p > 0.11). These experiments show that in addition to the well characterized G protein-coupled form of the FSHR, alternatively spliced variants of the FSHR may participate in follicular dynamics during the first follicular wave of the sheep estrous cycle. Furthermore, these results would indicate that an “alternatively” spliced form of the FSHR (FSHR-3) is the predominant form of the FSHR in the sheep.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

Thesis (MCur)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Background: Coronary artery disease is a form of cardiovascular disease (CVD) which manifests itself in three ways: angina pectoris, acute coronary syndrome and cardiac death. Thirty-three people die daily of a myocardial infarction (cardiac death) and 7.5 million deaths annually are caused by CVD (51% from strokes and 45% from coronary artery disease) worldwide. Globally, the CVD death rate is a mere 4% compared to South Africa which has a 42% death rate. It is predicted that by the year 2030 there will be 25 million deaths annually from CVD, mainly in the form of strokes and heart disease. The WHO compared the death rates of high-income countries to those of low- and middle-income countries, like South Africa, and the results show that CVD deaths are declining in high-income countries but rapidly increasing in low- and middle-income countries. Although there are several risk prediction tools in use worldwide, to predict ischemic risk, South Africa does not use any of these tools. Current practice in South Africa to diagnose acute coronary syndrome is the use of a physical examination, ECG changes and positive serum cardiac maker levels. Internationally the same practice is used to diagnose acute coronary syndrome but risk assessment tools are used additionally to this practise because of limitations of the ECG and serum cardiac markers when it comes to NSTE-ACS. Objective: The aim of this study was to systematically appraise evidence on the accuracy of acute coronary syndrome risk prediction tools in adults. Methods: An extensive literature search of studies published in English was undertaken. Electronic databases searched were Cochrane Library, MEDLINE, Embase and CINAHL. Other sources were also searched, and cross-sectional studies, cohort studies and randomised controlled trials were reviewed. All articles were screened for methodological quality by two reviewers independently with the QUADAS-2 tool which is a standardised instrument. Data was extracted using an adapted Cochrane data extraction tool. Data was entered in Review Manager 5.2 software for analysis. Sensitivity and specificity was calculated for each risk score and an SROC curve was created. This curve was used to evaluate and compare the prediction accuracy of each test. Results: A total of five studies met the inclusion criteria of this review. Two HEART studies and three GRACE studies were included. In all, 9 092 patients participated in the selected studies. Estimates of sensitivity for the HEART risks score (two studies, 3268 participants) were 0,51 (95% CI 0,46 to 0,56) and 0,68 (95% CI 0,60 to 0,75); specificity for the HEART risks score was 0,90 (95% CI 0,88 to 0,91) and 0,92 (95% CI 0,90 to 0,94). Estimates of sensitivity for the GRACE risk score (three studies, 5824 participants) were 0,03 (95% CI0,01 to 0,05); 0,20 (95% CI 0,14 to 0,29) and 0,79 (95% CI 0,58 to 0,93). The specificity was 1,00 (95% CI 0,99 to 1,00); 0,97 (95% CI 0,95 to 0,98) and 0,78 (95% CI 0,73 to 0,82). On the SROC curve analysis, there was a trend for the GRACE risk score to perform better than the HEART risk score in predicting acute coronary syndrome in adults. Conclusion: Both risk scores showed that they had value in accurately predicting the presence of acute coronary syndrome in adults. The GRACE showed a positive trend towards better prediction ability than the HEART risk score. / AFRIKAANSE OPSOMMING: Agtergrond: Koronêre bloedvatsiekte is ‘n vorm van kardiovaskulêre siekte. Koronêre hartsiekte manifesteer in drie maniere: angina pectoris, akute koronêre sindroom en hartdood. Drie-en-dertig mense sterf daagliks aan ‘n miokardiale infarksie (hartdood). Daar is 7,5 miljoen sterftes jaarliks as gevolg van kardiovaskulêre siektes (51% deur beroertes en 45% as gevolg van koronêre hartsiektes) wêreldwyd. Globaal is die sterfte syfer as gevolg van koronêre vaskulêre siekte net 4% in vergelyking met Suid Afrika, wat ‘n 42% sterfte syfer het. Dit word voorspel dat teen die jaar 2030 daar 25 miljoen sterfgevalle jaarliks sal wees, meestal toegeskryf aan kardiovaskulêre siektes. Die hoof oorsaak van sterfgevalle sal toegeskryf word aan beroertes en hart siektes. Die WHO het die sterf gevalle van hoeinkoms lande vergelyk met die van lae- en middel-inkoms lande, soos Suid Afrika, en die resultate het bewys dat sterf gevalle as gevolg van kardiovaskulêre siekte is besig om te daal in hoe-inkoms lande maar dit is besig om skerp te styg in lae- en middel-inkoms lande. Daar is verskeie risiko-voorspelling instrumente wat wêreldwyd gebruik word om isgemiese risiko te voorspel, maar Suid Afrika gebruik geen van die risiko-voorspelling instrumente nie. Huidiglik word akute koronêre sindroom gediagnoseer met die gebruik van n fisiese ondersoek, EKG verandering en positiewe serum kardiale merkers. Internationaal word die selfde gebruik maar risiko-voorspelling instrumente word aditioneel by gebruik omdat daar limitasies is met EKG en serum kardiale merkers as dit by NSTE-ACS kom. Doelwit: Die doel van hierdie sisematiese literatuuroorsig was om stelselmatig die bewyse te evalueer oor die akkuraatheid van akute koronêre sindroom risiko-voorspelling instrumente vir volwassenes. Metodes: 'n Uitgebreide literatuursoektog van studies wat in Engels gepubliseer is was onderneem. Cochrane biblioteek, MEDLINE, Embase en CINAHL databases was deursoek. Ander bronne is ook deursoek. Die tiepe studies ingesluit was deurnsee-studies, kohortstudies en verewekansigde gekontroleerde studies. Alle artikels is onafhanklik vir die metodologiese kwaliteit gekeur deur twee beoordeelaars met die gebruik van die QUADAS-2 instrument, ‘n gestandaardiseerde instrument. ‘n Aangepaste Cochrane data instrument is gebruik om data te onttrek. Data is opgeneem in Review Manager 5.2 sagteware vir ontleding. Sensitiwiteit en spesifisiteit is bereken vir elke risiko instrument en ‘n SROC kurwe is geskep. Die SROC kurwe is gebruik om die akkuraatheid van voorspelling van elke instrument te evalueer en te toets. Resultate: Twee HEART studies en drie GRACE studies is ingesluit. In total was daar 9 092 patiente wat deelgeneeem het in die gekose studies. Skattings van sensitiwiteit vir die HEART risiko instrument (twee studies, 3268 deelnemers) was 0,51 (95% CI 0,47 to 0,56) en 0,68 (95% CI 0,60 to 0,75) spesifisiteit vir die HEART risiko instrument was 0,89 (95% CI 0,88 to 0,91) en 0,92 (95% CI 0,90 to 0,94). Skattings van sensitiwiteit vir die GRACE risiko instrument (drie studies, 5824 deelnemers) was 0,28 (95% CI 0,13 to 0,53); 0,20 (95% CI 0,14 to 0,29) en 0,79 (95% CI 0,58 to 0,93). Die spesifisiteit vir die GRACE risiko instrument was 0,97 (95% CI 0,95 to 0,99); 0,97 (95% CI 0,95 to 0,98) en 0,78 (95% CI 0,73 to 0,82). Met die SROC kurwe ontleding was daar ‘n tendens vir die GRACE risiko instrument om beter te vaar as die HEART risiko instrument in die voorspelling van akute koronêre sindroom in volwassenes. Gevolgtrekking: Altwee risiko instrumente toon aan dat albei instrumente van waarde is. Albei het die vermoë om die teenwoordigheid van akute koronêre sindroom in volwassenes te voorspel. Die GRACE toon ‘n positiewe tendens teenoor beter voorspelling vermoë as die HEART risiko instrument.

Thesis (MMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Serum free light chains (FLC) are associated with imbalances in heavy and light chain production. Abnormal FLC ratios have been associated with risk of progression in certain diseases. Automated assays are available for their determination and they are used in the followup and management of patients with monoclonal gammopathies. Acceptable imprecision, specificity, accuracy and reproducibility between reagent batches is required to prevent under- or overestimation. Method validation is a standard process in every good laboratory to judge the acceptability of a new method. Reference intervals have been established in an older population, but it was considered important to verify these in our population. HIV is associated with B-cell dysfunction. As B-cell abnormalities are associated with disorders leading to monoclonal gammopathies, we postulated that the FLC levels and FLC ratio would be abnormal in HIV infected individuals. Methods and materials: Controls and pooled patient samples were used for the method validation study which included imprecision studies, linearity, recovery and interference studies, and method comparison studies, the latter compared our method to the same method used in another laboratory. For the reference interval study, blood was obtained from 120 healthy subjects. The following blood tests were performed: total protein, IgG, IgA, IgM, creatinine, protein electrophoresis, kappa FLC and lambda FLC. Using the kappa and lambda FLC results, a FLC ratio was determined. Three hundred and sixty-nine HIV positive subjects were then studied. The same tests were performed, as well as CD4+ counts and viral loads on the majority of them. Results: For the method validation study, precision, linearity and recovery was acceptable. Minimal interference was observed with haemolysis, lipaemia, bilirubin and rheumatoid factor. Our method showed comparable performance with the established method. For the reference interval study, all the creatinine values were normal, as were serum protein values. The serum protein electrophoreses were independently reviewed by 3 pathologists. Most were normal, with a few polyclonal increases seen, but no definite monoclonal bands. The 95% reference intervals for FLC’s as well as the FLC ratio were not statistically significantly different to the manufacturer’s recommendations. When examining the HIV positive study population, we found that FLC and FLC ratio were influenced by markers of HIV disease severity, such as CD4+ count, IgG, viral load, use of antiretroviral treatment and abnormal serum protein electrophoreses. Conclusion: The validation study of FLC showed excellent precision, acceptable bias, good linearity, good recovery and minimal interference, allowing routine introduction of the test. The 95% reference intervals obtained for our population were slightly higher than those recommended by the manufacturer. However, as most of the values fell within the manufacturer’s limits, we could accept the manufacturer’s recommended cut-offs. We found that FLC levels were definitely influenced by markers of HIV disease severity in our population and we postulate that they may be of use for follow-up of patients with HIV. / AFRIKAANSE OPSOMMING: Agtergrond: Serum vry ligte kettings (VLK) word geassosieer met ‘n wanbalans van ligte en swaar ketting produksie. Abnormale VLK ratios is geassosieer met ‘n risiko van verloop in sekere siektes. Geoutomatiseerde laboratorium toetse vir VLK is beskikbaar vir hul bepaling en word gebruik om pasiënte met monoklonale gammopatieë op te volg en te behandel. Aanvaarbare impresisie, spesifisiteit, akkuraatheid en herhaalbaarheid tussen reagens besendings is belangrik om onder- of oorbepaling te verhoed. Metode validasie is ’n standaard proses in elke goeie laboratorium om die aanvaarbaarheid van ’n nuwe metode te bepaal. Verwysingswaardes is al bepaal in ’n ouer populasie. Ons het besluit om die verwysingswaardes in ons populasie te bepaal. Mens-immuungebrekvirus (MIV) word geassosieer met B-sel disfunksie. Omdat B-sel abnormaliteite geassosieer word met afwykings wat tot monoklonale gammopatieë lei, het ons gepostuleer dat die VLK vlakke en VLK ratio abnormaal sal wees in MIV geïnfekteerde persone. Metodes en Materiale: Kontroles en pasiënt monsters is gebruik vir die metode validasie studie wat impresisie studies, lineariteit, herwinning, inmenging en metode korrelasie studies ingesluit het. In laasgenoemde geval is ons metode met dieselfde metode van ’n ander laboratorium vergelyk. Vir die verwysingswaardes studie is 120 gesonde persone se bloed gebruik. Die volgende toetse is bepaal: totale proteïen, IgG, IgA, IgM, kreatinien, proteïen elektroferese, kappa en lambda VLK. Die VLK ratio is bepaal deur die kappa en lambda resultate te gebruik. Driehonderd nege en sestig MIV-positiewe pasiente is gebruik vir die studie. Dieselfde toetse was gedoen, asook CD4+ tellings en virale ladings op die meerderheid van pasiente. Resultate: Vir die metode validasie studie, was presisie, lineariteit en herwinning aanvaarbaar. Minimale inmenging van hemolise, lipemie, bilirubien en rumatoïede factor is waargeneem. Ons metode het goed gekorreleer met die bepaalde metode. Die serum kreatinien en serum totale proteïen waardes was normaal tydens die verwysingswaardes studie. Die serum proteïen elektroferese was onafhanklik beoordeel deur 3 patoloë. Die meeste was normaal met enkele poliklonale verhogings, maar geen definitiewe monoklonale bande nie. Die 95% verwysings intervalle vir VLK en VLK ratio het nie statisties betekenisvol verskil van die vervaardiger se aanbevelings nie. In die studie van die MIV-positiewe studie populasie, het ons gevind dat VLK en VLK ratio beïnvloed word deur merkers van ernstige MIV siekte, soos CD4+ telling, IgG, virale lading, die gebruik van antiretrovale medikasie en abnormale serum proteïen elektroferese. Gevolgtrekking: Die validasie studie van VLK het uitstekende presisie, aanvaarbare partydigheid, goeie lineariteit, goeie herwinning en minimale inmenging gewys, wat die roetine instelling van die toets toegelaat het. Die 95% verwysingsintervalle wat vir ons populasie bepaal is, was effens hoër as die vervaardiger se aanbeveling. Die meeste van die waardes het egter binne die vervaardiger se limiete geval, dus kon ons die vervaardiger se afsnypunte aanvaar. Ons het gevind dat VLK vlakke definitief beïnvloed word deur merkers van die ernstigheidsgraad van MIV siekte in ons populasie en ons postuleer dat VLK van waarde kan wees met die opvolg van MIV pasiente. / NHLS / Harry Crossley for funding obtained

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Grape and wine phenols consist of flavanols which is the building blocks for tannins. These building blocks are called monomers which consist of catechins, epicatechins, epigallocatechins and epicatechin-gallate. Tannin is important in wine as it contributes to bitterness, mouth feel (astringency) and maturation potential of the wine. Futhermore it has a health benefit as an antioxidant. Anthocyanins are responsible for the colour of red wine. The anthocyanins combine with tannins to form stable polymeric pigments. Due to the importance of tannins and anthocyanins in wine, it is imperitative that different winemaking techniques are used to extract as much of these components as possible and that the analysis is done quickly and accurately. The aim of this study was to evaluate different winemaking techniques and their extraction of tannins and anthocyanins into the wine. Too much tannin extraction can have a negative effect on the sensory quality of the wine. Therefore a second aim was to evaluate the mouth feel properties of a Shiraz wine. A third aim was to compare the two tannin precipitation methods in terms of time efficiency, repeatability and the ease of practice. To investigate the amount of tannin concentration extracted by different winemaking techniques, two cultivars (Cabernet Sauvignon and Shiraz) were used. These treatments included the addition of an enzyme during fermentation [E], cold maceration [CM], post maceration [PM] and the combination of cold and post maceration [CM+PM]. The grapes were harvested in two different climatic areas during the 2008 and 2009 vintages. The two climatic areas were classified according to the Winkler scale as a III (Morgenster) and a IV (Plaisir de Merle). The grapes were harvested at two different ripeness levels in order to evaluate the effect of the different winemaking processes on the extraction of tannins and anthocyanins. One harvest was before (LB) and the other after (HB) the commercial harvest. The results of this study showed significant differences in the phenolic composition of the wines. It was found that the warmer area showed higher tannin concentrations than the cooler area for both cultivars. In the 2008 Cabernet Sauvignon the CM extracted higher concentrations of tannin from the cooler area at both ripeness levels. In the warmer area, CM extracted the highest tannin concentration HB, but the CM+PM extracted the highest tannin concentration from Cabernet Sauvignon at the LB and CM at the HB of the warmer area. In 2009 the PM extracted the highest concentration of tannin at the lower ripeness level, while the E treatment extracted the highest concentration from the warmer area. In the cooler area the CM+PM extracted the highest concentration of tannin at a lower ripeness level, while there were no siginicant differences between the different treatments at the higher ripeness level. The highest anthocyanin concentration was found in the cooler area. The CM treatment was found to have no effect on anthocyanin extraction. Different methods are available to quantify the tannin concentration in wine. Two of the most popular tannin analytical methods are the bovine serum albumin (BSA) and the methyl cellulose precipitable tannin (MCP) methods. The BSA method is a very complex method which uses at least 3 times more reagents than the MCP method. The MCP method only analyzes tannins, while the BSA method analyzes tannins, monomeric pigments (MP), small polymeric pigments (SPP) and large polymeric pigments (LPP). In this study a good correlation was found between the two tannin precipitation methods (R2 – 0.88). There is controversy regarding the variability of these methods. Some scientists found that the two methods show a good correlation with HPLC, while others found that there was no such correlation between the precipitation methods and the HPLC. The MCP method had a practical advantage as it could be performed in half the time required for the BSA method. This has a significant impact in scenarios where a high sample throughput is required although it only measures total tannin. The phenolic composition and mouth feel of the wine was strongly influenced by the climatic area. In the warmer area the effect of tannin concentration on mouth feel was much less than in the cooler area. The wine made of riper grapes, was more grippy, bitter and numbing than the wines made from greener grapes. The E treatment was especially associated with a dry, grippy sensation. / AFRIKAANSE OPSOMMING: Druif en wyn fenole bestaan uit flavanole wat weer die boublokke is van tanniene. Hierdie boublokke, wat bekend staan as monomere, betsaan uit katesjiene, epikatesjiene, epigallokatesjiene an epikatesjien-gallaat. Tanniene is belangrik in wyn aangesien dit bydra tot bitterheid, mondgevoel (vrankheid) asook die verouderingspotensiaal van wyn. As antioksidante hou dit ook gesondheidsvoordele in. Antosianiene dra by tot die kleur van rooiwyn. Antosianiene kombineer met tanniene om meer stabiele polimeriese pigmente te vorm. As gevolg van die belangrikheid van tanniene en antosianiene is dit van uiterse belang dat verskillende wynmaak tegnieke gebruik word om ekstraksie in die wyn te bevoordeel en dat die analitiese metode so vinnig en akkuraat as moontlik gedoen word. Die eerste doel van hierdie studie was om die ekstraksie van tanniene en antosianiene deur middel van verskillende wynmaak tegnieke te evalueer. Te veel tanniene in die wyn kan negatiewe sensoriese kwaliteit tot gevolg het. Daarom is die tweede doel om die sensoriese kwaliteit van Shiraz wyn te evalueer. Die derde doel van hierdie studie was die twee tannien presipitasie metodes met mekaar te vergelyk in terme van die moeilikheidsgraad van die metode, tyd doeltreffendheid en herhaalbaarheid. Verskillende wynmaak tegnieke (ensiem byvoegings [E], koue maserasie [CM], verlengde dopkontak [PM] en ‘n kombinasie van koue maserasie en verlengde dopkontak [CM+PM]) is vergelyk ten opsigte van tannien en antiosianien ekstraksie. In 2008 en 2009 is twee kultivars (Cabernet Sauvignon en Shiraz) in twee verskillende klimatologiese areas gepars. Hierdie areas is geklassifiseer in die Winklerskaal as ‘n IV (Plaisir de Merle) en ‘n III (Morgenster). Om die effek van die verskillende wynmaak tegnieke op die ekstraksie van antosianiene en tanniene te vergelyk, is hierdie twee kultivars by twee verskillende rypheidsgrade geoes. Die eerste oes was net voor kommersiële oes (LB) en die tweede oes het net na kommersiële oes (HB) plaasgevind. Die 2009 Shiraz wyn is organolepties beoordeel om die effek van die verskillende wynmaak tegnieke op die wyn se mondgevoel te vergelyk. Die resultate van hierdie studie toon beduidende verskille in die fenoliese samestelling van die wyne. Dit is gevind dat die warmer area hoër tannien konsentrasies het as die koeler area. In 2008 het die CM+PM die meeste tanniene uit die Cabernet Sauvignon geëkstraheer by LB en die CM by HB in die warmer area. Die CM het in die koeler area meer tanniene geëkstraheer by beide die LB en HB rypheidsgrade. In 2009 het PM die meeste tanniene geëkstraheer by LB terwyl E die meeste tanniene geëkstraheer in die warmer area. In die koeler area het CM+PM die meeste tanniene geëkstraheer, terwyl geen van die behandelings ‘n effek gehad het by HB. Die meeste antosianien konsentrasie was in die koeler area gevind as in die warmer area. In beide 2008 (LB en HB) en 2009 (LB) het CM die meeste antosianiene geëkstraheer, terwyl geen behandeling ‘n effek gehad het by HB. Twee van die mees populêre tannien analitiese metodes is die BSA (bovine serum albumien) en die MCP (metielsellulose presipitasie) metodes. Die BSA metode is ‘n baie meer ingewikkelde metode waarvoor drie keer meer reagense gebruik word as vir die MCP metode. Maar waar die MCP net tanniene ontleed, ontleed die BSA metode tanniene, monomere (MP), klein polimeriese pigmente (SPP) en groot polimeriese pigmente (LPP). Dit help indien daar gekyk wil word na die evolusie van polimeriese pigmente. In hierdie studie is bevind dat daar ‘n redelike korrelasie (R2 – 0.88) tussen die BSA en MCP metode bestaan. Die herhaalbaarheid van die metodes het redelike kontroversie veroorsaak, waar sommige navorsers bevind het dat die BSA metode nie so herhaalbaar is soos eers bevind is nie. Die MCP metode het ’n praktiese voordeel aangesien dit in die helfde van die tyd van die BSA metode uitgevoer kan word. Dit het ‘n groot impak indien ‘n groot hoeveelheid monsters ontleed moet word. Die fenoliese samestelling en mondgevoel word sterk beïnvloed deur die klimatologiese area. In die warmer area was die effek van tannien konsentrasie op mondgevoel kleiner as in die koeler area. Die wyn van ryper druiwe het meer harder, verdowingseffek en bitter nasmaak gehad as by die wyn van groener druiwe. Die ensiem behandeling was meer geassossieerd met droë mond gevoel.

In real life, immediately after joint replacement, the artificial joint is actually bathed in blood (and clotted blood) instead of synovial fluid. Blood contains large molecules and cells of size ~ 5 to 20 μm suspended in plasma and considered to be a non-Newtonian (pseudoplastic) fluid with density of 1060 Kg/m³ and viscosity ~ 0.01 Pas at shear rates of 3000 s⁻¹ (as obtained in this work). The effect of these properties on friction and lubrication is not fully understood and, so far to our knowledge, hardly any studies have been carried out regarding friction of metal-on-metal bearings with various clearances in the presence of lubricants such as blood or a fluid containing macromolecules such as hyaluronic acid (HA) which is a major component of synovial fluid increasing its viscosity and lubricating properties. In this work, therefore, we have investigated the frictional behaviour of a group of Smith and Nephew Birmingham Hip Resurfacing implants with a nominal diameter of 50mm and diametral clearances in the range ~ 80 μm to 300 μm, in the presence of blood (clotted and whole blood), a combination of bovine serum (BS) with hyaluronic acid (HA) and carboxymethyl cellulose (CMC, as gelling agent) adjusted to a range of viscosities (~0.001-0.2 Pas), and bovine serum with CMC adjusted to a similar range of viscosities. These results suggested that reduced clearance bearings have the potential to generate high friction especially in the presence of blood which is indeed the in vivo lubricant in the early weeks after implantation. Friction factors in higher clearance bearings were found to be lower than those of the lower clearance bearings using blood as the lubricant. Similar trends, i.e. increase in friction factor with reduction in diametral clearance, were found to be also the case using a combination of BS+CMC or BS+HA+CMC as lubricants having viscosities in the range 0.1-0.2 and 0.03-0.14 Pas, respectively. On the other hand, all the lubricants with lower viscosities in the range 0.001-0.0013 and 0.001-0.013 Pas for both BS+CMC and BS+HA+CMC, respectively, showed the opposite effect, i.e. caused an increase in friction factor with increase in diametral clearance. Another six large diameter (50mm nominal) BHR deflected prostheses with various clearances (~ 50-280μm after cup deflection) were friction tested in vitro in the presence of blood and clotted blood to study the effect of cup deflection on friction. It was found that the biological lubricants caused higher friction factors at the lower diametral clearances for blood and clotted blood as clearance decreased from 280μm to 50μm (after deflection). The result of this investigation has suggested strongly that the optimum clearance for the 50 mm diameter MOM BHR implants to be ≥150μm and <235μm when blood lubricant used, so as to avoid high frictions (i.e. avoid friction factors >0.2) and be able to accommodate a mixed lubrication mode and hence lower the risk of micro- or even macro-motion specially immediately after hip implantation. These suggested optimum clearances will also allow for low friction (i.e. friction factors of <0.2-0.07) and reasonable lubrication (dominantly mixed regime) for the likely cup deflection occurring as a result of press-fit fixation.