Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: A number of relevant brewing industry issues associated with malt quality were examined. These included beer foam quality, premature flocculation of yeast during fermentation and antimicrobial factors in malt. The cause of poor foam at a brewery relative to other similar breweries was identified as being related to the boiling temperature during wort preparation and the associated conformational changes of the abundant foam protein lipid transfer protein 1 (LTPl). The temperature range of 96 to 102°C was revealed to be critical. At the higher temperature the denaturation of LTP 1 was more extensive and its effectiveness as a foam protein was reduced. In addition, it was shown that the prominent role of LTPI with respect to foam was as a lipid binding protein, forming a lipid sink and protecting foam from lipid damage. The occurrence of malt associated premature yeast flocculation (PYF) during fermentation was induced in malt by the addition of extra-cellular fungal enzymes to the malt husk or by micro-malting barley in the presence of fungi. In addition, treating malt husk with commercial xylanase or adding commercial arabinoxylan to the fermentation also impacted on yeast flocculation. It was proposed that a range of molecular weight arabinoxylans formed by the enzymatic breakdown of the major barley husk component (arabinoxylan) resulted in PYF. Antimicrobial activity against brewing yeast (Saccharomyces cerevisiae), other fungi and bacteria was found in barley, malt and malt derived wort trub. Wort trub is the non-specific precipitate of protein, polyphenols and lipids formed during wort boiling and which is, to some extend, carried over in the wort to the fermentation. Antimicrobial activity appeared to increase during malting. The growth of brewery collected yeast was inhibited in the presence of brewery production wort when compared to the same wort filtered to remove the trub. Brewery yeast was found to be more sensitive to inhibition than laboratory propagated yeast of the same strain. Different strains of S. cerevisiae were also found to differ in their sensitivity to inhibition. Investigation revealed that the activity originated from the inside of the barley grain and impacted on yeast sugar uptake. However, there was no direct correlation detected between levels of antimicrobial activity in malt and fermentation performance. At high concentrations the factors were microcidal causing cell lysis. Partial characterisation of an antimicrobial extract from malt revealed the presence of a factor between 5 and 14 kDa, containing a cationic peptide component. The optimum pH stability was ±5 when it was also most cationic. The factor easily and irreversibly lost activity at extreme pH and when exposed to certain reagents but was heat resistant in accordance with its survival in wort trub. Preliminary results showed the presence of LTP1 associated with other peptides in the active cationic fraction from the one malt tested. The occurrence of malt related PYF and malt antimicrobial factors are associated with microbial contamination of the grain. The fungi generating the PYF factors from the barley husk while the barley's defence mechanism generates antimicrobial factors to cope with the pathogenic effect of the fungi. In addition there is a potential link between the foam protein LTP 1 and malt antimicrobial activity as LTP 1 or LTP 1 in association with another component(s) is potentially antimicrobial. / AFRIKAANSE OPSOMMING: 'n Aantal problematiese areas in die broubedryf, wat met mout geassosieer word, is ondersoek, naamlik bierskuimkwaliteit, voortydige flokkulering van gis tydens fermentasie en die invloed van antimikrobiese faktore in mout. Die oorsaak van swak bierskuim by 'n spesifieke brouery relatief tot ander soortgelyke brouerye was geidentifiseer as die moutekstrakkookpunt tydens moutekstrakbereiding. Tydens hierdie proses ondergaan dieskuimprotein, lipiedoordrag proteien 1 (lipid transfer protein 1, LTPI), 'n konformasieverandering. Die temperature tussen 96 to 102°C was kritiek t.o.v. ideale konformasieverandering vir skuimaktiwiteit. Denaturering van LTPI het by hoër temperature plaasgevind wat die skuimproteien se aktiwitiet verminder het. Daar is ook bewys dat LTPI 'n verdere rol in bierskuim speel aangesien dit 'n lipiedbindingsproteien is wat die skuimnegatiewe lipiede verwyder. Die voorkoms van moutgeassosieerde voortydige flokkulering van gis (PYF) tydens fermentasie is op twee maniere in mout geinduseer, naamlik: • deur die toevoeging van ekstrasellulêre swamensieme tot die moutdop • deur mikrovermouting van gars in die teenwoordigheid van swamme. Die behandeling van die moutdop met kommersiele xilanase of die toevoeging van kommersiele arabinoxilaan by fermentasies het ook die flokkulering van gis beinvloed. Die hipotese was dat PYF veroorsaak is deur 'n reeks arabinoxilane met verskillende molekulêre massas wat gevorm het tydens die ensimatiese afbraakproses van die primere moutdopkomponent (arabinoxilaan). Antimikrobiese aktiwiteit teenoor brouersgis (Saccharomyces cerevisiae), ander swamme en bakterie was teenwoordig in gars, mout en moutekstrakpresipitaat. Die presipitaat bestaan uit nie-spesifieke presipitate van proteien, polifenole en lipiede wat gedeeltelik in die gekookte moutekstrak agterbly. Daar is gevind dat antimikrobiese aktiwiteit tydens vermouting toe geneem het. Die groeiproses van brouersgis, gekollekteer by 'n brouery, was geinhibeer deur die teenwoordigheid van brouery-geproduseerde moutekstrak in vergelyking met dieselfde moutekstrak wat gefiltreer was om die presipitaat te verwyder. Die brouersgis was meer sensitief heens inhibisie in vergeleke met dieselfde gisstam wat opgegroei is in die laboratorium. Verskillende S. cerevisiae stamme het ook verskille in sensitiwiteit getoon t.o.v. the antimikrobiese komponente in die moutekstrakte. 'n Verdere ondersoek het getoon dat die oorprong van die inhiberende aktiwiteit die interne dele van die gars is, asook dat dit die gissuikeropname beinvloed. Daar was egter geen direkte verband tussen antimikrobiese aktiwiteit in mout en fermentasie effektiwiteit, soos gemeet onder laboratorium toestande, nie. Hoë konsentrasies van die faktore het egter gelei tot seldood weens sellise. 'n Kationiese peptiedbevattende fraksie tussen 5 en 14 kDa en 'n optimale pH stabliliteit van 5 is gevind deur gedeeltelike karakterisering van 'n antimikrobiese moutekstrak. Die aktiewe fraksie se aktiwiteit is onomkeerbaar vernietig by ekstreme pH en blootstelling aan sekere reagense. Die aktiewe verbinding(s) is egter hittebestand en resultate het getoon dat hierdie aktiwiteit die brouproses oorleef as deel van die moutektrakpresipitaat. Voorlopige resultate van die een mout wat getoets is het die teenwoordigheid van LTP 1 getoon, asook die moontlike assosiasie met ander peptiede of kleiner komponente in die aktiewe kationiese fraksie. Die voorkoms van moutgeassosieerde PYF en antimikrobiese faktore in mout word met die mikrobiologiese kontaminasie van gars verbind. Swamme produseer die PYF faktore vanuit die moutdopkomponente, terwyl die plant weer antimikrobiese faktore produseer as deel van 'n beskermingsmeganisme teen die patogene effek van die swamme. Daar is ook 'n potensieele verwantskap tussen bierskuimproteien LTP 1 en antimikrobiese faktore in mout, aangesien LTPI ofLTPl tesame met 'n ander verbinding(s) moontlik antimikrobies is.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This study describes: a) the isolation and purification of horseradish peroxidase isoenzymes from horseradish roots, b) the characterization of various forms and components of the enzyme by cation-exchange and reversed-phase high performance liquid chromatography, c) the preparation of antibodies against horseradish peroxidase isoenzymes, d) immunological studies for the development of an isoenzyme quantification method and e) the formation of an enzyme-melamine conjugate for use in a melamine quantification immunoassay. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: a) die isolering en suiwering van peperwortel-peroksidase-isoënsieme vanuit die peperwortel, b) die karakterisering van verskillende vorme en komponente van dié ensiem deur katioonuitruilings en omgekeerde-fase HPLC c) die voorbereiding van teenliggaampies vir peperwortel-peroksidase-isoënsieme, d) immunologiese studies vir die ontwikkeling van 'n isoënsiem-kwantifiseringsmetode; en e) die vorming van 'n ensiem-melamien-konjugaat vir gebruik in 'n melamienkwantifiseringsmetode.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The complexity of microbial systems has presented serious obstacles to the quantification of fermentation processes. Using computer modelling techniques progress has been made in monitoring, controlling and optimising microbial systems using material balancing techniques and empirical process models. The Monod equation is among the most commonly used models and is based on empirical findings with no mechanistic basis. Monod presents a simple model to describe the growth of a cell in a defined nutrient environment. The Monod equation is mathematically analogous to the formula that was proposed by Michaelis and Menten to describe enzyme kinetics. Both equations describe a hyperbolic function with a half-saturation constant (K_s in the monod equation and K_m in the Michaelis Menten equation) but the meaning of the two saturation constants K_s and K_m is different. In number of studies K_s and K_m are used as if they are equivalent. In contrast to Michaelis-Menten kinetics, which describes a process catalysed by a single enzyme, Monod kinetics describes an overall process involving thousands of enzymes. The Monod equation describes the specific growth rate of a microbial cell as the function of a limiting substrate concentration. The aim of this study was to test this principle, for Saccharomyces cerevisiae VIN13 under glucose limited aerobic chemostat conditions. The VIN13 was observed to follow the Monod description and when compared with other growth kinetic models gave one of the best fits to the data. A functional relationship between the half-saturation constant, K_s, and Michaelis Menten constant, K_m, was there after derived. This was achieved by using metabolic control analysis (MCA) to explain when K_m of the transporter becomes equal to the K_s. Using the deductions obtained from MCA a core kinetic model was then formulated to demonstrate that the K_s can either be smaller, equal or higher than the K_m of the transporter, depending on the flux control distribution in the model. / AFRIKAANSE OPSOMMING: Die kwantifisering van fermentasieprosesse word ernstig belemmer deur die kompleksiteit van mikrobiale sisteme. Deur gebruik te maak van rekenaar-ondersteunde modelleringstechnieke vir die opstelling van massa balans vergelykings en empiriese prosesmodelle is vordering gemaak in die waarneming, beheer en optimalisering van mikrobiale sisteme. Die Monod vergelyking is een van die mees gebruikte groeimodelle en is gebaseer op empiriese bevindings - die model het nie ‘n meganistiese grondslag nie. Die Monod vergelyking is wiskundig ekwivalent aan die vergelyking wat opgestel is deur Michaelis en Menten vir die beskrywing van ensiemkinetika. Beide vergelykings beskryf ‘n hyperboliese kurwe met ‘n konstante wat die halfversadigingswaarde aangee vir substraat (Ks in die Monod vergelyking en Km in die Michaelis-Menten vergelyking), maar die betekenis van die twee versadigingskonstantes is verskillend. In ‘n aantal studies word die Ks en Km waardes gebruik asof hulle gelyk is aan mekaar. In teenstelling met die Michaelis- Menten kinetika wat ‘n enkel ensiem-gekataliseerde reaksie beskryf, beskryf die Monod vergelyking ‘n proses wat duisende ensieme behels. Die Monod vergelyking beskryf die spesifieke groeitempo van ‘n bakteriële sel as ‘n funksie van die beperkende substraatkonsentrasie. Die doel van hierdie studie was om hierdie beginsel te toets vir Saccharomyces cerevisiae VIN13 wat onder glukose beperkte, aerobiese kondisies in ‘n chemostat gekweek word. Die VIN13 groei kon goed beskryf word met die Monod model, wat in vergelyking met ander groeimodelle een van die beste passings vir die meetpunte het gegee. Vervolgens is ‘n funksionele verwantskap afgelei tussen Ks en Km; deur gebruik te maak van metabole kontrole analise (MCA) kon verduidelik word wanneer die Ks gelyk is aan die Km van die transporter vir die beperkende substraat. Deur gebruik te maak van die MCA analise is ‘n eenvoudige kinetiese model opgestel om aan te toon dat die Ks kleiner, gelyk aan of groter kan wees as die Km van die transporter, afhanklik van die fluksie-kontrole verdeling in die model.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The African penguin, which occurs along the south-eastern and south-western shores of South-Africa and Namibia, has experienced a severe reduction in population numbers due to guano and egg collection in the first half of the 19th century, and oil pollution in the second half of the 19th century as a result of oil tankers rounding the Cape of Good Hope. The population would have been reduced by a further 19% had it not been for the rehabilitation of penguins at the South African National Council for the Conservation of Coastal Birds (SANCCOB) facility. Although this has been very successful, mortalities as a result of avian malaria infection have considerably reduced the efficiency of rehabilitation. In an effort to assess the role of immunity against malaria in combating the disease, an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody levels to avian malaria was developed. The ELISA was used to detect antibody levels to avian malaria of penguins on entry and during rehabilitation from October 2001 to January 2003. The aim of this study was to continue the determination of antibody levels to avian malaria of penguins entering the SANCCOB facility, in order to allow an evaluation of the antibody levels to avian malaria for two full calendar years. This investigation was combined with a polymerase chain reaction (PCR)-based method, capable of detecting any Plasmodium species in penguin serum. These two methods were also used to investigate avian malaria in several breeding colonies in order to assess the role avian malaria may play in the survival of the African penguin in the wild. Results indicated that the ability of penguins to produce anti-Plasmodium antibodies was not influenced by oiling and that infection with malaria was not due to recrudescence but rather due to infection via mosquitoes. This indicated a possible role of the SANCCOB facility in exposing the penguins to avian malaria. However a large number of penguins arrived at the facility previously infected with malaria, indicating that malaria was present in the breeding colonies. Investigations in the breeding colonies revealed extremely high avian malaria prevalence even though no sick birds or mortalities were observed. This raised the question whether different types of malaria are responsible for infection in the SANCCOB facility and breeding colonies. / AFRIKAANSE OPSOMMING: Die Afrika Pikkewyn kom langs die suid-oostelike en suid-westelike kus van Suid Afrika en Namibië voor. In die afgelope eeu het hierdie spesie ‘n geweldige afname in populasie getalle ondervind. Dit was hoofsaaklik die gevolg van die versameling van guano en pikkewyneiers in die eerste helfte van die 19de eeu en oliebesoedeling in die tweede helfde van die 19de eeu. Die “South African Foundation for Conservation of Coastal Birds” (SANCCOB) is ‘n seevoëlreddings- en rehabilitasiesentrum vir siek, beseerde en ge-oliede pikkewyne. Dit word geskat dat die Afrika Pikkewyn populasie met ‘n verdere 19% sou afgeneem het as dit nie vir die rehabilitasie by die SANCCOB sentrum was nie. Hierdie sentrum het egter aansienlike vrektes in die somer as gevolg van voëlmalaria, wat sodoende die effektiwiteit van die rehabilitasie verlaag. In ‘n poging om die rol van immuniteit teen malaria te bepaal is ‘n “enzyme-linked immunosorbent assay” (ELISA) ontwikkel vir die bepaling van antiliggaam vlakke teen malaria. Hierdie ELISA is gebruik vir die bepaling van die anti-Plasmodium antiliggaam vlakke van die pikkewyne by aankoms en ten tye van rehabilitasie by SANCCOB vanaf Oktober 2001 to Januarie 2003. Die doel van hierdie studie was eerstens om hierdie ELISA bepalings voort te sit om sodoende antiliggaam vlakke teen malaria oor twee kalender jare te kan evalueer. Hierdie ondersoek was gekombineer met ‘n polimerase ketting reaksie (PCR) metode, wat enige Plasmodium spesie in pikkewynserum sou kon opspoor. Hierdie twee metodes is ook gebruik vir ondersoeke in sommige broeikolonies, met die doel om te bepaal watter rol voëlmalaria in die oorlewing van die Afrika pikkewyn in die natuur speel. Resultate het getoon dat olie nie die vermoë van die pikkewyn beïnvloed om anti- Plasmodium antiliggame te vervaardig nie en dat malaria infeksie hoofsaaklik deur muskiete veroosaak word en nie deur heruitbraak van ‘n bestaande infeksie nie. Dit dui egter daarop dat pikkewyne blootgestel word aan voëlmalaria by die SANCCOB sentrum. Daar is ook gevind dat ‘n groot aantal pikkewyne met malaria infeksies by die sentrum opgedaag het wat dui op die voorkoms van malaria in die broeikolonies. Ondersoeke in die broeikolonies het ‘n besonder hoë voorkoms van malaria onthul. Geen vrektes of siek pikkewyne is in die broeikolonies waargeneem nie, wat moontlik kan beteken dat pikkewyne by SANCCOB met ‘n ander tipe malaria geïnfekteer word as in die broeikolonies.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Ostrich farming is of significant economical importance in South Africa. Three ostrich mycoplasmas, Ms01, Ms02 and Ms03 have been identified previously, and were provisionally named ‘Mycoplasma struthiolus’ (Ms) after their host Struthio camelus. Ostrich mycoplasmas are the major causative organisms of respiratory diseases, and they cause stock losses, reduced production and hatchability, and downgrading of carcasses and therefore lead to large economic losses to the industry. In order to be pathogenic to their host, they need to attach through an attachment organelle, the so-called tip structure. This structure has been identified in the poultry mycoplasma, M. gallisepticum, and is made up of the adhesin GapA and adhesin-related CrmA. Currently, no ostrich mycoplasma vaccine is commercially available and for this reason the need to develop one has arisen. Therefore the first part of this study was dedicated to the identification and isolation of vaccine candidate genes in the three ostrich mycoplasmas. Four primer approaches for polymerase chain reactions (PCR’s), cloning and sequencing, were used for the identification of adhesin or adhesin-related genes from Ms01, Ms02 and Ms03. The primer approaches revealed that the target genes could not be identified due to the high diversity of sequences that were generated. Therefore sequences were also compared with those of other mycoplasma species in BLAST searches. Results showed that the most significant hit was with the human pathogen M. hominis oppD, which is located in the same operon as the membrane protein P100 involved in adhesion. Other hits were with ABC transporters which may also play a role in cytadhesion. The second part of this study was aimed at testing whether two poultry mycoplasma vaccines, M. synoviae and M. gallisepticum, can be used in ostriches to elicit immune responses until an ostrich mycoplasma vaccine has been developed. Ostriches on three farms of different age groups in the Oudsthoorn district were therefore vaccinated with these vaccines in a vaccine trial. The enzymelinked immunosorbent assay (ELISA) was used to test the level of antibody response. Results showed that both vaccines elicited an immune response in all three age groups. A high percentage of the ostriches reacted positively, which indicates that both vaccines elicit antibody responses and may therefore give protection against ostrich mycoplasma infections. / AFRIKAANSE OPSOMMING: Volstruisboerdery is ‘n belangrike ekonomiese sektor in Suid-Afrika. Drie volstruismikoplasmas, Ms01, Ms02 en Ms03, is voorheen geïdentifiseer en voorlopig ‘Mycoplasma struthiolus’ (Ms) benaam na aanleiding van hul gasheer, Struthio camelus. Volstruismikoplasmas is die grootste oorsaaklike organismes van respiratoriese siektes, kudde verliese en die afgradering van karkasse wat lei tot groot ekonomiese verliese in die volstruisbedryf. Ten einde patogenies vir die gasheer te wees, moet mikoplasmas deur middel van ‘n aanhegtingsmeganisme vasheg – die sogenaamde puntvormige struktuur. Hierdie struktuur is in die pluimvee mikoplasma M. gallisepticum geïdentifiseer, en bestaan uit aanhegting proteïen GapA en die aanhegting verwante proteïen CrmA. Tans is geen volstruismikoplasma entstof kommersieel beskikbaar nie, en derhalwe het die behoefte ontstaan om so ‘n entstof te ontwikkel. Die eerste gedeelte van hierdie studie is dus gewy aan die identifisering en isolering van entstof kandidaat gene in al drie volstruismikoplasmas. Vier inleier benaderings vir polimerase ketting reaksies (PKR), klonering asook geenopeenvolging bepalings vir die identifisering van aanhegting of aanhegting verwante gene vanuit Ms01, Ms02 en Ms03 is gebruik. Die inleier benaderings het getoon dat die teikengene nie geïdentifiseer kon word nie as gevolg van hoë variasie in die gegenereerde geenopeenvolgings. Derhalwe is geenopeenvolgings met ander mikoplasma spesies deur middel van BLAST soektogte vergelyk. Resultate het getoon dat die betekenisvolste ooreenstemming dié met die menslike patogeen M. hominis oppD was, wat deel vorm van die membraan proteïen P100 operon wat betrokke is by aanhegting. Ander ooreenstemmings sluit ABC transporters in wat moontlik betrokke kan wees by aanhegting. Die tweede gedeelte van hierdie studie het ten doel gehad om te toets of twee pluimvee mikoplasma entstowwe, M. synoviae en M. gallisepticum, gebruik kan word in volstruise om immuunresponse te ontlok tot tyd en wyl ‘n volstruismikoplasma entstof ontwikkel is. Volstruise vanaf drie plase in verskillende ouderdomsgroepe in die Oudtshoorn distrik was ingeënt met hierdie entstowwe in ‘n entstof proefneming. Die ensiem-afhanklike immuno-absorpsie essaï (ELISA) was gebruik om antiliggaam response te toets. Die resultate het getoon dat beide entstowwe immuunresponse ontlok het in al drie ouderdomsgroepe. ‘n Groot persentasie van die volstruise het positief gereageer wat ‘n aanduiding is dat beide entstowwe immuunresponse ontlok het en kan dus beskerming bied teen volstruismikoplasma infeksies.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The interests in understanding the metabolic processes of microbial systems are numerous. The interest in the species Lactococcus lactis (L. lactis) lies in applications to the food industry and in studies comparing the metabolism of related organisms. The aim of this study was to perform in vivo supply-demand analysis on anaerobically fermenting L. lactis. This was done by perturbing both the supply and demand pathways, then measuring glycolytic flux (by means of 13C NMR spectroscopy) and intracellular ATP/ADP (by means of 31P NMR spectroscopy) at steady state – where the central metabolite, ATP, is produced at the same rate as it is consumed and its concentration thereby remains constant. The L. lactis reference strain MG1363 was supplemented with glucose and analysed “online” by 13C-NMR under anaerobically-fermentative conditions. The rates of glucose consumption and lactate production were determined from this 13C flux. Due to experimental difficulties with the online detection of 31P (possibly due to the low biomass yield and the choice of growth medium), ATP/ADP levels had to be determined offline: from the same batch cultures as the 13C samples, fermentations were performed and halted at time points when the cells had attained a steady state. These fermentation cultures were then subjected to cell lysis and centrifugation in order to extract intracellular metabolites. These cell extracts were analysed offline by 31P NMR in order to determined levels of phosphate metabolites, specifically ATP and ADP. Perturbation of the supply pathway was achieved by utilising a genetically modified strain (the CS8 strain with over-expressed las operon) and comparing it to the reference strain MG1363. This resulted in a slight increase in ATP/ADP, but also yielded a slightly reduced flux, which is contrary to expectations from a mutant with over expressed glycolytic enzymes. The demand pathway was perturbed by two methods: 1) utilisating a genetically modified strain (the BK1506 strain with over-expressed F1-ATPase) and comparing it to the reference strain MG1363, and 2) by treating wild-type MG1363 with sodium acetate and comparing flux and ATP/ADP values to the untreated wild-type. Sodium acetate dissociates in the cytoplasm and causes dissipation of the transmembrane proton motive force, which is re-established by upregulation of membrane-bound H+-translocating ATPases. While the use of genetically modified strains provided only one flux-vs-ATP/ADP data point to compare to the wild-type (not sufficient for complete supply-demand analysis), the treatment of the wild type with uncoupler yielded several data points where flux and ATP/ADP values differed according to the concentration of uncoupler added. The CS8 strain demonstrated a 19 % reduced glucose flux (24 % reduced lactate flux) with respect to the wild type MG1363. The BK1506 strain demonstrated a 72 % increase in glucose flux (33 % increase in lactate flux) with respect to the wild type. The treatment with 2 mM acetate resulted in a 72 % increase in glucose flux (123 % increase in lactate flux), whereas treatment with 4 mM acetate resulted in a 107 % and a 126 % increase in glucose and lactate fluxes, respectively. The treatment with different concentrations of acetate provided several data points with corresponding flux and ATP/ADP, enabling the calculation of the elasticity coefficient of the supply pathway to changes in ATP/ADP (εsupply ) which was found to be -5.6 and -6.3 for glucose and lactate, respectively. ATP/ADP The elasticity coefficient was high compared with values obtained in similar studies on other organisms. Considering that at steady state the supply and demand fluxes are equal, the high supply elasticity (which is easier to measure), when incorporated into control coefficient summation theorems, gives the indication that: 1) a greater amount of control may reside in the ATP demand pathway (the elasticity of which is more difficult to determine experimentally, but which may well be lower than the supply elasticity), and 2) ATP/ADP homeostasis is good, as indicated by a high elasticity of the supply pathway to ATP/ADP. This study represents a basis for further supply-demand analysis with non-growing batch cultures of L. lactis. / AFRIKAANSE OPSOMMING: Daar is groot belangstelling daarin om die metaboliese prosesse van mikrobiese sisteme beter te verstaan. Die belang van die spesie Lactococcus lactis (L. lactis) lê beide in die toepassing in die voedselbedryf en in studies wat die metabolisme van verskeie organismes vergelyk. Die doel van hierdie studie was om in vivo vraag-aanbod analise uit te voer op anaerobies-fermenterende L. lactis. Dit was gedoen deur beide die aanbod en vraag reaksie-blokke te moduleer en dan die glikolitiese fluksie (d.m.v. 13C KMR spektroskopie) en die intrasellulêre ATP/ADP (d.m.v. 31P KMR spektroskopie) in ’n bestendige toestand te meet (wanneer die sentrale metaboliet, ATP, teen dieselfde tempo geproduseer en verbruik word en sy konsentrasie daardeur konstant bly). Die L. lactis verwysing-stam MG1363 is met glukose aangevul en 13C fluksie is aanlyn onder anaerobies-fermenterende kondisies gemeet. Die tempo van glukose verbruik en laktaat produksie is vanaf die 13C fluksie bereken. Eksperimentele probleme met die aanlyn bepaling van 31P (dalk as gevolg van lae biomassa en/of die keuse van groeimedium) moes ATP/ADP vlakke af-lyn indirek bepaal word: fermentasies van dieselfde lot-kulture as die 13C monsters is opgestel en by sekere tydpunte gestop wanneer ‘n bestendige toestand bereik was (waar ATP/ADP konstant bly). Hierdie fermenterende kulture is blootgestel aan sel-lise en sentrifugasie om intrasellulêre metaboliete te onttrek. Dié sel-ekstrakte is deur 31P KMR geanaliseer om die vlakke van fosfaat metaboliete, spesifiek ATP en ADP, te bepaal. Die aanbod blok is gemoduleer deur gebruik te maak van ‘n geneties-gemodifiseerde stam (die CS8 stam met ‘n ooruitgedrukte las operon) en met die verwysing stam MG1363 te vergelyk. Dié gemuteerde stam het ’n effense toename in ATP/ADP getoon, maar het gelyktydig ook ’n afname in glikolitiese fluksie getoon, wat onverwags is vir ’n stam met ooruitgedrukte glikolitiese ensieme. Die vraag blok is met twee metodes gemoduleer: 1) deur gebruik te maak van ‘n geneties-gemodifiseerde stam (die BK1506 stam met ‘n ooruitgedrukte F1-ATPase), en 2) deur die wildetipe MG1363 met natrium asetaat te behandel en daardeur ATP verbruik van biomassa produksie te ontkoppel en die vraag na ATP te vermeerder. Daarna word die fluksie en ATP/ADP waardes met die onbehandelde wildetipe vergelyk. Natrium asetaat dissosieer in die sitoplasma en verswak die transmembraan elektriese potensiaal, wat dan weer versterk word deur membraan-gekoppelde H+-ATPase ensieme wat protone uit die sitoplasma uit pomp. Terwyl die gebruik van geneties-gemodifiseerde stamme net een fluksie-tot-ATP/ADP datapunt voorsien om met die wildetipe te vergelyk (wat nie voldoende is vir totale vraag-aanbod analise nie), het die behandeling van die wildetipe met ontkoppelaar meerdere datapunte voorsien waar fluksie en ATP/ADP waardes verskil volgens die konsentrasie van ontkoppelaar wat bygevoeg is. Die CS8 stam het ’n 19 % verminderde glukose fluskie getoon, asook ’n 23 % verminderde laktaat fluksie, in vergelyking met die wilde tipe MG1363. Die BK1506 stam het ’n 73 % toename in glukose fluskie getoon, asook ’n 34 % toename in laktaat fluksie, in vergelyking met die wilde tipe. Behandeling met 2 mM natrium asetaat het ’n 64 % toename in glukose fluksie veroorsaak, sowel as ’n 124 % toename in laktaat fluksie, en behandeling met 4 mM natrium asetaat het 108 % toename in glukose fluksie en 127 % toename in laktaat fluskie veroorsaak. Behandeling met verskillende konsentrasies natrium asetaat het genoeg data punte (fluksies met toepaslike ATP/ADP waardes) verskaf om die berekening van elastisiteits-koëffisiënt van die aanbod reaksie-blok tot veranderinge in ATP/ADP (εsupply ) te bereken. Die waardes was -5.6 vir glukose fluksie en -6.3 vir laktaat fluksie. ATP/ADP Die elastisiteits koëffisiënt was relatief hoog in vergelyking met waardes wat in soorteglyke studies op ander organismes bepaal is. Aangesien die fluksies van die aanbod en vraag reaksie blokke by ’n bestendige toestand dieselfde tempo het, kan die hoë waarde van die aanbod elastisiteits-koëffisiënt (wat die makliker een is om te meet) na die volgende afleidings lei: 1) meer kontrole mag in die ATP verbruikende reaksie-blok geleë wees (die elastisiteits-koëffisiënt is moeiliker om eksperimenteel te bepaal maar mag wel laer as die aanbod-elastisiteit wees), en 2) ATP/ADP homeostase word goed gehandhaaf, soos aangetoon deur die hoë aanbod-elastisiteit. Hierdie studie dien as ’n basis vir verdere vraag-aanbod analise in nie-groeiende L. lactis lot-kulture.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Progress in computational systems biology depends crucially on the availability of generic rate equations that accurately describe the behaviour and regulation of catalysed processes over a wide range of conditions. Such equations for ordinary enzyme-catalysed reactions have been developed in our group and have proved extremely useful in modelling metabolic networks. However, these networks link to growth and reproduction processes through template-directed synthesis of macromolecules such as polynucleotides and polypeptides. Lack of an equation that captures such a relationship led us to derive a generic rate equation that describes catalysed, template-directed polymerisation reactions with varying monomer stoichiometry and varying chain length. A model describing the mechanism of a generic template-directed polymerisation process in terms of elementary reactions with mass action kinetics was developed. Maxima, a computational algebraic solver, was used to determine analytical expressions for the steady-state concentrations of the species in the equation system from which a steady-state rate equation could be derived. Using PySCeS, a numerical simulation platform developed in our group, we calculated the time-dependent evolution and the steadystates of the species in the catalytic mechanisms used in the derivation of the rate equations. The rate equation was robust in terms of being accurately derived, and in comparison with the rates determined with PySCeS. Addition of more elongation steps to the mechanism allowed the generalisation of the rate equation to an arbitrary number of elongations steps and an arbitrary number of monomer types. To test the regulatory design of the system we incorporated the generic rate equation in a computational model describing a metabolic system consisting of multiple monomer supplies linked by a template-directed demand reaction. Rate characteristics were chosen to demonstrate the utility of the simplified generic rate equation. The rate characteristics provided a visual representation of the control and regulation profile of the system and showed how this profile changes under varying conditions. / AFRIKAANSE OPSOMMING: Die beskikbaarheid van generiese snelheidsvergelykings wat die gedrag en regulering van gekataliseerde prosesse akkuraat oor ’n wye reeks omstandighede beskryf is van kardinale belang vir vooruitgang in rekenaarmatige sisteembiologie. Sulke vergelykings is in ons groep ontwikkel vir gewone ensiem-gekataliseerde reaksies en blyk uiters nuttig te wees vir die modellering van metaboliese netwerke. Hierdie netwerke skakel egter deur templaat-gerigte sintese van makromolekule soos polinukleotiede en polipeptiede aan groei- en voorplantingsprosesse. Die gebrek aan vergelykings wat sulke verwantskappe beskryf het ons genoop om ’n generiese snelheidsvergelyking af te lei wat gekataliseerde, templaatgerigte polimerisasie-reaksies met wisselende monomeerstoigiometrie en kettinglengte beskryf. ’n Model wat die meganisme van ’n generiese templaat-gerigte polimerisasie-proses in terme van elementêre reaksies met massa-aksiekinetika beskryf is ontwikkel. Maxima, ’n rekenaarmatige algebraïese oplosser, is gebruik om analitiese uitdrukkings vir die bestendige- toestand konsentrasies van die spesies in die vergelyking-stelsel te vind. Hierdie uitdrukkings is gebruik om ’n bestendige-toestand snelheidsvergelyking af te lei. Ons het die tyd-afhanklike progressie en die bestendige toestande bereken van die spesies in die katalitiese meganismes wat gebruik is in die afleiding van die snelheidsvergelykings. Die rekenaarprogram PySCeS is ’n numeriese simulasieplatform wat in ons groep ontwikkel is. Die snelheidsvergelyking blyk akkuraat afgelei te wees en is in ooreenstemming met snelhede deur PySCeS bereken. Die toevoeging van verdere verlengingstappe tot die meganisme het dit moontlik gemaak om die snelheidsvergelyking te veralgemeen tot ’n arbitrêre hoeveelheid verlengingstappe en monomeertipes. Om die regulatoriese ontwerp van die sisteem te toets het ons die generiese snelheidsvergelyking in ’n rekenaarmatige model geïnkorporeer wat ’n metaboliese sisteem bestaande uit verskeie monomeer-aanbodblokke en ’n templaatgerigte aanvraagblok beskryf. Snelheidskenmerkanalise is gekies om die nut van die vereenvoudigde generiese snelheidsvergelyking te demonstreer. Met hierdie snelheidskenmerke kon ons die kontrole- en reguleringsprofiel van die stelsel visualiseer en wys hoe hierdie profiel verander onder wisselende omstandighede.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Fermentation in Zymomonas mobilis has been described as a catabolic highway, with 50 % of soluble protein comprising glycolytic and fermentative enzymes. In conjunction with one of the fastest observed fermentations, the conversion of glucose to ethanol forms one of the least efficient energy extractions found in nature. The low energy yield of fermentation in Z. mobilis is a result of the usage of the Entner-Doudoroff glycolytic pathway, which has half the energy yield per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway. The work presented in this thesis forms part of a larger project to compare glycolytic regulation in different micro-organisms (i.e., Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus lactis). These organisms were chosen based on their usage of different glycolytic mechanisms. By using supply-demand analysis for quantifying glycolytic regulation as well as similar experimental conditions (e.g. using non-growing cell cultures), we can compare the regulatory behaviour of mechanistically distinct freeenergy supplies. The aim of this thesis was to quantify the importance of anaerobic free-energy generation for the regulation of the Entner-Doudoroff glycolytic pathway in Z. mobilis. We used metabolic control analysis (MCA) and supply-demand analysis to realize this goal. The central message of MCA is that when a metabolic parameter (e.g., a conserved metabolic moiety) is deemed important for affecting a particular steady-state variable (i.e., fermentation flux), its effect on the steady state variable should be tested. An extension to MCA, supply-demand analysis, provides a quantitative framework for analyzing the regulatory importance of cellular commodities such as anaerobic free-energy. This is done through comparing the elasticities of anaerobic free-energy supply and demand, which yields the degree to which the respective reaction blocks control the flux through anaerobic free-energy metabolism, as well as determine the cellular free-energy state (ATP/ADP ratio). The regulation of anaerobic free-energy metabolism in Z. mobilis was investigated with an experimental approach. The key features of our experimental setup were the use of NMR spectroscopy for detecting metabolites, as well as employing non-growing conditions for supply-demand experiments. With NMR spectroscopy metabolites could be detected in real time without using invasive sampling techniques; the use of nongrowing conditions further simplified the analysis by enabling us to correlate fermentative behaviour exclusively with the anaerobic free-energy state. Fermentation of glucose was investigated in the wild type Z. mobilis, a recombinant containing a non-expressing plasmid, or expressing plasmids for over-expressing the glucose facilitator (TCDB 2.A.1.1.4) or glucose-6-phosphate dehydrogenase (EC 1.1.1.49). In addition, ATP demand in the non-expressing recombinant and wild type was perturbed by titrating with the uncoupler acetic acid. Our results show that the anaerobic free-energy demand, the glucose facilitator and glucose-6-phospate dehydrogenase all control the flux of ethanol production in Z. mobilis. The Entner-Doudoroff glycolytic supply activity was found to be sensitive to changes in the ratios of ATP/ADP (elasticity varied between –0.31 and –0.49) and NTP/NDP (elasticity varied between –0.31 and – 0.50). / AFRIKAANSE OPSOMMING: Fermentasie in Zymomonas mobilis word beskryf as ‘n kataboliese snelweg, waar glikolitiese en fermentatiewe ensieme 50% van totale oplosbare proteïene in die sel uitmaak. Hoewel dié fermentasie een van die vinnigstes is wat tot op hede waargeneem is, is die omskakeling van glukose na etanol een van die mees ondoeltreffende energieekstraksies in die natuur. Dié lae energie-opbrengs, soos waarneembaar in fermentasie in Zymomonas mobilis, kan toegeskryf word aan die Entner-Doudoroff metaboliese pad. Hierdie metaboliese pad lewer slegs die helfte van die energie-opbrengs per mol substraat vergeleke met die meer bekende Embden-Meyerhof-Parnas glikolitiese pad. Die navorsing in hierdie tesis is deel van ‘n omvattende projek wat poog om die regulering van glikolise in verskillende mikro-organismes (Z. mobilis, Escherichia coli, Saccharomyces cerevisiae en Lactococcus lactis) te vergelyk. Dié organismes is gekies op grond van die uiteenlopende glikolitiese meganismes waarvan hulle gebruik maak. Ten einde die reguleringsgedrag van meganisties verskillende vry-energie produksieweë m.b.v. vraag-aanbod analise te vergelyk, moet glikolitiese regulering eers onder eenderse eksperimentele kondisies (b.v. nie-groeiende selkulture) gekwantifiseer kan word. Die hoofdoel van hierdie tesis was om die belang van anaerobiese vry-energie produksie vir die regulering van die Entner-Doudoroff glikolitiese pad in Z. mobilis te kwantifiseer. Hiervoor is van Metaboliese kontrole-analise (MKA) en vraag-aanbodanalise (‘n uitbreiding van MKA) gebruik gemaak. MKA is ‘n tegniek waarmee die effek wat ‘n metaboliese parameter (soos metaboliese deel-konservering) op ‘n spesifieke bestendige toestand-veranderlike (soos fermentasiefluksie) het, gekwantifiseer kan word. Vraagaanbodanalise daarenteen, bied ‘n kwantitatiewe raamwerk waardeur die regulatoriese belang van sellulêre kommoditeite (byvoorbeeld anaerobiese vry-energie) geanaliseer kan word. Tydens laasgenoemde proses word die elastisiteit van die anaerobiese vry-energie aanbod en die elastisiteit van die vraag vergelyk. Op hierdie manier kan die mate van beheer wat die onderskeie reaksieblokkie oor die fluksie deur anaerobiese vry-energie metaboliese paaie, sowel as oor die sellulêre vry-energie toestand (ATP/ADP verhouding), bepaal word. In hierdie werk is die regulering van anaerobiese vry-energie metabolisme in Z. mobilis ondersoek deur van ‘n eksperimentele benadering gebruik te maak. Die sleuteleienskappe van dié benadering was om kernmagnetiese-resonansiespektroskopie (KMR spektroskopie) te gebruik om metabolietkonsentrasies te meet, en om van niegroeiende kondisies gebruik te maak vir die vraag-aanbod eksperimente. Metabolietkonsenstrasies kon aaneenlopend bepaal word sonder die gebruik van monsternemingstegnieke wat die reaksie sou kon beïnvloed. Eksterne invloede op die fermentasiegedrag kon ook uitgesluit word deur van nie-groeiende kondisies gebruik te maak, sodat die waargenome fermentasiegedrag uitsluitelik aan die anaerobiese vryenergie toestand toegeskryf kan word. Glukose fermentasie was ondersoek in wilde tipe Z. mobilis, en in drie rekombinante wat onderskeidelik ‘n glukose fasiliteerder ooruitdrukkingsplasmied (TCDB 2.A.1.1.4), ‘n glukose-6-fosfaat dehidrogenase ooruitdrukkingsplasmied (EC 1.1.1.49), en ‘n nieuitdrukkingsplasmied bevat het. Die ATP vraag in die wilde tipe en die nieuitdrukkingsrekombinant is geperturbeer deur titrasies met asynsuur as ontkoppelaar. Die resultate toon dan die anaerobiese vry-energievraag, sowel as die glukose fasiliteerder en glukose-6-fosfaat dehidrogenase, die fluksie van etanolproduksie in Z. mobilis beheer. Die Entner-Doudoroff glikolitiese produksie-aktiwiteit was sensitief vir veranderinge in die ATP/ADP verhouding (elastisiteite was tussen -0.31 en -0.49) en die NTP/NDP verhouding (elastisiteite was tussen -0.31 en -0.50).

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The aim of this study was to build a mathematical model that describes the binding of 17- -estradiol (E2) to estrogen receptor (ER- ) and the influence the sex hormone binding globulin (SHBG) has on this interaction. The influence of SHBG on the transactivation of an estrogen response element, via ligand bound ER- , was also studied. COS-1 cells, derived from the kidney of a green african monkey, were used to study the binding of E2 to ER- in the absence of SHBG. The influence of SHBG on the binding of E2 to ER- was studied using Hep89 cells, human hepatacoma carcinoma, which express SHBG endogenously and are stably transfected with the ER- gene. Human pregnancy plasma was used to study the interaction of E2 with SHBG in the absence of ER- . The results of this study have shown that the Kd (E2) for ER- was determined as between 3.4nM and 4.4nM in the absence of SHBG. With respect to the binding of E2 to ER- it was not possible to determine the Kd app and Bmax for ER- using the Hep89 experimental system. The Kd (E2) for SHBG was not determined using the human pregnancy plasma experimental system. With the aid of mathematical modelling, a model of the Hep89 and human pregnancy plasma experimental systems, was built. The results of the numerical modelling, using mathematical modelling, showed that the presence of albumin together with SHBG was the reason that the Kd app (E2) could not be determined in the Hep89 experimental system. With respect to the use of human pregnancy plasma to determine the Kd (E2) for SHBG it was shown that if the plasma was diluted 200 times it would have been possible to determine the Kd app (E2) for SHBG, in the presence of albumin. Ligand independent transactivation of an estrogen response element was shown to be a problem in the COS-1 cell system when promoter reporter gene assays were undertaken. As COS-1 cells were used as a control for the absence of SHBG no further promoter reporter gene assays were undertaken using the Hep89 experimental system. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was die bou van ‘n wiskundige model wat die verbinding van E2 met die estrogeenreseptor (ER- ) en die invloed wat die geslagshormoon-verbindingglobulien (SHBG) op hierdie interaksie het, beskryf. Die effek van SHBG op die transaktivering van ‘n estrogeen responselement, via die ligandverbonde ER- , is ook bestudeer. COS-1-selle uit die nier van ‘n groen afrika-aap is gebruik om die verbinding van E2 met ER- in die afwesigheid van SHBG te bestudeer. Die invloed van SHBG op die verbinding van E2 met ER- , is bestudeer deur gebruik te maak van Hep89-selle, die menslike lewergeswelkarsinoom, wat SHBG uitwendig afgee en wat stabiel getransfesteer kan word met die ER- geen. Menslike swangerskapplasma is gebruik om die interaksie van E2 met SHBG in die afwesigheid van ER- te bestudeer. Die uitslag van hierdie studie toon aan dat die Kd (E2) vir ER- vasgestel tussen 3.4nM en 4.4nM in die afwesigheid van SHBG. Met betrekking tot die verbinding van E2 met ER- , was dit nie moontlik om die Kd (E2) en Bmax app vir ER- met die gebruik van die Hep89 eksperimentele stelsel vas te stel nie. Die Kd (E2) vir SHBG is nie vasgestel deur die gebruik van die menslike swangerskapplasma eksperimentele stelsel nie. ‘n Model van die Hep89 en menslike swangerskapplasma eksperimentele stelsels is met behulp van wiskundige modellering gebou. Die uitslag van die numeriese modellering, met gebruik van wiskundige modellering, toon dat die teenwoordigheid van albumien, saam met SHBG, die rede was dat die Kd app (E2) nie in die Hep89 eksperimentele stelsel vasgestel kon word nie. Wat betref die gebruik van menslike swangerskapplasma om die Kd (E2) vir SHBG vas te stel, is daar aangetoon dat, indien die plasma 200 maal verdun was, dit moontlik sou gewees het om die Kd app (E2) vir SHBG in die teenwoordigheid van albumien vas te stel. Promotor verkilkkergeen toetse het ligandonafhanklike transaktiveering van ‘n estrogeen responselement aangetoon as ‘n probleem in die COS-1-selle stelsel. Omdat COS-1-selle gebruik is as ‘n kontrole vir die afwesigheid van SHBG, is geen verdere promotor verkilkkergeen toetse onderneem met die gebruik van die Hep89 eksperimentele stelsel nie.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Dichloro-diphenyl-trichloroethane (DDT) was extensively used in agriculture pest control and is still used for indoor residual spraying to control malaria. The lipophylicity of DDT and its breakdown product dichloro-diphenyl-dichloroethylene (DDE) dictates that they associate with membranes, lipids and hydrophobic proteins in the biological environment. Their poor degradable nature causes DDT and DDE to persist for decades in the environment and in individuals who are or were in contact with the pesticide. In many countries the synchronised resistance of the mosquito vector to insecticides and the malaria parasite towards antimalarial drugs led to a drastic rise in malaria cases and to malaria epidemics. This study assesses the influence of low level exposure of DDT and DDE on chloroquine (CQ) resistance of the dire human malaria parasite, Plasmodium falciparum. The in vitro activity of p,p’-DDT and p,p’-DDE towards blood stages of chloroquine sensitive (CQS) P. falciparum D10 and chloroquine resistant (CQR) P. falciparum Dd2 was determined using two complementary in vitro assays (Malstat and SYBR Green 1). The 50% inhibition concentrations (IC50s) of p,p’-DDT and p,p’-DDE were found to be ±14 to 38 μM (5-12 μg/mL) and highly similar towards CQS and CQR P. falciparum strains. This result indicated that the proteins involved in CQ resistance have no effect on the activity of the insecticide DDT and it breakdown product DDE. In order to assess the influence of DDT and DDE on CQ activity, in vitro fixed ratio drug combination assays were performed, as well as isobologram analysis. We found that CQ works in synergy with p,p’-DDT and p,p’-DDE against CQS P. falciparum D10. However, both p,p’-DDT and p,p’-DDE were antagonistic toward CQ activity in CQR P. falciparum Dd2. This indicated that p,p’-DDT and p,p’-DDE do have an effect on CQ resistance or on the action of CQ via a target other than hemozoin polymerization. The observation of reciprocal synergism of p,p’-DDT and p,p’-DDE with CQ against CQS D10 and antagonism against CQR Dd2 strain is highly significant and strongly indicates selection of CQ resistant strains in the presence of p,p’-DDT and p,p’-DDE. People who have low levels of circulating DDE and/or DDT could be at a high risk of contracting CQR malaria. However, medium term (nine days) DDE exposure of CQS P. falciparum D10 did not induce resistance, as no significant change in activity of CQ, p,p’-DDT and p,p’-DDE towards blood stages the CQS strain was observed. This exposure was, however, shorter than expected for a malaria infection and would be addressed in future studies. From our results on the interaction of CQ with p,p’-DDT and p,p’-DDE, it was important to assess the residual DDT and DDE variable and how much of residual p,p’-DDT and/or p,p’- DDE would enter into or remain in the different compartments (the RPMI media, erythrocytes and infected erythrocytes) over time. In combination with liquid-liquid extraction, we developed a sensitive GC-MS analyses method and a novel HPLC-UV analysis method for measuring DDT and DDE levels in malaria culturing blood and media. Whilst the HPLC-UV method was relatively cheaper, faster, and effective in determining high DDT and DDE concentrations, the optimised GC-MS method proved to be effective in detecting levels as low as 78 pg/mL (ppt) DDE and 7.8 ng/mL (ppb) DDT in biological media. Using both the HPLC and GC-MS methods we observed that malaria parasites influence distribution of the compounds between the erythrocytic and media fractions. P. falciparum D10 infection at ±10% parasitemia lead to must faster equilibration (less than 8 hours) between compartments. Equimolar distribution of p,p’-DDE was observed, but the parasites lead to trapping of the largest fraction of p,p’-DDT in the erythrocyte compartment. These results indicate that a substantial amount would reach the intra-erythrocytic parasite and could influence the parasite directly, possibly leading to either synergistic or antagonistic drug interactions. This study is the first to illustrate the “good and bad” of the insecticide DDT in terms of CQ resistance and sensitivity toward the human malaria parasite P. falciparum. These results will hopefully have an important influence on how future policies on malaria control and treatment particularly in endemic areas will be addressed and could also have an impact on the anti-malarial drug discovery approach. / AFRIKAANSE OPSOMMING: Dichlorodifenieltrichloroetaan (DDT) is op groot skaal in landbouplaagbeheer gebruik en word nog steeds gebruik vir binnenshuise oppervlakbespuiting om malaria te beheer. Die lipofilisiteit van DDT en sy afbraakproduk dichlorodifenieldichloroetileen (DDE) dikteer dat hulle met membrane, lipiede en hidrofobiese proteïene in die biologiese omgewing assosieer. Stadige afbraak veroorsaak dat DDT en DDE vir dekades in die omgewing agterbly, asook in individue wat in kontak is, of was met die insekdoder. In baie lande het gesinkroniseerde weerstand van die muskietvektor teenoor insekdoders en die malariaparasiet teenoor antimalariamiddels gelei tot 'n drastiese styging in malariagevalle en tot malariaepidemies. In hierdie studie word die invloed van lae vlak blootstelling van DDT en DDE op chlorokien (CQ) weerstand van die mens malariaparasiet, Plasmodium falciparum, geëvalueer. Die in vitro aktiwiteit van p,p'-DDT en p,p'-DDE teenoor die bloedstadia van chlorokiensensitiewe (CQS) P. falciparum D10 en chlorokien-weerstandbiedende (CQW) P. falciparum Dd2 is bepaal deur gebruik te maak van twee komplementêre in vitro toetse (Malstat en SYBR Groen toetse). Die 50% inhibisie konsentrasies (IC50s) van p,p'-DDT en p,p'-DDE is bepaal as ±14 to 38 μM (5-12 μg/mL) en was hoogs vergelykbaar tussen CQS en CQW P. falciparum stamme. Hierdie resultaat het aangedui dat die proteïene betrokke by CQ weerstand geen effek op die aktiwiteit van die insekdoder DDT en die afbraakproduk DDE het nie. Om die invloed van DDT en DDE op CQ aktiwiteit te evalueer, is die aktiwiteit van kombinasies van die verbindings in vaste verhoudings getoets, tesame met isobologram ontleding. Ons het gevind dat CQ sinergisties saam met p, p'-DDT en p, p'-DDE teen CQS P. falciparum D10 werk. Daarteenoor het beide p, p'-DDT en p, p'-DDE antagonistiese werking getoon teenoor CQ aktiwiteit met CQW P. falciparum Dd2 as teiken. Dit het aangedui dat p,p'-DDT en p, p'-DDE wel 'n invloed op CQ weerstand het of ‘n aktiwiteit van CQ, anders as hemozoin polimerisasie, beïnvloed. Die waarneming van resiproke sinergisme en antagonisme van p, p'-DDT en p, p'-DDE in kombinasie met CQ teenoor die CQS D10 en CQW DD2 stamme respektiewelik, is hoogs betekenisvol en dui op seleksie van CQweerstandige stamme in die teenwoordigheid van p, p'- DDT en p, p'-DDE. Mense wat lae vlakke van sirkulerende DDE/DDT het, het dus 'n hoër risiko om CQW malaria te kry. Verder is gevind dat medium termyn (nege dae) DDE blootstelling van CQS P. falciparum D10 nie weerstand nie veroorsaak nie, want geen beduidende verandering in die aktiwiteit van CQ, p,p'-DDT en p,p'-DDE teenoor die bloed stadiums van die CQS stam is waargeneem nie. Hierdie blootstelling is egter korter as in 'n malaria-infeksie en sal verder bestudeer word in toekomstige studies. Vanuit die interaksie resultate van CQ met p, p'-DDT en p, p'-DDE was dit belangrik om die residuele DDT en DDE veranderlike te evalueer, asook die distribusie van p,p'-DDT en p,p'- DDE tussen die verskillende kompartemente (die kultuurmedium, eritrosiete en geïnfekteerde rooibloedselle) oor verloop van tyd. In kombinasie met vloeistof-vloeistof ekstraksie, het ons 'n sensitiewe GC-MS en nuwe HPLC-UV analisemetode ontwikkel vir die meet van DDT en DDE-vlakke in bloed (normale en geïnfekteerde eritrosiete) en die kultuurmedium. Terwyl die HPLC-UV metode relatief goedkoper, vinniger en effektief in die bepaling van hoë DDT en DDE-konsentrasies is, was die geoptimaliseerde GC-MS metode doeltreffend in die opsporing van vlakke so laag as 78 pg/mL (dpt) DDE en 7.8 ng/mL (dpb) DDT in biologiese media. Met behulp van beide die HPLC-UV en GC-MS metodes is waargeneem dat die malariaparasiet die ekwilibrasie van die verbindings tussen die eritrosiet- en media kompartemente beïnvloed. P. falciparum D10 infeksie met ± 10% parasitemia lei tot vinniger ekwilibrasie (minder as 8 uur) tussen die kompartemente. Ekwimolêre verspreiding van p,p'- DDE is waargeneem, maar die parasiete het die grooste fraksie van p,p'-DDT in die eritrosiet kompartement vasgevang. Hierdie resultate wys dat 'n aansienlike fraksie die intraeritrositiese parasiet kan bereik en sodoende die parasiet direk kan beïnvloed en moontlik kan lei tot sinergistiese of antagonistiese middel interaksies. Hierdie studie is die eerste om die "goed en sleg" van die insekdoder DDT in terme van CQ weerstand en sensitiwiteit teenoor die menslike malariaparasiet P. falciparum te illustreer. Hierdie resultate sal hopelik 'n belangrike invloed hê op die toekomstige beleid oor die beheer van malaria en behandeling, veral in endemiese gebiede, en mag ook 'n impak hê op die antimalariamiddel navorsing.