Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a need for identifying new diagnostic antigens for the serological diagnosis of trypanosomiasis, a disease of humans and animals in Africa caused by protozoa belonging to the genus Trypanosoma. Invariant surface glycoproteins (ISGs) are present in most strains of the parasite and have the potential to replace the variable surface glycoproteins as diagnostic antigens. In order to avoid the challenges of in vivo culturing of bloodstream form (BSF) trypanosomes in laboratory animals, ISG65 and ISG75, the two most common ISGs were heterologously expressed in Escherichia coli and Pichia pastoris expression systems. The extracellular domains of ISG65 and ISG75 of both T. b. brucei and T. b. gambiense were amplified by PCR from genomic DNA using appropriate primers to give inserts of 1121 bp and 1342 bp sizes. These were sub-cloned into the pGEX-4T1 and pET28a expression vectors. Chemically competent E. coli BL21 (DE3) were transformed using the resultant plasmids and the transformed E. coli cells were used for heterologous protein expression. The expressed proteins were purified by three phase partitioning (TPP), nickel or glutathione affinity and molecular exclusion chromatography and analysed by reducing SDS-PAGE. The glycosylation status of ISG65 and ISG75 expressed in the M5 strain of P. pastoris, which has an engineered N-glycosylation pathway that produces glycosylated proteins similar to what is obtained in trypanosomes, was determined. The enzymatic action of Endoglycosidase H resulted in a shift in the electrophoretic migration of ISG65 but not ISG75 on SDS-PAGE, confirming N-glycosylation. Anti-ISG65 and anti-ISG75 antibodies were produced in chickens and affinity purified using the respective recombinant proteins immobilised on affinity matrices. The antibodies recognised native ISG65 and ISG75 respectively in western blots of lysates of T. b. brucei parasites cultured in vitro. Similar recognition of the native ISGs by the anti-recombinant ISG antibodies was also obtained using immunofluorescence microscopy of fixed T. b. brucei parasites. The results obtained demonstrate the potential of application of the recombinant ISG65 and ISG75 and their respective antibodies in the diagnosis of African trypanosomiasis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Effect of c-Ha-ras(V12) on cathepsin trafficking in invasive breast cancer cells. Various mutations of Ha-ras together with lack of p53-related control over cell cycle progression, result in an immortal, tumorigenic phenotype in 50% human epithelial cancers. Unmutated Ha-Ras transiently mediates external growth factor-related signaling, initiating downstream kinase activity that is normally terminated by p53. This protects the cell from immortalization, i.e. uncontrolled proliferation. An MCF10A breast epithelial cell line, derived from a fibrocystic breast mastectomy specimen, spontaneously immortalized in culture, due to a chromosomal deletion (9p21-/-). This gave rise to a non-malignant and non-invasive cell line in which the effects of deletion of upstream control of both p53 and the cell cycle and c-Haras( V12) transfection may be studied. Transfection of this cell line with the c-Haras( V12) oncogene gave rise to the invasive MCF10AneoT premalignant derivate, in which distribution of cathepsin B (CB), cathepsin L (CL) and cathepsin D (CD), membrane-type 1 matrix metalloprotease (MT1-MMP), a membrane-bound collagenase, is altered. The possible role of these proteases in the premalignant invasive phenotype, as well as the role of the V12 mutation and the effect of p53 on vesicle trafficking, was explored. In the MCF10AneoT cell line lack of negative feedback by p53 and other Ha-Ras effectors such as Rac, Rho and CDC42, seems to result in lack of control over the cytoskeleton and thus cell polarity during growth stimulus-related migration. Luminal alkalinization, especially of vesicles distant from the perinuclear region, as well as degradative efficiency seem affected, possibly as functional assembly of the acidifying vacuolar-ATPase proton pump on these vesicles is compromized. In normal cells CB and CD seem discretely located, while a spread of proteases was noted in transfected cells, from a perinuclear position to along the basal plane. Increased association of CB with lysosome-associated membrane protein-2 (LAMP- 2), and of CD with an acidic juxta-nuclear structure (JNS) was also noted, while this structure was observed in two sites in transfected cells, compared to only one in normal cells. In invasive cancers increased levels of both CB and MT1-MMP have been found to correlate with accelerated pathological degradation and invasion of the underlying basement membrane (BM) barrier and extracellular matrix (ECM). MT1- MMP is known to regulate BM turnover, while the manner in which the association of CB with the plasma membrane (PM) supports such turnover, ECM degradation and migration, is not yet clear. The current investigation showed altered distribution of PM-associated CB and MT1-MMP in transformed cells, compared to normal. This phenotype seems explained in terms of the effects of the mutationally activated c-Ha- Ras(V12) on its downstream effectors, Rac and PI3K and their effectors, on cytoskeletal organization and vesicle trafficking, increased calcium and, via Rho, cytoplasmic alkalinization due to proton extrusion by an activated NHE-1 membraneassociated proton pump. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Satellite cells are known as the definitive muscle stem cells and are responsible for skeletal muscle maintenance and repair. The capacity of these satellite cells to participate in myogenesis decreases with age and as a result, muscle repair and maintenance in an aging organism is characterized by fibrosis, lipid accumulation and atrophy, a process known as sarcopenia. Recent parabiotic studies have shown that satellite cells with reduced myogenic capability in aging muscle can be rejuvenated to undergo effective myogenesis when exposed to a young environment. Further analysis has suggested that the Wnt family of signaling proteins identified in serum is pivotal in regulating cell fate, proliferation and differentiation, during aging. Wnt3a is known to regulate fibrogenensis, Wnt10b adipogenesis and Wnt7 myogenesis. In the current study, we aim to determine the cytosolic and secreted expression profiles of the three Wnt isoforms, Wnt3a, 7 and 10b, during myogenesis of early and late passage C2C12 myoblasts. We then extend our analysis to determine whether conditioned media could improve the myogenic capacity of late passage cells. Late passage C2C12 cells had elevated Wnt3a cytosolic levels along with reduced differentiation capacity and a rapidly declining Wnt7 levels, in comparison to early passage cells. The elevated Wnt3a suggests an elevated fibrogenic predisposition, whereas the declining Wnt7 cytosolic levels, a decrease in myogenic capacity. Furthermore, analysis of the secreted vs. cytosolic ratio in Wnt7 levels revealed a more rapid decline in late vs. early passage cells during differentiation, supporting the observed decreased myogenic ability. Moreover, late passage cells also showed lower Wnt10b levels compared to early passage cells. This low level of Wnt10b is likely associated with an increase in adipogenic predisposition. The results obtained in the cross-over experiments indicated that conditioned media from early passage cells did not improve the differentiation of late passage cells by the low levels of Myogenin and MHC. However, early passage cells treated with conditioned media from late passage cells surprisingly showed a marginal increase in both Myogenin and MHC levels. Interestingly, cytosolic Wnt3a and 7 in late passage cells treated with ‘young media’ were increased compared to control whereas early passage cells treated with ‘old’ media showed significantly decreased levels of Wnt3a and 7. Furthermore, early passage cells acquired a declining expression when treated with ‘young’ media whereas late passage cells had an increasing level when treated with ‘old’ media. This indicates a possible improvement in differentiation in late passage cells. Taken together, our results support a role for Wnt7 and Wnt10b in promoting myogenesis while Wnt3a may decrease myogenesis. With the increase in passage numbers, the reduced myogenic predisposition is regulated by reduced Wnt10b, 7 and elevated Wnt3a levels, respectively. Moreover, we speculate that the lack of myogenic improvement in the cross-over experiment could be the presence of unknown secreted factors in ‘young’ media that impedes myogenesis. Finally, cell lines are known to be biologically different to primary myoblasts through the accumulation of mutations which could render the cells less sensitive to growth factors. Therefore, it is imperative that the current study is repeated with primary culture myoblasts. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

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).