Doctoral Degrees (Biochemistry)

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Altered lipid metabolism as a possible mechanism in fumonisin-induced hepatocarcinogenesis in rats and investigations into risk assessment in humans
(Stellenbosch : Stellenbosch University, 2013-12) Burger, Hester Maria; Gelderblom, W. C. A.; Swart, P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Exposure to food contaminates such as mycotoxins have been associated with a variety of animal and human diseases worldwide. In South Africa, maize is the most To further refine risk assessment in the socio-demographic heterogeneous population of South Africa, the development and evaluation of a sensitive and interactive model the Mycotoxin Risk Assessment Model (MYCORAM) proofed to be more sensitive compared to the classical probable daily intake (PDI). The development of the MYCORAM was based on mycotoxin distribution during dry milling of maize in milling fractions intended for human consumption which was superimposed on the maize intake profiles of the South African population. Although dry milling, including a degerming step, is an effective way to reduce mycotoxins, risk and exposure assessment are influenced by maize dietary intakes, gender and ethnicity. This became evident when considering FB dietary exposure in rural maize subsistence farming communities in the Eastern Cape Province, South Africa confirmed the vulnerability of this subpopulation to risk of fumonisin exposure. Specific maximum tolerated maximum levels (MTL) to safeguard these communities fall outside the international regulatory processes and need to be urgently addressed. With the complex nature of cancer development in mind, integration of basic science and nutritional epidemiology will be important to contribute to our understanding of the adverse effects of FB and to define relevant risk assessment parameters. important commercial grain crop not just economically but also as a local food commodity both commercially and in subsistence rural farming communities. In order to control and manage mycotoxin contamination in food, evidence-based risk assessment is needed that includes mechanistic and human exposure studies. From this perspective the current study was conducted and aimed in further unravelling fumonisin B1 (FB1) mycotoxin induced hepatocarcinogenesis via the disruption of the lipid metabolism. The study also critically evaluates aspects of human risk assessment due to its relevance and importance to food safety known to impact on food security. This entails mycotoxin distribution during maize dry milling and the assessment of mycotoxin exposure in the South African population and vulnerable rural communities at risk. Fumonisin B1 affects the integrity of biological membranes by altering key lipid and fatty acid parameter in plasma, microsomal, mitochondrial and nuclear subcellular membrane fractions in rat liver. Changes in the major lipid constituents entailing an increase in cholesterol (CHOL) and phosphatidylethanolamine (PE) whilst sphingomyelin (SM) and phosphatidylcholine (PC) tended to decrease. Isolated plasma membrane lipid rafts, from rat primary hepatocytes exposed to FB1 augments the intricate effects exerted on the lipid metabolism regarding CHOL, SM and PE. The disruption of lipid and fatty acid constituents, such as arachidonic acid and ceramide, are likely to be key determinants affecting growth regulatory signaling pathways relevant to the critical balance between cell proliferation and apoptosis during cancer promotion. These changes provide further evidence that FB1 induce cancer promotion by differential inhibition and/or stimulation process whereby a few resistant “initiated” hepatocytes proliferate in an environment where the growth of normal cells is inhibited. A specific lipogenic phenotype is effected by FB1 which is closely associated with cancer development and considered to occur via an epigenetic-type of mechanism. These effects are not adequately addressed in defining risk assessment parameters.
An assessment of the diversity and pathogenicity of Potato leafroll virus in South Africa
(Stellenbosch : Stellenbosch University, 2018-03) Roos, Wiets Gideon; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Potato production in South Africa has steadily intensified through improved pivot irrigation. Since potatoes are vegetatively propagated the potato industry is continuously threatened by Potato leafroll virus (PLRV) which is responsible for increasing yield losses in South Africa. Effective management of PLRV is dependent on its accurate detection in seed potato stocks. PLRV is a small spherical plant virus consisting of a protein capsid and an RNA genome of approximately 5900 bases. This virus is phloem restricted and is vectored by, most notably, by the green peach aphid. Plant RNA viruses pose a threat due to their high mutation rate and the ability to adapt rapidly to a changing environment. To effectively manage PLRV infection, detection of virus infection in seed potatoes is paramount. In this study, five field trials were carried out in potato production fields, to compare the commonly used DAS-ELISA with RT-PCR for PLRV detection. From the results obtained it was concluded that DAS-ELISA detection greatly underestimates the number of infected samples when compared to RT-PCR. The hotter climate of the Sandveld region appears to inhibit PLRV accumulation in infected plants and these infections then remain undetected by DAS-ELISA. PLRV isolates were sequenced and phylogenetic and bioinformatic analyses were performed to identify and characterise local variants of PLRV. PLRV isolates found in the Sandveld region were closely related to PLRV isolates from Australia. Some of these isolates had recombined with variants commonly found in Europe, Asia and the Americas as well as with those similar to PLRV isolates from Peru and Germany. Three locally produced cultivars were graft-inoculated with two PLRV isolates that represent the two main variant groups found to assess symptom development and yield reduction. Symptom development in locally produced cultivars was typical for PLRV. A yield loss resulted from this infection with no difference between the Australian type and the European/Australian recombinant type. The proteins produced by the newly sequenced isolates were further analysed in comparison to other isolates found worldwide. The variation in the proteins produced by the newly sequenced isolates was mainly due to recombination between distinct groups of PLRV in the 5’ half of the genome and through mutation in the 3’region of the genome. A differential RT-PCR was designed to distinguish, in a single reaction, between the Australian type and the European/Australian recombinant type of PLRV. This revealed that simultaneous infections with both types occurred commonly, and could explain why recombination has occurred.
Bioaffinity separation using ligand-modified pluronic and synthetic membranes
(Stellenbosch : University of Stellenbosch, 2011-10) Govender, Selvakumaran; Swart, P.; Jacobs, E. P.; Bredenkamp, M. W.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: A new membrane based affinity separation system that is bio-specific, biocompatible, well characterised and capable of being regenerated or re-used is described. The amphiphilic non-ionic surfactant Pluronic® F108, was covalently derivatised to form two novel bioligands (Pluronic-Biotin and Pluronic-DMDDO) for the bio-specific immobilisation of avidin conjugated proteins and histidine tagged proteins respectively. Pluronic was also used to non-covalently functionalise nonporous membranes for ligand attachment and to simultaneously shield the surfaces from non-specific protein adsorption. Each component of this bioaffinity system (from the membrane matrix to the elution/desorption of the ligate/ligand system) was studied with the aim of producing a well characterised system and key quantitative data for the development of a robust, reliable, re-usable and scalable technology. Specifically, this study describes: 1. The fabrication and partial characterisation of nonporous planar and capillary membranes as model affinity matrices. 2. The development and evaluation of a robust protocol for solvent desorption and accurate colorimetric quantification of Pluronic® F108 and its derivatives. 3. Interfacial analysis of Pluronic adsorption onto nonporous affinity membranes, including the direct solid-state analysis of model, halogenated Pluronic derivatives using nuclear microprobe analysis. 4. Development of a surfactant based protocol for affinity membrane regeneration and re-use. 5. Specific bioaffinity immobilisation of avidin conjugated peroxidase onto biotinylated membranes in the presence of model protein foulants. 6. Cloning and expression of C-terminal hex-histidine tagged human cytochrome b5 into the bacterial expression system E. coli BL-21 DE3. 7. Development and characterisation of an immobilised metal affinity membrane system for metal chelation (Ni2+, Cu2+ and Zn2+) using a new chelator Pluronic- N,N-dicarboxymethyl-3,6-diazaoctanedioate and the bio-specific immobilisation of N-terminal hex-histidine tagged pantothenate kinase.
A biochemical study of the effect of ultraviolet treatment on bovine milk and Cheddar cheese
(Stellenbosch : Stellenbosch University, 2015-12) Cilliers, Frans Pieter; Swart, Pieter; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study describes: 1. The evaluation of a novel, patented thin-film, turbulent-flow Ultravioletdisinfection system as an alternative processing method to thermal pasteurisationfor the disinfection of bovine milk. 2. The microbial, biochemical and sensory characterization of bovine milk treated by heat and Ultraviolet light and then used for the commercial production of Cheddarcheese. 3. The microbial, biochemical and sensory characterization of commercial Cheddarcheese produced from bovine milk treated by heat and Ultraviolet light.
The biochemical study of the R- and S-enantiomers of 2-(4-acetoxyphenyl)-2-chloro-Nmethylethylammonium chloride (Compound A).
(Stellenbosch : Stellenbosch University, 2017-03) Swart, Liezel Maria; Swart, Pieter; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The study describes: . The synthesis of a racemic mixture of Compound A (2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride), a synthetic analogue of the active compound isolated from the African shrub, Salsola tuberculataformis Botschantzev. . The development of a strategy to separate the R- and S-enantiomers of Compound A. . The isolation and purification of a highly purified substrate-free cytochrome P450 11β-hydroxylase (CYP11B1) from ovine adrenals. . The isolation of a partially purified mixture of the mitochondrial electron transport chain, adrenodoxin reductase and adrenodoxin from ovine adrenals. . The investigation into the mechanism of action of the R- and S-enantiomers of Compound A on the spectroscopic properties of substrate-free CYP11B1. . The investigation into the influence of the R- and S-enantiomers of Compound A on the mitochondrial electron transport chain by using cytochrome c as an electron acceptor.
A biochemical study of tissue type plasminogen activator in bovine milk
(Stellenbosch : Stellenbosch University, 2007-03) Cilliers, Frans Pieter; Swart, Pieter; Hofmeyr, Jannie; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study describes: 1. The isolation and the purification of tissue type plasminogen activator and urokinase plasminogen activator in bovine milk. 2. The biochemical characterisation of tissue type plasminogen activator in bovine milk. 3. An investigation of the influence of the addition of purified tissue type plasminogen activator to ultra high temperature milk, Gouda cheese and yoghurt.
Biofilms as multifunctional surface coatings and adaptive systems: a biomimetic approach
(Stellenbosch : Stellenbosch University, 2016-12) Loots, Ruenda; Cloete, Thomas Eugene; Swart, Pieter; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Biomimicry is an emerging scientific discipline that promotes nature-inspired innovation for sustainable solutions. Several patterns and survival strategies are repeated in Nature and these have been extrapolated into a hierarchical set of biomimetic principles that can be used to investigate the complexity of natural systems. A biomimetic approach was used to review biofilm literature and create a novel framework based on these principles to describe microbial biofilms on a molecular, structural and systems level. By reinterpreting current biofilm knowledge within a biomimetic framework, this study demonstrates that microorganisms use life-friendly chemistry to integrate biofilm development with growth, giving rise to resource-efficient systems. Furthermore, these structured microbial communities are responsive to their local environment, adapt to changes and, ultimately, evolve to survive. Subsequently, the application of biomimetic principles to biofilms was investigated using various analytical techniques. Two gfp-labelled Pseudomonas strains and an environmental multi-species community were selected for this study. Microscopic and spectroscopic techniques were used for biochemical investigations of single-species biofilm composition and structure. The distribution of biomolecules in Pseudomonas biofilms was investigated using protein- and glycoconjugate-specific fluorescent stains and confocal laser scanning microscopy (CLSM). CLSM was also used to investigate structural adaptations of Pseudomonas biofilms to changes in nutrient availability and hydrodynamic conditions. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to explore biochemical adaptations of single- and multi-species biofilms cultivated in different nutrient media. ATR-FTIR spectra, visual observations and the quantification of biofilm parameters by digital image analysis of CLSM images support the hypothesis that biofilms are resource-efficient, self-organised systems that are built from the bottom up using life-friendly chemical principles. Both Pseudomonas strains adapted to environmental conditions by changing the three-dimensional structure of their biofilms, specifically in terms of biomass, substratum area coverage, average thickness and the surface area of biofilms exposed to the bulk liquid. In order to study biofilms as a system and investigate the responsiveness of a biofilm community as a whole, a relatively new approach was used to monitor biofilm responses in real time by measuring CO2 production as an indication of whole-biofilm metabolism. A CO2 evolution measurement system (CEMS) was combined with metabolic assays and direct plate count methods to monitor biofilm metabolism and biofilm-derived planktonic cell yield in response to environmental changes, i.e. changes in nutrient source and concentration or exposure to antimicrobial compounds (either streptomycin or a solution containing isothiazolone). The metabolic responses of biofilms, measured as CO2 production rates, showed that both single- and multi-species biofilms are able to respond rapidly to changes in nutrient availability or exposure to biocides and antibiotics. Multi-species biofilms generally recover faster after environmental changes or antimicrobial exposures, indicating that diversity adds to biofilm resilience and adaptability. Regardless of the conditions, single- and multi-species biofilms are able to maintain some level of metabolic activity, as well as release high numbers of planktonic cells into the effluent. The maintenance of biofilm-derived planktonic cell yield supports the hypothesis that biofilms are active proliferation sites in order to ensure survival – a feature of biofilms that is often overlooked in biofilm research. This study contributes to the growing field of biomimicry by applying biomimetic principles in biofilm research for the first time. A biomimetic approach can inform novel anti-biofilm strategies, promote biofilm-inspired innovation and explain complex microbial ecological phenomena. Within a biomimetic framework, the increasing degrees of complexity in biofilms are organised in a new way, demonstrating that the biochemical, structural and functional complexity of microbial communities are interconnected and need to be considered together in biofilm studies. To this end, the usefulness of CEMS as a non-destructive technique to study real-time biofilm responses is demonstrated.
Cancer modulating properties of unique South African herbal teas (rooibos and honeybush) in short term in vitro and in vivo carcinogenesis assays
(Stellenbosch : Stellenbosch University, 2004-12) Marnewick, Jeanine Lucasta; Gelderblom, W. C. A.; Swart, P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This thesis provides the first scientific evidence on the cancer modulating properties of two unique South African herbal teas, rooibos (Aspalathus Iinearis) and honeybush (Cyclopia intermedia) utilizing in vitro as well as in vivo carcinogenesis assays by: • Demonstrating the in vitro antimutagenic activity of aqueous extracts of the herbal teas against the metabolic activated mutagens, 2-acetylaminofluorene (2- AAF) and the mycotoxin, aflatoxin B1 (AFB,) as well as, to a certain extent, against the direct acting mutagen, hydrogen peroxide, utilizing the Salmonella typhimurium mutagenicity assay. • Increasing the activity of hepatic drug metabolizing enzymes, glutathione Stransferase alpha and UPD-glucuronosyl transferase, and reduced the oxidative stress by stabilizing the level of reduced glutathione (GSH) resulting in an increased hepatic reduced to oxidized glutathione ratio (GSG:GSSG). No toxic effects were noticed in rats consuming the herbal teas for 10 weeks as their sole source of drinking fluid. • Demonstrating the ex vivo modulation of 2-AAF- and AFB1-induced mutagenesis by sub- cellular hepatic fractions of rats consuming the herbal teas in the Salmonella mutagenicity assay. Hepatic cytosolic fractions protected against mutagenesis of both mutagens, while the microsomal fractions exhibited a reduced capacity to metabolize AFB1 to its active mutagenic metabolite. • Providing evidence for the in vivo modulation of tumour promotion using the liver as well as the two-stage skin carcinogenesis animal models. The unprocessed herbal teas arrested proliferation of the placental form of glutathione-Stransferase (GSTP+) altered cells as well as reduced the total number of enzyme altered foci in the liver of rats. Topical application of polyphenolic fractions of the various herbal teas prior to 12-0-tetra-decanoylphorbol-13-acetate (TPA) tumour promotion, reduced tumour formation in mouse skin initiated with 7,12-dimethylbenz[ ajanthracene (DMBA). The protective effect was illustrated by a decreased tumour incidence, a reduction in tumour volume as well as a delayed onset of tumour development. The f1avanol/proanthocyanidin content of the fractions could playa major role in the protection against skin tumour promotion. • Proposing possible mechanisms whereby rooibos and honeybush herbal teas could exert their cancer modulating properties with respect to in vitro and ex vivo antimutagenicity, in vivo oxidative status and reduced tumour promotion. • Providing evidence that the herbal teas mimic the cancer modulating properties of green and black teas although differences exist, presumably due to differences in the polyphenolic constituents. • Suggesting that rooibos and honeybush herbal teas may play an important role as chemopreventive agents in the modulation of cancer.
Cape baboon Cytochrome P450 11β-hydroxylases : the characterization of two functional enzymes
(Stellenbosch : Stellenbosch University, 2007-03) Brown, Natasja; Swart, Amanda C.; Swart, P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: This study: 1. Describes the localization of CYP11B1 in the Cape baboon adrenal gland using Western blot analysis. CYP11B1 was localized to the adrenal cortex and medulla. 2. Describes the catalytic activity of CYP11B1 towards 11-deoxycorticosterone and corticosterone in adrenal cortical- and medullary tissue homogenates. Aldosterone formation in the adrenal medulla was identified using an atmospheric pressure chemical ionization-mass spectrometry method, which was developed in our department. 3. Compares the catalytic activity of three recombinant Cape baboon CYP11B1 cDNAs, expressed in COS-1 cells, towards 11-deoxycorticosterone and 11-deoxycortisol. 4. Describes the determination of the Michaelis-Menten constants and maximum reaction rates of 11-deoxycorticosterone and 11-deoxycortisol utilization by two functional recombinant Cape baboon CYP11B1 cDNAs, respectively. 11-Deoxycorticosterone metabolites were quantified using an enzyme immunoassay kit. 11-Deoxycortisol metabolites were quantified using a liquid chromatography-mass spectrometry method, which was developed in our department. 5. Describes the homology modeling of two isoforms of Cape baboon CYP11B1 using CYP102 and CYP2C5 as structural templates. The influence of three amino acid residue substitutions, located in the predicted D-E helix, on the catalytic activity of the two CYP11B1 isoforms was examined.
Changes in cell surface and metabolism associated with strains of Listeria monocytogenes displaying different sensitivities to class IIa bacteriocins
(Stellenbosch : Stellenbosch University, 2003-12) Vadyvaloo, Viveka; Hastings, J. W.; Stellenbosch University. Faculty of Science . Dept. of Biochemistry.
ENGLISH ABSTRACT: The possible use of the bacterially produced antimicrobial peptides, and in particular class IIa bacteriocins as food preservatives is a motivating factor in studies on resistance to them by food-borne pathogens like Listeria monocytogenes. The high frequencies of resistance to class Ha bacteriocins have however sparked concern regarding their adequacy as potential biopreservatives. Activity of these cationic peptides was reported to occur by membrane permeabilisation due to pore formation, which results in the leakage of the intracellular contents followed by cell death. The cell envelope (cell wall and cell membrane) is therefore envisaged as a key site of modification in suscepti bility of bacteria to class Ha bacteriocins. Mutants of the L. monocytogenes 873 isolate, resistant to the class IIa bacteriocin, leucocin A, were generated at the start of the study to complement the existing array of L. monocytagenes wild-type and resistant isolates obtained from other sources. The fifty percent inhibitory concentrations using a highly sensitive and reproducible bioassay were determined. This allowed categorisation of the mutants into intermediate and highly resistant phenotypes. Analysis of the growth patterns of all these strains showed decreased growth rates and higher growth yields for all the resistant strains in general. This provided evidence for possible effects of membrane adaptation and metabolic changes in the resistant strains and prompted further investigation. The major focus of the study on the class Ha resistant mutants were: (1) analysis of membrane compositional changes and factors influencing cell surface charge; (2) assessment of physical changes in the membrane and bacteriocin itself using circular dichroism and fourier transform infrared spectroscopy; (3) and, determination of changes in glucose metabolism. Electrospray mass spectrometry analysis of the major listerial phospholipid, phosphatidylglycerol, revealed that membranes of resistant strains had increased levels of unsaturated and short-acyl-chain phosphatidylglycerol molecular species, indicating more fluid membranes. In addition, treatment with a desaturase inhibitor resulted in increased sensitivity of only the intermediate resistant strains to the class na bacteriocin, leucocin A. This indicated the influence of membrane adaptation in only lower levels of resistance. It is conceivable that more fluid membranes could also impact on decreased stability of pore formation by the bacteriocin. Complementary biophysical studies using fourier transform infrared spectroscopy indicated the possible occurrence of greater membrane fluidity of resistant cells, by the notable shift in the anti symmetric CH2 stretching vibration from 2921 cm-I to 2922 cm-I. Additionally, circular dichroism revealed a decreased a-helical and increased random structure of leucocin A in the presence of listerial liposomes derived from highly resistant cell membrane extracts. It is possible that this may result in reduced activity of the bacteriocin in resistant cell membranes as a-helical stucture is a critical feature for membrane insertion of cationic antimicrobial peptides. Cell surface charge was determined by quantification of alanine and lysine esterification of the anionic cell surface polymer, teichoic acid, and membrane phospholipids respectively. Increased D-alanine, which causes neutralisation of the cell surface, was observed in all resistant cells. A tendency for greater lysine content in membrane phospholipids, which also impacts on neutralisation of the anionic phospholipid of listerial membranes, was observed in highly resistant strains only. This neutralisation of the negative charge of the cell surface may interfere with initial electrostatic interaction of bacteriocin with the cell, and subsequent interactions required for permeabilisation of the cell membrane. These differences in alanine and lysine esterification were not the result of increased expression of certain associated genes (d/tA and /mo1695) and may be the result of post-transcriptional regulation. It was, however, found that all resistant L. monocytogenes strains, including the intermediate resistant strains, exhibited decreased expression of a putative docking molecule, the mannose-specific phosphotransferase system EIIAB subunit (EIlABMan).A clear correlation existed between the levels of resistance and EIIABMandown-regulation. Finally, analysis of the glucose metabolism in highly resistant and wild-type strains, indicated a more efficient metabolism with regards to higher growth yields and ATP yield, in contrast to a lower specific growth rate in a spontaneous and genetically defined (EIlABMan inactivated) highly resistant mutant. The switch in metabolic end-product observed, was attributed to the loss of the glucose transporter, EIlABMan,and may cast doubts on the feasibility of the use of class Ha bacteriocins as food preservatives in light of a stable and efficient resistant phenotype.
Characterisation of small cyclic peptides with antilisterial and antimalarial activity
(Stellenbosch : Stellenbosch University, 2014-04) Leussa, Nyango-Nkeh Adrienne; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new antimicrobial drugs especially with the rise in resistance to almost all available drugs by public health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat to public health. One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research found that the six major Trcs were active against Listeria monocytogenes and Plasmodium falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide unit has an important role in activity. We set out to extend the qualitative structure to activity relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either naturally or by chemical synthesis produced and characterised in terms of chemical character and biological activity. The Trcs were significantly more active than RW peptides, although much more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an aromatic amino acid residue for selective activity towards the leucocin A resistant L. monocytogenes B73-MR1. However, structural properties favouring a tighter membrane interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There was also need for an amphipathic balance between hydrophobicity and size/steric parameters for optimal activity. From our QSAR studies we predict as lead peptide for a future library of antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport system or bacterial dehydrogenases. We confirmed, as previously found, that the Trcs have potent antimalarial activity that is sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in activity. From these results a proposed lead peptide for future research is cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages. This indicated that membrane structures containing neutral lipids, as well as chromatin may be targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic towards each other’s activity. This indicated a shared target and we propose the food vacuole as another of the Trc targets in P. falciparum.
Characteristics and adaptation of skeletal muscle to endurance exercise
(Stellenbosch : University of Stellenbosch, 2011-10) Kohn, Tertius A.; Myburgh, Kathryn H.; Rautenbach, Marina; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Skeletal muscle adapts to stimuli by modifying structural and metabolic protein expression. Furthermore, a muscle group may vary within itself to accommodate specialisation in regions. Structural and metabolic characteristics of an individual are regulated partly by genotype, but contraction duration and intensity may play a greater role in muscle phenotype. The aims of this dissertation were to investigate: structural and metabolic regionalisation in a muscle group, possible relationships between training volume and intensity and hybrid fibres, muscle characteristics of athletes from two different ethnic groups, and muscle adaptation in already well-trained athletes subjected to high intensity interval training. Myosin heavy chain (MHC) isoform content and citrate synthase (CS) activities were measured in the Quadriceps femoris (QF) muscle of 18 female rats. Muscle was divided into superficial, middle and deep, distal, central and proximal parts. MHC IIb and IIx were more abundant in superficial regions (P < 0.05) with low CS activities compared to deeper parts. Isoform content varied along the length of deep regions. This study showed that the QF has regional specialisation. Therefore, standardisation of sampling site is important. Hybrid fibre proportions in muscle biopsies of 12 middle distance runners and 12 non-runners were investigated. MHC IIa/IIx correlated with training volume/week in runners (r = -0.66, P < 0.05) and MHC IIa/IIx correlated with exercise hours/week in non-runners (r = -0.72, P < 0.01). Average preferred racing distance (PRDA) correlated better with MHC IIa/IIx in runners (r = -0.85, P < 0.001). MHC IIa/IIx may therefore be more closely related to exercise intensity than previously thought. Fibre type characteristics and performance markers were investigated in 13 Xhosa and 13 Caucasian distance runners, matched for performance, training volume and PRDA. Xhosa runners had less MHC I and more MHC IIa fibres in muscle biopsies than Caucasian runners (P < 0.05). Xhosa runners had lower plasma lactate at 80% peak treadmill speed (PTS) (P < 0.05), but higher lactate dehydrogenase (LDH) (P < 0.01) and phosphofructokinase (P = 0.07) activities in homogenate muscle samples. LDH activities in MHC I (P = 0.05) and IIa (P < 0.05) fibre pools were higher in Xhosa runners. Xhosa athletes may thus have a genetic advantage or they may have adapted to running at a higher intensity. Six weeks of individually standardised high intensity interval treadmill training (HIIT) were investigated in 15 well-trained runners. PTS increased after HIIT (P < 0.01), while maximum oxygen consumption (VO2max) only showed a tendency to have increased as a result of HIIT (P = 0.06). Sub-maximal tests showed lower plasma lactate at 64% PTS (P = 0.06), with lower heart rates at workloads from 64% to 80% PTS (P < 0.01) after HIIT. No changes were observed for cross-sectional area, capillary supply and enzyme activities in homogenates muscle samples. LDH activity showed a trend (P = 0.06) to have increased in MHC IIa pools after HIIT. Higher HIIT speed was related to decreases in MHC I fibres, but increases in MHC IIa/IIx fibres (r = -0.70 and r = 0.68, respectively, P < 0.05). Therefore, HIIT may alter muscle fibre composition in well-trained runners, with a concomitant improvement in performance markers.
Characterization of prokaryotic pantothenate kinase enzymes and the development of type-specific inhibitors
(Stellenbosch : Stellenbosch University, 2011-12) Koekemoer, Lizbe; Strauss, Erick; Stellenbosch University. Faculty of Sciences. Dept. of Biochemistry.
ENGLISH ABSTRACT: Pantothenate kinase (PanK) enzymes catalyze the first reaction in the five step biosynthesis of the essential cofactor coenzyme A. Enzymes representing each of the three identified PanK types have been studied and characterized and these PanK types exhibits a unique diversity between different organisms, therefore highlighting them as potential drug targets. In this study the type III PanK of specifically pathogenic bacteria were characterized with the goal of developing type-specific inhibitors. Several questions about the activity of the Mycobacterium tuberculosis enzyme was answered, which addresses the contradicting results achieved in related PanK studies performed to date. Furthermore the first inhibitors, that are competitive to the pantothenate binding site, were designed, synthesized and tested against the Pseudomonas aeruginosa enzyme. This resulted in the discovery of the most potent inhibitors of the type III PanKs to date.
Chemopreventive properties of South African herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp) : mechanisms against skin carcinogenesis
(Stellenbosch : Stellenbosch University, 2013-12) Magcwebeba, Tandeka Unathi; Gelderblom, W. C. A.; Joubert, E.; Swart, P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry .
ENGLISH ABSTRACT: The present study employed a two-phased approach to investigate the possible mechanisms involved in the chemopreventive properties of rooibos (Aspalathus linearis) and different honeybush species (Cyclopia spp.) in vitro. In the first phase, the effect of unfermented methanol and aqueous herbal tea extracts against the growth parameters (cell viability, proliferation and apoptosis) of normal (CRL 7761); premalignant (HaCaT); and malignant (CRL 7762) skin cells was evaluated and compared to green tea extracts. The predictive potential of polyphenol content (total polyphenol and flavanol/proanthocyanidins) and antioxidant properties (ABTS; ORAC; FRAP and LPO) in the biological activity of extracts in cells was also assessed. Of the herbal teas, the methanol extract of rooibos was the most active and it inhibited the growth of skin cells presumably by inducing mitochondrial dysfunction via membrane depolarisation. At lower concentrations, this activity was associated with inhibition of cell proliferation that was selective for cancer cells whilst higher concentrations induced apoptosis that was more prominent in premalignant cells. The strong antioxidant properties of the extracts implicated the role of pro-oxidative polyphenol/iron interactions involving monomeric flavonoids and polymeric proanthocyanidins in the cytotoxic effects of rooibos. The strong relationship between total polyphenolic and flavanol/proanthocyanidins content, antioxidant properties and reduction of cell viability indicated that these parameters (polyphenols and antioxidant properties) can serve as predictive tools for the cytotoxic effects of rooibos in vitro. The aqueous extracts of honeybush species, although weaker, displayed similar effects to rooibos extracts in cells with C. genistoides being the most effective at selectively inhibiting the proliferation of cancer cells whilst the pro-apoptotic activity of C. subternata and C. intermedia was more prominent in premalignant cells. The underlying mechanisms are also likely to result from pro-oxidative mechanisms resulting from polyphenol/iron interactions that mainly involve polymeric flavanol-like proanthocyanidin compounds in honeybush. In contrast, the methanol extracts exhibited weaker cytotoxic effects and protected cancer cells from going into apoptosis. The cytoprotective effects of honeybush species are possibly mediated by the major monomeric compounds such as mangiferin and hesperidin through antioxidant mechanisms that result in reduction of oxidative stress. Due to the possible dual role of the monomeric and polymeric compounds in the honeybush extracts, the total polyphenolic content of these herbal teas may not be a good indicator of biological activity in vitro. However, as aqueous extracts displayed high flavanol/proanthocyanidins content and exceptional activity in the ABTS assay, these parameters may be considered as indicators of cytotoxicity. On the other hand, methanol extracts, particularly from the xanthone-rich species (C. genistoides and C. longifolia) which exhibited the weakest cytotoxic effects, were more active in the ORAC thus this assay may be a useful predictor for cytoprotective activity. In the second phase, an in vitro UVB/HaCaT model which used IL-1α as a biomarker for early inflammation was developed and validated with known anti-inflammatory compounds, dexamethasone and ibuprofen. It was used to determine the specific mechanisms involved in the modulatory effects of the herbal tea extracts against inflammation. Rooibos extracts and the aqueous extract of honeybush enhanced the cytotoxic effects of UVB in the model and exhibited indirect anti-inflammatory effects as they removed icIL-1α containing cells via apoptosis. In contrast, methanol extracts of honeybush exacerbated icIL-1α by protecting UVB stimulated cells from undergoing apoptosis. In conclusion, methanol extract of rooibos and aqueous extracts of honeybush species may be useful in protecting the skin after UVB exposure. These herbal tea extracts may block initiation and delay the promotion stage during skin carcinogenesis by removing premalignant cells via apoptosis and preventing onset of inflammation. In contrast, due to their cytoprotective effects, methanol extracts of honeybush may be more effective at preventing oxidative stress in skin before UVB exposure. Future studies should focus on the effects of extracts and polyphenolic fractions on the oxidative status of the cells and development of biomarkers of chemoprevention that can be utilised in vivo and in human skin.
Cloning and identification of genes involved in the interaction between the bacterial stone fruit pathogen Pseudomonas syringae pv. syringae strain NV and plum trees
(Stellenbosch : Stellenbosch University, 2001-03) Appel, Maryke; Bellstedt, D. U.; Mansvelt, E. L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Bacterial canker of stone fruit, caused by Pseudomonas syringae pv. syringae, is one of the most destructive crop diseases in South Africa. Chemical control has failed completely and effective long-term management strategies will have to rely on the breeding of resistant host trees. To assist in such breeding programmes, investigations into the molecular basis of the interaction between P. s. pv. syringae and stone fruit trees have been undertaken in collaboration with the ARC-Fruit, Wine and Vine Research Institute in Stellenbosch. The aim of this dissertation was to clone and identify genes that are involved in interaction between the bacterial canker pathogen and stone fruit trees. In the first part of the study, the harpin encoding gene of a local strain of the pathogen, P. s. pv. syringae NV, was amplified in a polymerase chain reaction (PCR) strategy with primers based on the hrpAZB sequences of the bean pathogen, P. s. pv. syringae 61. Sequencing of this hrpZpssNvgene revealed a high degree of homology (96%) between the harpin encoding genes and harpin proteins of the two strains. The hrpZpssNvgene was subsequently cloned into the pMAL-c2 expression vector and expressed in Escherichia co/i. This system was used for the production of purified, biologically active, recombinant HrpZpSSNV protein. In the second part of the study, differential display (DD) technology was used to identify genes that are induced in stone fruit trees in response to P. s. pv. syringae and/or its harpin elicitor. For this purpose, actively growing shoots of two Prunus sa/icina cultivars, the moderately resistant cv. 'Laetitia' and the highly susceptible cv. 'Songold' were treated with recombinant harpinpssNvprotein or live P. s. pv. syringae NV bacteria. An untreated control and wounding control was included in the experiment. Total RNA was isolated for comparative mRNA analysis 24 hours after treatment. DD profiles were generated with fifteen primer combinations. Eight candidate bands were re-amplified, cloned and sequenced. Reverse transcription PCR was employed to verify the expression patterns of the cloned bands in the original RNA sample set. Two bands, DDc and DD4 were shown to be differentially expressed between treatments and/or cultivars, while no differences in the expression levels of the remaining six bands (DDa, DDe, DD3, DD5, DD6 and DD7) were observed. BLAST similarity searches yielded significant matches for DDe, DD4 and DD7 with plant defense-related genes.
Coenzyme A biosynthesis and Coenzyme A-dependent redox processes as targets for anti-staphylococcal drug development
(Stellenbosch : Stellenbosch University, 2015-12) Moolman, Wessel Johannes Albertus; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH SUMMARY: Staphylococcus aureus, the bacterium that causes most hospital-acquired in humans is rapidly becoming more prevalent in the community and, alarmingly, increasingly resistant to the current arsenal of available antibacterial agents. More than ever, new treatments are urgently needed to combat this threat. In this study we proposed an alternative strategy to current drug development methodologies that entails the identification and targeting of processes that are essential to the survival of pathogenic bacteria in their human host, i.e. where they need to counter the defences of the human immune system. In particular, the focus of this study is the importance of the central metabolic cofactor coenzyme A (CoA) in the defence mechanisms that S. aureus employs under such circumstances, and therefore on the targeting of CoA biosynthesis and enzymology as potential antistaphylococcal targets. The viability of coenzyme A disulfide reductase (CoADR) as a potential antistaphylococcal drug target was evaluated. The S. aureus CoADR (SaCoADR) enzyme structures in complex with mechanism-based Michael acceptor-containing inhibitors were examined; specifically how its interaction with these compounds relates to the observed differences in activity between them. Consequently, the observed enzyme inhibition could be adequately explained when taking into account the chemical properties of the inhibitors in combination with their interactions with SaCoADR. Also, the structural data in the study provided a strong starting point for future inhibitor design. The reasons for the poor correlation between the in vitro inhibition of SaCoADR by the Michael acceptor-containing CoA analogues and the whole cell inhibition of S. aureus by their corresponding pantothenamide precursors were investigated and these results led us to the conclusion that the poor correlation is due to SaCoADR not being essential under normal growth conditions. However, our results suggest that under conditions where CoA levels are sufficiently reduced, CoADR might become relevant, even under normal growth conditions. This opens the door for studies on the possible synergistic effects of CoADR inhibitors and compounds that reduce CoA levels; such combinations most likely hold the most potential for work focused on CoADR as a drug target. The mechanism of inhibition of phosphopantothenoylcysteine synthetase (PPCS) enzymes by 4’-phospho- CJ-15,801-cytidylate (PCJ-CMP) was investigated by determining the basis for the apparent stability of the inhibitor. We showed that the PPCS protein itself plays no role in the mechanism of inhibition by PCJ-CMP, but that the introduction of the double bond in the β-alanine moiety of the substrate with its extra π-electrons renders the acyl phosphate resistant to nucleophilic attack by introducing new, stable resonance forms. This mechanism of apparent stabilisation via resonance was also applied to an unrelated system and we were able to convert substrates of human VNN1 pantetheinase into inhibitors of the enzyme. These studies allowed us to rationalise the tight-binding inhibition observed for PCJ-CMP. Additionally, we uncovered a new strategy whereby β-alanine-containing compounds can be rendered resistant to hydrolysis and/or acyl transfer; this strategy can likely have wide-ranging applications in the design of such small molecule inhibitors and therapeutics.
Coenzyme a biosynthesis and utilization in Plasmodium falciparum : drug targets for antimalarial chemotherapy
(Stellenbosch : Stellenbosch University, 2014-12) Macuamule, Cristiano Joao; Strauss, Erick; Saliba, Kevin J.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Pantothenate (also known as Vitamin B5) is the sole precursor of the essential enzyme cofactor coenzyme A (CoA) that is required in several metabolic reactions virtually in all living organisms including the human malaria parasite Plasmodium falciparum. While the parasite has the capacity to generate CoA from pantothenate, it cannot produce this nutrient de novo, and as a result depends on external supplies. Processes in the CoA metabolic pathway have been identified as possible targets for drug development and pantothenate analogues as agents that can interfere with those processes to block parasite development. In this dissertation it is shown that the class of pantothenate analogues known as Nsubstituted pantothenamides (PanAms) and N‐substituted pantoyltauramides, inhibit the growth of intraerythrocytic‐stage P. falciparum parasites at sub‐ and low micromolar concentrations respectively. In both cases, the compounds inhibited parasite proliferation through inhibition of pantothenate‐dependent processes. It is also shown that the antiplasmodial potency of PanAms can be strengthened through structural modifications rendering the compounds less susceptible to degradation by enzymes known as pantetheinases, which occur natural and ubiquitously in mammals, particularly in the serum. Finally it is also shown that the antiplasmodial mode of action of PanAms results from the compounds serving as alternative substrates for pantothenate kinase (PanK), the first enzyme intervening in the CoA biosynthesis pathway, thus interfering with the phosphorylation of the natural substrate – pantothenate. In addition, it negatively affects the production of functional CoA and acyl carrier proteins (ACPs) which are required in various cellular metabolic processes.
A comparative analysis of CoA biosynthesis in selected organisms: a metabolite study
(Stellenbosch : Stellenbosch University, 2016-03) Goosen, Rene; Strauss, Erick; Snoep, Jacky L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH SUMMARY: This study investigated the biochemical regulation of CoA production because it is an essential pathway that presents an important target for antimicrobial drug discovery studies. Currently, the specific life-sustaining functions of CoA are not clearly defined and a better understanding of the regulation of the CoA biosynthesis pathway would aid in the understanding of the relevance of maintaining specific CoA levels for survival. Regulation of CoA production was investigated on two levels. First, it was determined if production is up-regulated under conditions predicted to be associated with increased demand in S. aureus. Second, regulation of production of CoA by the salvage pathway in E. coli was investigated. In S. aureus it was found that CoA production is up-regulated under conditions of oxidative stress by an as yet unidentified mechanism. This led to an investigation of the regulation in the CoA biosynthesis pathway to understand how production is controlled. At present, the regulation of CoA production is thought to occur at a single, rate-limiting step identified as the first enzyme in the pathway, pantothenate kinase (PanK). Failure of inhibition of PanK to result in growth inhibition suggested that a re-evaluation of this premise is required. To this end, a systems analysis approach was taken in this study to elucidate the control of CoA production by the salvage pathway. Previously, a lack of analytical tools to measure the intermediates of CoA biosynthesis hampered investigations into regulation of the pathway and a holistic study has not been performed to elucidate the control profile. Consequently a method was also developed for the quantification of all the intermediates of the CoA salvage pathway based on derivatization with a fluorescent thiol probe and HPLC analysis. This method allowed for time course analysis of the reconstituted pathway to be performed to provide a holistic interpretation of CoA production. A kinetic model of the pathway was constructed from rate equations parameterized with a combination of experimentally determined values and values reported in the literature. Time course profiles were used to validate the model for subsequent control analyses. Both time course profiles and predictions made by the model indicated that PanK is unlikely to control the rate of CoA production under most conditions, and that it is in fact dephospho-CoA kinase (DPCK), the last enzyme in the pathway, that controls the rate under physiological conditions. This implies that DPCK is the best target for inhibition of the CoA biosynthetic pathway because it is far more likely to be in control of the rate of CoA production under physiological conditions. This finding is significant to antimicrobial drug development efforts because it suggests that the target focus should be shifted from PanK to DPCK. Therefore the findings of this study represent a major shift in our current understanding of the regulation of the rate of CoA production. It also highlights the importance of conducting a detailed systems analysis when studying metabolic pathways from both regulatory and drug development perspectives.
A comparative analysis of the G1/S transition control in kinetic models of the eukaryotic cell cycle
(Stellenbosch : University of Stellenbosch, 2009-12) Conradie, Riaan; Snoep, Jacky L.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The multiplication of cells proceeds through consecutive phases of growth and division (G1, S, G2 and M phases), in a process known as the cell cycle. The transition between these phases is regulated by so-called checkpoints, which are important to ensure proper functioning of the cell cycle. For instance, mutations leading to faulty regulation of the G1/S transition point are seen as one of the main causes of cancer. Traditionally, models for biological systems that show rich dynamic behavior, such as the cell cycle, are studied using dynamical systems analysis. However, using this analysis method one cannot quantify the extent of control of an individual process in the system. To understand system properties at the process level, one needs to employ methods such as metabolic control analysis (MCA). MCA was, however, developed for steady-state systems, and is thus limited to the analysis of such systems, unless the necessary extensions would be made to the framework. The central question of this thesis focuses on quantifying the control in mathematical models of the G1/S transition by the individual cell cycle processes. Since MCA was never applied to the cell cycle, several new methods needed to be added to the framework. The most important extension made it possible to follow and quantify, during a single cell cycle, the control properties of the individual system processes. Subsequently, these newly developed methods were used to determine the control by the individual processes of an important checkpoint in mammalian cells, the restriction point. The positioning of the restriction point in the cell cycle was distributed over numerous system processes, but the following processes carried most of the control: reactions involved in the interplay between retinoblastoma protein (Rb) and E2F transcription factor, reactions responsible for the synthesis of Delayed Response Genes and Cyclin D/Cdk4 in response to growth signals, the E2F dependent Cyclin E/Cdk2 synthesis reaction, as well as the reactions involved in p27 formation. In addition it was shown that these reactions exhibited their control on the restriction point via the Cyclin E/Cdk2/p27 complex. Any perturbation of the system leading to a change in the restriction point could be explained via its e ect on the Cyclin E/Cdk2/p27 complex, showing a causal relation between restriction point positioning and the concentration of the Cyclin E/Cdk2/p27 complex. Finally, we applied the new methods, with a modular approach, to compare a number of cell cycle models for Saccharomyces cerevisiae (budding yeast) and mammalian cells with respect to the existence of a mass checkpoint. Such a checkpoint ensures that cells would have a critical mass at the G1/S transition point. Indeed, in budding yeast, a correction mechanism was observed in the G1 phase, which stabilizes the size of cells at the G1/S transition point, irrespective of changes in the specific growth rate. This in contrast to the mammalian cell cycle models in which no such mass checkpoint could be observed in the G1 phase. In this thesis it is shown that by casting specific questions on the regulation and control of cell cycle transition points in the here extended framework of MCA, it is possible to derive consensus answers for subsets of mathematical models.
Comparative cross-species analysis of detailed kinetic models of glycolysis
(Stellenbosch : University of Stellenbosch, 2009-12) Du Preez, Franco B.; Snoep, Jacky L.; Rohwer, J. M.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: With the recent advances in the field of molecular biology, there is an increased need to integrate data on the various constituents of the cell in kinetic models that can predict and describe cellular behavior. When working towards a description of the entire cell using such kinetic models, the question arises: How do we compare different models for a given biological network? This is the central question addressed in my thesis and I developed and applied mathematical and computational methods for comparing dozens of existing models of erythrocyte and yeast glycolysis. To compare the steady-state behavior in models of erythrocyte glycolysis, I focussed on the function of the pathway, which is to supply the cell with Gibbs-free energy (γ- phosphate of ATP). I used supply-demand analysis in the framework of metabolic control analysis to make this comparison, which revealed that the ATP concentrations were homeostatically buffered at varying supply rates. I also applied this approach to compare steady-state behavior in models of yeast glycolysis, finding that they were not necessarily optimized for homeostatic maintenance of the ATP concentration and that in models for this organism the rate of ATP production is often determined by the supply reactions of glycolysis. In addition, I tested whether a kinetic model can describe novel behavior if it is adjusted to conditions different from those for which the model was originally constructed. More specifically, using a model of steady-state yeast glycolysis, I showed that small adjustments to the original enzyme concentrations are enough to obtain an oscillating model, which shows a remarkable resemblance to the experimentally observed oscillations. Importantly, some of these enzyme concentrations changes are known to occur during the pre-treatment of the cells which is necessary to obtain oscillatory behavior. To the best of my knowledge, the resulting model is the first detailed kinetic model that describes the experimentally observed strong synchronization of glycolytic oscillations in yeast populations. To analyze the dynamic behavior of yeast glycolytic models and to compare different models in terms of dynamics, I introduced a framework used in physics and engineering to create a vector based, two dimensional graphical representation of the oscillating metabolites and reactions of glycolysis. Not only was it possible to make a concise comparison of the set of models, but with the method I could also quantify the contribution of the interactions in the network to the transduction of the oscillations. Furthermore I could distinguish between different mechanisms of oscillation for each of the models, and demonstrated how the framework can be used to create such representations for experimental data sets.

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