Nitric oxide (NO), a vasoprotective and ubiquitous signaling molecule generated from the endothelial cells (EC) by the enzyme endothelial nitric oxide synthase (eNOS) have a vital role in regulation of vascular function and integrity. However, a significant attenuation of eNOS and NO leads to endothelial dysfunction (ED) and increased risk of cardiovascular disease (CVD) in diabetes. Lipoproteins particularly LDL, undergo glycation in diabetic patients and turns it into pro-atherogenic glycated LDL (glyLDL). However, the impact of glyLDL on eNOS, the transmembrane signalling events, involvement of mitochondrial and endoplasmic reticulum (ER) stress in EC remains unclear. Also, literatures reveal impaired platelet mitochondrial function in diabetes patients; however, the impact of family history of diabetes on platelet mitochondrial bioenergetics still remains unknown. In the present study, we had provided the evidence for diabetes-associated metabolic stress involving glyLDL can attenuate eNOS protein, gene and activity in EC, as well as glyLDL and high glucose attenuates eNOS content in EC. Receptor of advanced glycation end products (RAGE) and H-Ras pathway are implicated in the upstream signalling events in the downregulation of eNOS in EC. In addition, ER stress, impaired mitochondrial function due to significant reduction of complex-specific oxygen consumption and bioenergetics were identified in glyLDL-treated EC. Further, we have also detected significant impairment in platelet mitochondrial bioenergetics in healthy individuals with familial history of diabetes. Identifying the mechanisms involved in diabetes associated metabolic stress induced signaling in EC and early detection of mitochondrial impairment in healthy individuals will help to find new targets for the prevention and treatment of diabetic cardiovascular complications and improve quality of life in diabetic patients. / May 2015

Les organismes unicellulaires sont très vulnérables aux changements rapides de leur environnement puisqu’ils sont en contact direct avec celui-ci. Les organismes unicellulaires utilisent plusieurs stratagèmes pour réguler l’expression génique et par conséquent, les diverses voies métaboliques nécessaires aux différentes conditions environnementales auxquelles ils sont confrontés. On sait maintenant que certains ARN, dont les riborégulateurs, ont également un grand rôle à jouer dans les processus de régulation de l’expression du génome. En général, les riborégulateurs sont des éléments génétiques situés dans les régions 5’ non traduites (5’ UTR) de l’ARN messager bactérien. Ces éléments possèdent une structure tertiaire bien définie qui est conservée à travers l'évolution. Les riborégulateurs subissent un changement de conformation en s’associant à un ligand spécifique et modulent l’expression des gènes qu’ils contrôlent en aval. Le rôle des riborégulateurs en relation avec le métabolisme des eubactéries peut être d’une grande importance. C’est le cas de Clostridium difficile qui voit son génome régulé par au moins une quarantaine de riborégulateurs. Étant un pathogène nosocomial, causant des infections contractées dans le milieu hospitalier, bien connu pour engendrer des complications au niveau de l’intestin, il est intéressant d’étudier la relation gène-riborégulateur-métabolisme chez C. difficile. De plus, les riborégulateurs ont montré un potentiel intéressant comme cible antibiotique pour combattre certaines infections. C. difficile possède quatre riborégulateurs guanine transcriptionnels qui contrôlent quatre gènes intervenant dans la voie de biosynthèse de la guanosine monophosphate (GMP). Deux de ces gènes interviennent directement dans la voie de synthèse du GMP soit une guanosine-monophosphate synthase (guaA) et une xanthine phosphoribosyltransférase (XPRTase) (xpt) ainsi que deux transporteurs de précurseur nommés CD630_21070 codant pour une perméase uracile/xanthine et CD630_ 27040 une perméase putative. Bien que quelques études ont démontré l’importance que pourrait avoir le gène guaA chez d’autres espèces bactériennes (Staphylococcus aureus, Escherichia coli, Streptococcus suis et, Salmonella thyphimurium), aucune étude de C. difficile n'a élucidé la relation entre les quatre riborégulateurs guanine et les gènes qu’ils contrôlent, de même que leur rôle au sein du métabolisme du GMP dans un contexte in vivo. Le présent mémoire porte sur l’étude de ces quatre riborégulateurs guanine chez C. difficile, les gènes régulés par ces riborégulateurs et leurs effets sur la biologie de C. difficile. Une fois que les recherches bio-informatiques ont été entreprises, le projet s’est divisé en deux grandes parties soit l’efficacité de régulation des riborégulateurs guanine et l’importance des quatre gènes qui sont sous le contrôle de ces riborégulateurs chez C. difficile 630. Afin de vérifier si les riborégulateurs guanine avaient une affinité acceptable pour leur ligand (guanine), des essais de cartographies chimiques ont été faits et des Kd ont été déterminés. Ces Kd se retrouvent tous dans le bas nanomolaire (nM) de 2,61 ± 1,29 nM pour le riborégulateur guaA et des Kd de 1,78 ± 0,95 nM, de 3,06 ± 0,29 nM et de 4,44 ± 2,75 nM pour les riborégulateurs xpt, CD_21070 et CD_27040 respectivement. Pour la deuxième partie du projet, quatre mutants d’inactivation de gène par insertion ont été conçus grâce à l’utilisation du système ClosTron. Ces quatre mutants, correspondant aux gènes guaA, xpt, CD630_21070 et CD630_27040, ont ensuite été analysés lors d’essais de croissance afin de vérifier les phénotypes associés aux inactivations de gènes. À la suite des résultats d’essais de croissance des divers mutants d’inactivation, une emphase a été mise sur le gène guaA et son riborégulateur puisque ce dernier montrait un phénotype d’inhibition de croissance. L’efficacité avec laquelle le riborégulateur guaA pouvait réprimer l’expression génique a été déterminée lors des essais de gène rapporteur gusA et de PCR quantitative en temps réel. Une baisse de l’expression ligand-dépendante a été observée dans des conditions variant en concentration de guanine. Le mutant de délétion par inactivation du gène guaA a aussi démontré une baisse dans sa capacité d’infecter un modèle murin. En effet, lors d’une étude d’infection en compétition avec le type sauvage, les décomptes cellulaires du mutant guaA étaient diminués de 4 logs. Ces résultats indiquent un rôle fondamental du gène guaA sur le pouvoir infectieux de C. difficile 630 et mettent en évidence l’importance du riborégulateur qui contrôle l’expression de ce gène. Toutes les expériences faites dans le cadre du projet ont été entreprises chez C. difficile 630 afin de garder un contexte plus naturel au cours des analyses. Les résultats présentés ici mettent en évidence le potentiel des riborégulateurs comme cibles antibiotiques. Des travaux futurs devront être effectués afin de vérifier l’effet que pourraient avoir certains analogues de ligands qui ciblent les riborégulateurs guanine sur la viabilité de C. difficile.

Dans le but de mieux comprendre le mode d’action et de nous permettre de déterminer la cible de la tomatidine, nous avons dans un premier temps tenté de mieux circonscrire le spectre d’activité de la tomatidine. Grâce à ces travaux, nous sommes, en effet, maintenant en mesure de dire que la tomatidine possède une activité antibactérienne contre les espèces de la division des Firmicutes et plus précisément contre les bactéries de l’ordre des Bacillales dont font partie les genres Bacillus, Staphylococcus et Listeria. Nous avons également découvert, grâce à des expériences en collaboration avec le laboratoire d’Éric Marsault, qu’un analogue de la tomatidine (FC04-100) avait non seulement des propriétés similaires à la molécule naturelle, mais démontrait une activité par lui-même contre S. aureus à phénotype normal alors que la tomatidine possède uniquement une activité contre les « small colony variants ». De plus, alors que la tomatidine possède plutôt une activité bactériostatique contre la forme SCV de L. monocytogenes, le nouveau composé (FC04-100) démontre quant à lui, une forte activité bactéricide contre cette souche, tout comme contre la forme SCV des autres Bacillales. Parallèlement, et toujours dans le but de rechercher le mode d’action et la cible de la tomatidine, nous avons obtenu, par passages successifs dans un milieu avec antibiotiques, des mutants de S. aureus à phénotype normal et des SCV résistants à la tomatidine ou à la combinaison tomatidine et gentamicine. Après le séquençage de ces mutants, l’étude de la position de ces mutations, à l’aide de différents logiciels de bio-informatique, nous a permis d’émettre un modèle-hypothèse quant au mode d’action et à la cible de la tomatidine. Selon les résultats que nous avons à ce stade-ci, la cible de la tomatidine chez S. aureus serait la sous-unité c de l’ATP synthase. Cependant, son mode d’action serait également dépendant de la fonctionnalité de la chaine de transport des électrons et donc de la polarisation membranaire et de la production de ROS intracellulaire, ce qui expliquerait la différence d’activité entre les souches à phénotype normal et les SCV.

Dihydrodipicolinate Synthase (EC 4.3.3.7; DHDPS), the product of the dapA gene, is an enzyme that catalyzes the condensation of pyruvate and S-aspartate-β-semialdehyde (ASA) into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS catalyzes the first committed step in the biosynthesis of ʟ-lysine and meso-diaminopimelate; each of which is a necessary cross-linking component between peptidoglycan heteropolysacharide chains of bacterial cell walls. Therefore, strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and ʟ-lysine biosynthesis in bacteria leading to decreased bacterial growth and cell lysis. Much attention has been given to targeting the active site for inhibition; however DHDPS is subject to natural feedback inhibition by ʟ-lysine at an allosteric site. In DHDPS from Campylobacter jejuni ʟ-lysine is known to act as a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. Little is known about how the protein structure facilitates the natural inhibition mechanism and mode of allosteric signal transduction. This work presents ten high resolution crystal structures of Cj-DHDPS and the mutant Y110F-DHDPS with various substrates and inhibitors, including the first reported structure of DHDPS with ASA bound to the active site. As a body of work these structures reveal residues and conformational changes which contribute to the inhibition of the enzyme. Understanding these structure function relationships will be valuable for the design of future antibiotic lead compounds. When an inhibitor binds to the allosteric site there is meaningful shrinkage in the solvent accessible volume between 33% and 49% proportional to the strength of inhibition. Meanwhile at the active site the solvent accessible volume increases between 5% and 35% proportional to the strength of inhibition. Furthermore, inhibitor binding at the allosteric site consistently alters the distance between hydroxyls of the catalytic triad (Y137-T47-Y111') which is likely to affect local pKa's. Changes in active site volume and modification of the catalytic triad would inhibit the enzyme during the binding and condensation of ASA. The residues H56, E88, R60 form a network of hydrogen bonds to close the allosteric site around the inhibitor and act as a lid. Comparison of ʟ-lysine and bislysine bound to wt-DHDPS and Y110F-DHDPS indicates that enhanced inhibition of bislysine is most likely due to increased binding strength rather than altering the mechanism of inhibition. When ASA binds to the active site the network of hydrogen bonds among H56, E88 and R60 is disrupted and the solvent accessible volume of the allosteric site expands by 46%. This observation provides some explanation for the reduced affinity of ʟ-lysine in high ASA concentrations. ʟ-Lysine, but not other inhibitors, is found to induce dynamic domain movements in the wt-DHDPS. These domain movements do not appear to be essential to the inhibition of the enzyme but may play a role in cooperativity between monomers or governing protein dynamics. The moving domain connects the allosteric site to the dimer-dimer interface. Several residues at the weak dimer interface have been identified as potentially involved in dimer-dimer communication including: I172, D173, V176, I194, Y196, S200, N201, K234, D238, Y241, N242 and K245. These residues are not among any previously identified as important for formation of the quaternary structure.

Subcellular sequestration of proteins within membrane bound compartments is widely acknowledged to be an important mode of enzymatic regulation. Recently a novel paradigm for metabolic enzyme compartmentalisation has become apparent with the identification of several proteins which are able to form filamentous structures in vivo. Multiple studies independently identified the essential de novo pyrimidine biosynthesis enzyme CTP synthetase as a major constituent of a novel filamentous structure which has been termed “the cytoophidium”. Cytoophidia have been observed to form in multiple organisms including bacteria (C. crescentus), yeast (S. cerevisiae) and fruit fly (D. melanogaster) as well as in human cultured cells. In this thesis I describe the development and results of a high throughput genomescale screen to identify factors involved in cytoophidia biogenesis. Observations of tissue specific CTPS distribution lead to the identification of the well-conserved growth regulator dm/dMyc as an essential factor for CTPS regulation in vivo. These results provide new insights into the coordination of cellular growth and metabolic regulation during normal development and indicate the potential of CTPS/cytoophidia as a future therapeutic target.

Alstroemeria is an important cut flower and its breeding has been developed focused on aesthetic characteristics and vase life longevity, but little is known about its scent. Five different genotypes were assessed including the non scented cultivars ‘Rebecca’ and ‘Samora’ and the scented cultivars, ‘Sweet Laura’, ‘Ajax’ and the species A. caryophyllaea. The scented Alstroemerias emitted the terpenoids: isocaryophyllene and ocimene as the major floral volatile compounds. Characterization of an Alstroemeria TPS (ALSTER) was based on four ESTs previously found in A. cv ‘Rebecca’. Rapid amplification of cDNA ends (RACE) was performed and the full length ORF was used for characterizations of the genomic organization and amino acid sequences (phylogenetic analysis). ALSTER genomic region contains five introns and six exons. This unique genomic organization classified ALSTER as a member of the class III with a merged 5-6 exon. The deduced amino acid sequence was classified into the subfamily TPS-b. A functional analysis showed enzymatic activity of ALSTER with geranyl diphosphate (GPP) and the monoterpene myrcene was the only product obtained. Gene expression evaluated through real time and semi q RT-PCR on eight different stages of development (SO to S7) showed high expression of ALSTER at around S2 - S4 in the scented Alstroemerias, coinciding with high scent emission perceived and also with the maturation of reproductive organs. Evaluations through surveys focused on level of liking of floral scent, were performed finding positive correlations between floral scent liking and floral appearance liking and between floral scent liking and floral scent intensity. Finally, 17 new lines of A. caryophyllaea were evaluated in terms of their morphology, phenology and productivity. Although none of them were suitable for the market because of their low productivity, short stems and small flowers, they were all scented and identified as promising starting points for breeding purposes.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The worldwide escalation in the incidence of obesity and its strong association with insulin resistance, type 2 diabetes and the cardiovascular complications that accompany these disease states have elicited interest in the underlying mechanisms of these pathologies. Preliminary data generated in our laboratory showed that obesity is associated with abnormalities in the insulin signalling pathway. Specifically, we found a down-regulation of protein kinase B (PKB/Akt), which is known to mediate the metabolic effects of insulin. One of the downstream targets of PKB/Akt is glycogen synthase kinase-3 (GSK-3), which is inhibited by this phosphorylation. Detrimental effects of unopposed activity of GSK-3 have recently been described. This may play a pivotal role in some of the adverse consequences of insulin resistance in the heart. Hypothesis: Chronic inhibition of GSK-3 will induce myocardial hypertrophy or exacerbate the development of existing hypertrophy in a pre-diabetic model of diet induced obesity and insulin resistance. Objectives: (1) Assess the extent of the development of myocardial hypertrophy in a rat model of diet induced obesity (DIO) and insulin resistance. (2) Assess the effect of inhibition of GSK-3 protein on the development of myocardial hypertrophy. Methods: Two groups of age-matched male Wistar rats were used. Control animals received standard rat chow, while obese animals received a high caloric diet for 20 weeks. After 12 weeks, half of the animals in both groups received GSK-3 inhibitor treatment (CHIR118637, 30mg/kg/day, Novartis). At the end of 20 weeks, three series of experiments were conducted. (i) The animals were subjected to echocardiography to determine in vivo myocardial function, and biometric, metabolic and biochemical parameters were evaluated. (ii) The ability of the cardiomyocytes to accumulate deoxy-glucose after stimulation with insulin was determined, and (iii) the localization of key proteins was monitored using fluorescence microscopy and cell size was determined using light microscopy and flow activated cell sorter analysis. Results and discussion: The high caloric diet increased body weight (p<0.005) and intraperitoneal fat mass (p<0.01) when compared to controls. Complications associated with obesity, such as impaired glucose tolerance (p<0.05), hyperinsulinemia (p<0.0005) and an increased HOMA-IR index (p<0.01) were observed. Additionally, cardiomyocytes from the DIO animals had a significantly impaired response to insulin, specifically when 10nM (p<0.05) and 100nM (p<0.05) of insulin were used as stimulus. We also found a dysregulation in PKB/Akt, indicated by a down-regulation of phosphorylated PKB/Akt (p<0.01). The diet promoted the development of myocardial hypertrophy, since the ventricular weight (p<0.05) and ventricular weight to tibia length ratio were increased (p<0.01). Echocardiography experiments showed an increase in end diastolic diameter in the DIO animals (p<0.05). Additionally, there was an increase in the cardiomyocyte cell width in the DIO rats (p<0.0001) and a tendency for peri-nuclear localization of NFATc3. GSK-3 inhibition promoted the development of insulin resistance in control animals, as indicated by an increase in the body weight (p<0.05), serum insulin levels (p<0.01) and HOMA-IR index (p<0.01). In the DIO animals, the GSK-3 inhibitor treatment improved insulin resistance, as a decrease in serum insulin concentration (p<0.05) was observed. The cardiomyocytes from the treated DIO animals also showed an increase in glucose uptake (p<0.05) when stimulated with 100nM of insulin. The GSK-3 inhibitor promoted the development of myocardial hypertrophy in the control animals, indicated by an increase in ventricular weight (p<0.05) and cardiomyocyte cell width (p<0.0001), but did not exacerbate hypertrophy in the DIO animals. Conclusion: Both the high caloric diet and the GSK-3 inhibitor promoted the development of insulin resistance and myocardial hypertrophy in the rats. In the DIO animals the GSK-3 inhibitor treatment ameliorated insulin resistance and did not promote the further development of myocardial hypertrophy. / AFRIKAANSE OPSOMMING: Inleiding: Die huidige styging in vetsugtigheid en die sterk assosiasie daarvan met insulien weerstandigheid, tipe 2 diabetes en kardiovaskulêre komplikasies soos hipertrofie, het ‘n belangstelling in die onderliggende meganismes van hierdie siektetoestande ontlok. Voorlopige data uit ons laboratorium het getoon dat vetsug geassosieerd is met abnormaliteite in die insulien seintransduksie-pad soos byvoorbeeld ‘n afregulering van miokardiale proteïen kinase B (PKB/Akt), wat bekend is om die metaboliese effekte van insulien te medieer. Een van die proteïene wat deur PKB/Akt gefosforileer en daardeur geïnhibeer word, is glikogeen sintase kinase-3 (GSK-3). Negatiewe effekte van onge-opponeerde aktiwiteit van GSK-3 is beskryf en dit mag ‘n sleutelrol speel in sommige van die nadelige gevolge van insulien weerstandigheid in die hart. Hipotese: Chroniese onderdrukking van GSK-3 sal miokardiale hipertrofie ontlok of die bestaande hipertrofie in ‘n pre-diabetiese model van dieet-geïnduseerde vetsug en insulien weerstandigheid vererger. Doelstellings: (1) Om die omvang van die ontwikkeling van miokardiale hipertrofie in ‘n rotmodel van dieet-geïnduseerde vetsug te ondersoek en (2) om die effek van inhibisie van GSK-3 op die ontwikkeling van hipertrofie te ondersoek. Metodes: Ouderdomsgepaarde manlike Wistarrotte is in hierdie studie gebruik. Die diere is vir ‘n periode van 20 weke aan verskillende diëte onderwerp, naamlik standaard kommersiële rotkos vir die kontrole diere en ‘n hoë kalorie dieet vir die eksperimenteel vet diere (DIO). Helfte van elke groep diere is vir 8 weke met ‘n GSK-3 inhibitor behandel (CHIR118637, 30mg/kg/day, Novartis). Na die 20 weke is 3 eksperimentele reekse uitgevoer: (i) Die diere is eggokardiografies ondersoek om in vivo miokardiale funksie te bepaal en biometriese, metaboliese en biochemiese parameters is evalueer. (ii) Die vermoë van kardiomiosiete om de-oksiglukose na insulien stimulasie te akkumuleer, is bepaal, en (iii) die lokalisering van sleutelproteïene is met behulp van fluoressensie mikroskopie en die selgrootte met behulp van ligmikroskopie bepaal. Resultate en bespreking: Die hoë kalorie dieet het gepaard gegaan met ‘n beduidende toename in liggaamsgewig (p<0.005) en intraperitoneale vetmassa (p<0.01) in vergelyking met diere op die kontrole dieet. Newe-effekte geassosieerd met vetsug nl. onderdrukte glucose toleransie (p<0.05), hiperinsulinemie (p<0.0005) en ‘n verhoogde HOMA-IR index (p<0.01) is ook waargeneem. Daar was ook ‘n beduidend ingekorte respons van glukose opname deur kardiomiosiete van die vet diere na stimulasie met 10nM (p<0.05) en 100nM (p<0.05) insulien. Disregulering van PKB/Akt is gevind in die vorm van ‘n afregulering van die fosforilering van die proteïen (p<0.01). Die dieet het ook gelei tot die ontwikkeling van miokardiale hipertrofie aangesien die ventrikulêre gewig (p<0.05) asook die verhouding van die ventrikulêre gewig teenoor tibia lengte beduidend toegeneem het (p<0.01). Eggokardiografie het ‘n toename in ventrikulêre end-diastoliese dimensie in die DIO diere aangetoon (p<0.05). Tesame hiermee het die breedte van kardiomiosiete van die DIO diere toegeneem (p<0.0001) en daar was ook ‘n peri-nukluêre lokalisering van NFATc3. Behandeling van kontrole diere met ‘n GSK-3 inhibitor het insulienweerstandigheid ontlok soos afgelei uit ‘n verhoging in liggaamsgewig (p<0.05), serum insulien-vlakke (p<0.01) en die HOMA-IR waarde (p<0.01). In teenstelling het behandeling van die DIO diere met die GSK-3 inhibitor tot ‘n verbetering van insulienweerstandigheid gelei aangesien ‘n verlaging in serum insulien konsentrasies gevind is (p<0.05). Kardiomiosiete vanaf die behandelde DIO diere het ook ‘n verhoogde insulien-gestimuleerde glukose opname met 100nM insulien getoon (p<0.05). Behandeling met die GSK-3 inhibitor het die ontwikkeling van miokardiale hipertrofie in die kontrole diere teweeggebring, soos aangetoon deur ‘n toename in die ventrikulêre gewig (p<0.05) en ‘n groter selwydte in kardiomiosiete terwyl dit geen invloed op die bestaande hipertrofie van die vet diere gehad het nie. Gevolgtrekking: Die huidige studie het getoon dat die betrokke dieet asook behandeling met ‘n GSK-3 inhibitor insulienweerstandigheid sowel as die ontwikkelling van miokardiale hipertrofie in rotte ontlok. In die DIO diere het die behandeling met die GSK-3 inhibitor bloedglukose en insulien-vlakke verlaag en het nie hipertrofie vererger nie.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: It is widely accepted that insufficient insulin-stimulated activation of muscle glycogen synthesis is one of the major components of non-insulin-dependent (type 2) diabetes mellitus. Glycogen synthase, a key enzyme in glycogen synthesis, is extensively regulated, both allosterically (by glucose-6-phosphate, ATP, and other ligands) and covalently (by phosphorylation). Although glycogen synthase has been a topic of intense study for more than 50 years, its kinetic characterization has been confounded by its large number of phosphorylation states. Questions remain regarding the function of glycogen synthase regulation and the relative importance of allosteric and covalent modification in fulfilling this function. The regulation of glycogen synthase and glycogen phosphorylase, the enzyme that catalyses the degradation of glycogen chains, are reciprocal in many respects. In the present research, using mathematical modelling, we aim to establish the function of the allosteric and covalent regulation of glycogen synthase and glycogen phosphorylase in muscle and, in the case of glycogen synthase, the relative importance of these two mechanisms in performing this function. In order to realize these aims it is essential that a detailed kinetic model of glycogen metabolism is constructed. We begin with a thorough review of the kinetics and regulation of glycogen synthase inwhich we propose that both allosteric and covalent modification of glycogen synthase can be described by a Monod-Wyman-Changeux model in terms of apparent changes to L0, the equilibrium constant between the T and R conformers. We then proceed to develop a rate equation according to the proposed Monod-Wyman-Changeux model and determine values for its kinetic parameters from published experimental data using non-linear least-squares regression. We show that the application of the Monod-Wyman-Changeux model to glycogen synthase kinetics also has important implications for the rate equations of enzymes that catalyse the phosphorylation and dephosphorylation of glycogen synthase. We formalize these implications for a generic protein that follows Monod-Wyman-Changeux-type conformational change and then also show how the findings apply to glycogen synthase. Taking into account the kinetic model of glycogen synthase and how it also influences the covalent regulation of the enzyme, we proceed to construct a detailed mathematical model of glycogen synthesis that includes the glycogen synthase phosphorylation cascade. A variation of this model in which glycogen synthase phosphorylation is described with a single parameter is also provided. We reuse an existing model of muscle glycogenolysis and also combine these models in an overall model of glycogen metabolism. Finally, we employ the theoretical frameworks of metabolic control analysis, supply-demand analysis, and co-response analysis to investigate the function of glycogen synthase and glycogen phosphorylase regulation. We show that the function of glycogen synthase regulation is not flux control, as assumed in the textbook view, but rather the maintenance of glucose-6-phosphate within a narrow range far from equilibrium. Similarly, we show that regulation of glycogen phosphorylase functions to minimize variation in cellular energy charge in the face of highly variable energy demand. We conclude with an appeal for a renewed interest in the enzyme kinetics of muscle glycogen metabolism. / AFRIKAANSE OPSOMMING: Daar word wyd aanvaar dat onvoldoende insulien-gestimuleerde aktivering van spierglikogeensintese een van die hoofkomponente van insulien-onafhanklike (tipe 2) diabetes mellitus is. Glikogeensintase, ’n sleutelensiem in glikogeensintese is onderworpe aan breedvoerige regulering, beide allosteries (deur glukose-6-fosfaat, ATP, en ander ligande) en kovalent (deur fosforilering). Alhoewel glikogeensintase reeds vir meer as 50 jaar deeglik bestudeer word, word die kinetiese karakterisering daarvan bemoeilik deur die groot aantal fosforilasiestate waarin die ensiem voorkom. Daar is steeds vrae betreffende die funksie van die regulering van glikogeensintase en die relatiewe bydrae van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Die regulering van glikogeensintase en glikogeenfosforilase, die ensiem wat die afbraak van glikogeenkettings kataliseer, is in baie opsigte resiprook. In hierdie studie beoog ons om met die hulp van wiskundige modellering vas te stel watter funksie die regulering van glikogeensintase en glikogeenfosforilase vervul en, in die geval van glikogeensintase, wat die relatiewe belang is van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Om hierdie oogmerke te verwesentlik is dit nodig dat ’n kinetiese model van glikogeenmetabolisme ontwikkel word. Ons begin met ’n omvattende oorsig van die kinetika en regulering van glikogeensintase waarin ons voorstel dat beide die allosteriese en kovalente regulering van glikogeensintase beskryf kan word met die Monod-Wyman-Changeux model in terme van oënskynlike veranderings aan L0, die ekwilibriumkonstante tussen die T en R konformasies. Ons gaan dan voort om ’n snelheidsvergelyking te ontwikkel volgens die voorgestelde Monod-Wyman-Changuex-model en bepaal ook die waardes van hierdie vergelyking se parameters vanaf gepubliseerde eksperimentele data deur middel van nie-lineêre kleinste-vierkantsregressie. Ons wys dat die toepassing van die Monod-Wyman-Changuex-model op glikogeensintase-kinetika belangrike gevolge het vir die snelheidsvergelykings van die ensieme wat die fosforilering en defosforilering van glikogeensintase kataliseer. Ons formaliseer hierdie gevolge vir ’n generiese Monod-Wyman-Changeux-tipe proteïen en wys dan ook hoe die bevindings op glikogeensintase van toepassing is. Met inagneming van die kinetiese model vir glikogeensintase en hoe dit die kovalente regulering van die ensiem be¨ınvloed, gaan ons voort om ’n gedetaileerde wiskundige model van glikogeensintese, wat ook die glikogeensintase-fosforileringskaskade insluit, te ontwikkel. ’n Variasie op hierdie model waarin die fosforilering van glikogeensintase deur ’n enkele parameter beskryf word, word ook voorsien. Ons herbruik ’n bestaande model van spierglikogenolise en kombineer ook hierdie modelle in ’n oorkoepelende model van glikogeenmetabolisme. Uiteindelik span ons die teoretiese raamwerke van metaboliese kontrole-analise, vraag-aanbod-analise, en ko-responsanalise in om die funksie van die regulering van glikogeensintase en glikogeenfosforilase te ondersoek. Ons wys dat die funksie van die regulering van glikogeensintase nie fluksiekontrole, soos algemeen in handboeke aangeneem word, is nie, maar liewer dat dit glukose-6-fosfaat handhaaf binne ’n noue band ver vanaf ekwilibrium. Insgelyks wys ons dat die regulering van glikogeenfosforilase funksioneer om variasie in sellulˆere energielading te beperk ten spyte van hoogs wisselende vlakke van energie-aanvraag. Ons sluit af met ’n pleidooi vir hernieude belangstelling in die ensiemkinetika van glikogeenmetabolisme in die spier. / National Research Foundation

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.