Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants. / Graduate Studies, College of (Okanagan) / Graduate

The diversity of biosynthetic pathways in prokaryotes and eukaryotes has led to numerous bioactive natural products (NPs) which occupy a vast chemical space. Despite the current challenges in NP research, these molecules are still relevant today as they are a major source of human medicine as well as being useful biological tools. The elucidation of their biosynthetic pathways has also provided information about the biochemical and biophysical properties of fascinating enzyme families such as the α- oxoamine synthases (AOSs). The AOSs are an expanding group of pyridoxal 5’- phosphate (PLP)-dependent enzymes, which are involved in the biosynthesis of several important NP, including those essential for life. This study reports the characterization and structural analysis of a unique AOS denoted as TamD from Pseudoalteromonas tunicata. This enzyme plays a major role in tambjamine biosynthesis and consists of both an acyl carrier protein (ACP) domain and a PLP-binding catalytic domain. UV/vis spectroscopy and mass spectrometry (MS) of the recombinant TamD purified from E. coli revealed that the enzyme forms a Schiff base with PLP via Lys380, which is responsible for its characteristic yellow colour. It binds L-serine as a natural substrate with a Kd of 5.01 ± 0.64 mM. This thesis also reports structural data for TamD from xray crystallography at a resolution of 4.98 Å, which shows four molecules in the asymmetric unit (ASU) suggesting the enzyme exist as a dimer. The absence of the Nterminal region where the ACP domain is located in the crystal strucuture also suggests intrinsic flexibility and disorder within that region. With the increasing demand for new anti-infective therapies, investigations of the molecular interactions between NPs and their protein targets are vital in understanding the inhibition or activation properties of these molecules. The cysteine transpeptidases known as sortases produced by Gram positive bacteria have been identified as attractive targets for NP inhibitors. In this thesis, the molecular basis for the inhibition of Streptococcus mutans sortase A (SrtA) by the plant flavonoid, trans-chalcone is explored, using a combination of MS, enzyme kinetics, molecular modelling and x-ray crystallography. This study reports the first high resolution crystal structure of the H139A mutant of S. mutans SrtA, which reveals a unique N-terminal α-helix domain. Trans-chalcone was found to inhibit the in vitro activity of S. mutans SrtA in a slow, tight–binding manner, with a half maximal inhibitory concentration (IC50) of 5.0 ± 0.6 μM. The interaction resulted in a covalent adduct with the active site cysteine residue (Cys205) via a Michael addition mechanism. Additionally, trans-chalcone showed evidence of S. mutans anti-biofilm activity in a concentration dependent manner up to 250 μM with an efficacy cut-off point at higher concentations. These results indicate that chalcone flavonoids are worth further investigation as potential antibiofilm inhibitors. A renewed interest in plant NPs has also led to a collaborative investigation on the antimicrobial potential of garlic-derived allicin, against Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. Allicin is the principal antibacterial agent in fresh preparations of garlic extracts. This investigation reports the first evidence that allicin and allicin-contaning garlic extracts possess inhibitory and bactericidal activities against Bcc. The minimum inhibitory concentrations (MICs) of aqueous garlic extract (AGE) against 38 Bcc isolates ranged from 0.5 to 3% (v/v). An investigation into the possible molecular mechanisms of allicin with a recombinant thiol-dependent peroxiredoxin (BCP) from B. cenocepacia revealed that allicin and AGE modify an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit further investigation as adjuncts to existing antibiotics.

The increasing spectrum of multidrug-resistant bacteria has caused a major global public health concern, necessitating the discovery of novel antimicrobial agents. Additionally, recent advancements in the use of microbial cells for the scalable production of industrial enzymes has encouraged the screening of new environments for efficient microbial cell factories. The unique ecological niche of the Red Sea points to the promising metabolic and biosynthetic potential of its microbial system. Here, ten sequenced Bacilli strains, that are isolated from microbial mat and mangrove mud samples from the Red Sea, were evaluated for their use as platforms for protein production and biosynthesis of bioactive compounds. Two of the species (B.paralicheniformis Bac48 and B. litoralis Bac94) were found to secrete twice as much protein as Bacillus subtilis 168, and B. litoralis Bac94 had complete Tat and Sec protein secretion systems. Additionally, four Red Sea Species (B. paralicheniformis Bac48, Virgibacillus sp. Bac330, B. vallismortis Bac111, B. amyloliquefaciens Bac57) showed capabilities for genetic transformation and possessed competence genes. More specifically, the distinctive biosynthetic potential evident in the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 was assessed and compared to nine available complete genomes of B. licheniformis and three genomes of B. paralicheniformis. A uniquely-structured trans-acyltransferase (trans-AT) polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) cluster in strains of this species was identified in the genome of B. paralicheniformis 48. In total, the two B. paralicheniformis Red Sea strains were found to be more enriched in modular clusters compared to B. licheniformis strains and B. paralicheniformis strains from other environments. These findings provided more insights into the potential of B. paralicheniformis 48 as a microbial cell factory and encouraged further focus on the strain’s metabolism at the system level. Accordingly, a draft metabolic model for B. paralicheniformis Bac48 (iPARA1056) was reconstructed, refined, and validated using growth rate and growth phenotypes under different substrates, generated using high-throughput Phenotype Microarray technology. The presented studies indicate that several of the isolated strains represent promising chassis for the development of cell factories for enzyme production and also point to the richness of their genomes with specific modules of secondary metabolism that have likely evolved in Red Sea Bacilli due to environmental adaptation.

Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

Thesis (Ph.D.(Microbiology)) -- University of Limpopo, 2022 / Respiratory tract infections (RTIs) are frequent ailments among humans and are a high burden to public health. One strategy for the development of new therapies against pathogenic bacteria such as Mycobacterium tuberculosis is to target essential biosynthetic pathways of its metabolism. The aim of this study was to evaluate and target the biosynthesis of aromatic amino acids (shikimate pathway) of Mycobacterial spp using medicinal plant extracts. The selection of the plants in this study was based on their ethnopharmacological use for the treatment of tuberculosis infections and related symptoms. The leaves were dried at ambient temperatures and ground to fine powder. The powdered material was extracted with hexane, dichloromethane, acetone, methanol and water. Phytochemical screening was done using standard protocols that tested for tannins, saponins, terpenoids, alkaloids, flavonoids, steroids, anthraquinones, phlobatannins, quinones, and betacynins. Phytochemical fingerprints were established using thin layer chromatography (TLC) where three mobile phases varying in polarity were used to develop the chromatograms. Total Phenolics, flavonoids, flavonols, tannins, alkaloids and proanthocyanidin contents were quantified using UV/Vis spectrometry. Spectrometric quantification of the free radical (DPPH) scavenging activity and ferric (potassium ferricyanide) reducing power were performed. The heat-dependent bovine serum albumin and egg albumin denaturation assays were used to evaluate anti-inflammatory activity. Antimycobacterial activity was screened using bioautography assay in qualitative analysis. Quantitatively, broth microdilution assay was used to determine the minimal inhibitory concentrations. The Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 interference genetic editing technique was used to evaluate and validate the essentiality of the aromatic amino acids in Mycobacteria to further determine the vulnerability and draggability of the transketolase (tkt) and DAHPs (aroG) genes. Plasmid, PLJR962, was used for the CRISPRi/dCas9 gene knockdown experiments. The integrating CRISPRi plasmid expressed both sgRNA with the targeting region (for tkt or aroG) and the dCas9 handle which is under control of the anhydrotetracycline (ATC) inducible promoters. The spot assay and growth curves were used to for phenotypic characterisation and gene knockdown experiments. RNA microarray (qPCR) was used to evaluate the level of expression inhibition of tkt gene . Mechanism of action of plants extracts bioactive components were predicted based on synergy between gene knockdown, shikimate inhibitors and the plant extracts. To evaluate whether the shikimate intermediates may rescue gene depleted M. smegmatis hypomorphs, the cultures were grown in L-tryptophan, L-phenylalanine, L-tyrosine and shikimic acid and growth curves constructed. Cytotoxicity of the extracts was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero cell lines and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages. Phytochemical analysis showed that the various extracts had various polar and non-polar compounds which belonged to phenolics, saponins, steroids, terpenoids, alkaloids, cardiac glycosides and resins. Numerous non-polar compounds from Gardernia volkensii, Senna petersiana, Ficus sur had antimycobacterial activity against M. smegmatis in bioautography. Remarkably, acetone extracts from S. petersiana, Acacia senegal, Carissa bispinosa, P. africanum and C. gratissimus that had moderate to low antimycobacterial activity against wild type M. smegmatis (mc2 155) demonstrated improved inhibitory activity against the tkt PAM1 M. smegmatis CRISPRi mutant. Only the acetone Clerodendrum glabrum, Croton gratissimus, Peltophorum africanum and Gardenia volkensii demonstrated activity against M. tuberculosis H37Rv. These results suggest that the employment of CRISPRi in M. tuberculosis to develop screening models may increase changes of obtaining bioactive chemical species because the tkt gene knockdown was showed to possess the ability to potentiate the antimycobacterial activity of the plant extracts. An added advantage of the plant extracts is their antioxidant and anti-inflammatory activities which may benefit the host immune system during treatment of infection by reducing free radicals and pro-inflammatory agents that perpetuate the infection. Non polar compounds were found to generally have higher anti-inflammatory activity than the polar counterpart for all the plant extracts. These results suggest that the non-polar compounds from the tested extracts may not only confer antimycobacterial effects, but also anti-inflammatory activities. A. senegal, G. volkensii, F. sur, S. petersiana and C. glabrum were found to be toxic to the Vero cell line. However, purification techniques may circumvent their toxic effects. This study demonstrated that the amino acid biosynthesis is a potential antimycobacterial drug target because it was found to be essential, vulnerable and druggable by medicinal plant extracts / University of Limpopo and National Research Foundation (NRF-DAAD In-Country Doctoral Scholarship Programme)

<p>  </p> <p>Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated, with many natural products being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. These elicitors can be natural products, including antibiotics or hormones, or synthetic compounds. The use of small molecule elicitors to induce the production of secondary metabolites has several advantages. First, addition of elicitors to fermentation media can result in increased titers of known natural products. Second, elicitors can enable the discovery of novel natural products typically produced at undetectable levels. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of “silent” natural products. In this work, we sought to expand on the method of chemical induction by utilizing oxytetracycline at a sub-MIC concentration to induce secondary metabolite production in Streptomyces. We have shown that translation-inhibiting antibiotics, specifically oxytetracycline, have a profound effect on the production of coeliomycin P1, actinorhodin, and calcium-dependent antibiotics (CDAs) in S. coelicolor and S. lividans. The expression of actinorhodin in S. lividans under these conditions is unique, unlike its counterpart, S. coelicolor, which can produce actinorhodin under standard conditions. In addition to the increased production of known secondary metabolites, we have also demonstrated the induction of BGCs in several other strains of Streptomyces, which were observed via LC-MS. </p> <p>In addition to exploring antibiotics as elicitors we have explored the traditional approach of natural product discovery by taking an bioactivity guided approach. Several strain that we isolated from soil collect of Hawaii were screened for activity against several pathogenic strains primarily looking for which strain will inhibit the growth of a. baumannii, which is an intriguing target because the rate of resistance to common antibacterial medication is rising and it’s membrane composition is vastly different compared to other gram negative bacterium like E.coli. From this preliminary screening 1 strain (Streptomyces sp. CS62) out of the 8 that tested exhibited the desired biological activity. The supernatant of Streptomyces sp .CS62 was processed and screen by LC-MS to gain insight on the type of molecules that Streptomyces CS62 could produce. Upon our initial screening process none of the masses observed in the mass spec were matched to knowns. However, after 2D NMR analysis and genomic analysis it was unveiled that Streptomyces sp. CS62 produces factumycin a known antibacterial agent that targets A.baumannii .This unfourtunate turn of events illustrates the issues with natural product discovery and the need to improve natural product databases.</p> <p>In conjunction to discovering a novel producer of factumycin we are also investigating the production of antifungal compounds from Staphylococcus lugdunensis  a commensal strain that modulates the microbiome by producing lugdunin. The supernatant collected of Staphylococcus lugdunensis  is exclusively being test against Candida auris due to the immense health risk it possess to society because of its innate resistance to many antifungal drugs and its ability to rapidly gain resistance to other classes of antifungals.</p> <p>In addition to exploring the influence of antibiotics on secondary metabolite production and using bioactivity as a guide to discovering antibiotics. We are evaluating the soils collected from unique environments as potential sources for novel natural products. Specifically, we are evaluating the biosynthetic potential of bacteria from ore-forming environments, specifically fluorspar and topaz mines. Soils from ore-forming environments tend have low pH, high saline content, low water holding capacity, and poor nutrient availability. Therefore, ore-forming environments pose a hostile environment for life. To date, no one has explored the natural product potential, or the bacterial diversity, exhibited in these harsh environments. To assess the bacterial diversity, bacteria were isolated from various ore-forming environments using a procedure that is selective for actinobacteria. Following bacterial isolation, genomic DNA was isolated and 16s rRNA gene sequencing was performed to gauge the type of bacteria that were isolated. To stimulate secondary metabolite production, bacteria were then subjected to 7 different media conditions. The supernatant collected from these media conditions were tested against ESKAPE pathogens utilizing the CTSI broth microdilution assay. LC-MS MS analysis was performed for samples exhibiting biological activity. GNPS molecular networking was then utilized to determine potential molecules present in each sample.  Through this process we were able to identify one strain, which we named Streptomyces sp. S1A that exhibited a board range of biological activity (anticancer and antibacterial) and possess a wide array of biosynthetic gene clusters ranging complex macrolides (PKS and NRPS) to terpenes. </p> <p>In summary this multifaced approach to natural product discovery may lead to the discovery of novel antibiotics, enable us to increase production of known or unknown antibiotics through chemical induction, and the characterization of metabolites from Streptomyces sp. S1A will shed insight on the biochemical potential of organisms that inhabit ore-forming environments </p>

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The cardiac isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid (FA) uptake; thus increased ACCβ activity decreases fatty acid utilization thereby potentially leading to intracellular myocardial lipid accumulation and insulin resistance (IR). Previous studies show that greater flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of IR. In light of this, we hypothesize that increased HBP flux induces ACCβ gene expression thereby contributing to the onset of IR. Our initial work focused on ACCβ gene promoter regulation and suggest that the HBP modulates upstream stimulatory factor 2 (USF2) thereby inducing ACCβ gene expression. Here, we further investigated HBP-mediated regulation of ACCβ gene expression by transiently transfecting cardiac-derived H9c2 cells with an expression vector encoding the rate-limiting HBP enzyme (GFAT) ± the full length ACCβ and 4 truncated promoter-luciferase constructs, respectively. GFAT overexpression increased ACCβ gene promoter activity for the full length and 3 larger deletion constructs (p<0.001 vs. controls). However, GFAT-mediated and USF2-mediated ACCβ promoter induction was blunted when co-transfected with the -38/+65 deletion construct suggesting that USF2 binds to the proximal promoter region (near start codon). Further investigation proves that USF2 binds to ACCβ promoter and activates it, but that USF2 is not O-GlcNAc modified even though there is a strong correlation between increased O-GlcNac levels and USF2 activation of ACCβ. This would suggest that there is another O-GlcNac modified factor involved in this regulatory pathway. Our study demonstrates that increased HBP flux induces ACCβ gene promoter activity via HBP modulation of USF2. We propose that ACCβ induction reduces fatty acid oxidation, thereby leading to intracellular lipid accumulation (FA uptake>>FA oxidation) and the onset of cardiac IR. / AFRIKAANSE OPSOMMING: Die kardiale isoform van asetiel-CoA karboksilase (ACCβ) produseer maloniel-CoA, ‘n kragtige inhibeerder van mitochondriale vetsuur (VS) opname, en om hierdie rede sal verhoogde ACCβ aktiwiteit, vetsuur gebruik verlaag en potensieël aanleiding gee tot intrasellulêre miokardiale lipiedophoping en insulienweerstand (IW). Vorige studies toon dat groter fluks deur die heksosamienbiosintetiese weg (HBW) bydra tot die ontwikkeling van IW. In die lig hiervan hipotetiseer ons dat verhoogde HBW fluks, ACCβ geenuitdrukking induseer, en sodoende tot die onstaan van IW bydra. Ons aanvanglike werk het op ACCβ geenpromotorregulering gefokus, en voorgestel dat die HBW die opstroom stimuleringsfaktor 2 (USF2) moduleer en dus ACCβ geen uitdrukking induseer. Hier het ons verder die HBW-gemedieërde regulering van ACCβ-geenuitdrukking deur kortstondige tranfeksie van kardiaalverkrygde H9c2 selle met ‘n uitdrukkingsvektor wat kodeer vir die tempo-bepalende HBW ensiem (GFAT) ± die volle lengte ACCβ, en vier afgestompte promotor-lusiferase konstrukte onderskeidelik, te ondersoek. GFAT ooruidrukking het ACCβ geenpromotor aktiwiteit vir die volle lengte, en drie groter uitwissingskonstrukte verhoog (p<0.001 vs. kontrole). Hoewel GFAT- en USF2-gemedieërde ACCβ promotorinduksie tydens ko-transfeksie van die -38/+65 uitwissingskonstruk versag was, is dit voorgestel dat USF2 aan die proksimale promotor area (naby die beginkodon) bind. Verdere ondersoek bewys ook dat USF2 aan die ACCβ promotor bind en dit aktiveer, maar dat USF2 nie O-GlcNAc gemodifiseer word nie ten spyte van ‘n sterk korrelasie tussen verhoogde O-GlcNac vlakke en USF2 aktivering van ACCβ. Dit kan dus voogestel word dat daar ‘n alternatiewe O-GlcNac gemodifiseerde faktor betrokke is in hierdie reguleringsweg. Ons studie demonstreer dat verhoogde HBW fluks ACCβ geenpromotor aktiwiteit via HBW modulering van USF2 veroorsaak. Ons stel voor dat ACCβ induksie vetsuuroksidasie verlaag en so tot intrasellulêre lipiedophoping (VS opname >> VS oksidasie) en die onstaan van kardiale IW lei.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background:Obesity is a growing global burden; current studies have projected the prevalence of obese / overweight individuals to increase to ~1.35 billion by 2030. A number of factors contribute to cardiovascular diseases, of which the focus of this study is what effect an increased level of free fatty acids has on the flux through the hexosamine biosynthetic pathway (HBP). It has been widely proven that an increased flux through the HBP causes an increase in protein O-GlcNAcylation, which leads to increased reactive oxygen species (ROS) production as well as an increase in cell death (apoptosis). Methods: For the purpose of this study a cell model was used. H9c2 cardiomyoblasts were cultured in 5ml Dulbecco‟s Modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin. The cells were then exposed to 0.25mM monounsaturated fatty acid (oleic acid) for 24, 48 and 72 hours respectively. The cultured cells were then evaluated to assess the degree ROS production, overall O-GlcNAcylation and cell death (apoptosis and necrosis), using flow cytometry and immunofluorescence microscopy. Results: We found that oleic acid causes a significant decrease in ROS production at the 48 hour time point when analysed on the flow cytometer, which indicates that oleic acid is metabolized by the cells in a independent manner. Oleic acid also caused a significant decrease in cell death at all the time intervals. With regard to the HBP, oleic acid activates this pathway but causes downstream cardioprotective effects that do not necessarily occur along this pathway. Conclusion: This study explored whether a monounsaturated fatty acid, oleic acid, is able to act as a novel cardioprotective agent. The in vitro data supports this concept and we showed that it is able to blunt oxidative stress and cell death. It was also found that although oleic acid activated the HBP, it did not mediate its protective effects via this pathway only. / AFRIKAANSE OPSOMMING: Agtergrond: Vetsug is 'n groeiende wêreldlas; huidige studies voorspel dat die voorkoms van vetsugtige / oorgewig individue toe sal neem tot ~1.35 biljoen teen 2030. Alhoewel verskeie faktore tot kardiovaskulêre siektes bydra is die fokus van hierdie studie om die effek van verhoogde vryvetsuurvlakke op die fluks deur die heksosamienbiosintestiese weg (HBW) te ondersoek. Dit is reeds bewys dat verhoogde fluks deur die HBW 'n verhoging in proteïen O-GlcNAsilering lei, wat verder tot verhoogde reaktiewe suusrtofspesies (ROS) vorming aanleiding gee en ook seldood (apoptose) verhoog. Metodes:'n Selmodel is vir die doel van hierdie studie gebruik. H9c2 kardiomioblaste is in 5ml Dulbecco's Modified Eagles Medium (DMEM) gekweek en gesupplementeer met 10% fetale beesserum en 1% penisillien-streptomysien. Die selle is blootgestel aan 'n 0.25mM mono onversadigde vetsuur (oleïensuur ) vir 24, 48 en 72 uur onderskeidelik. Die gekweekte selle is gevolglik ondersoek vir die graad van ROS ontwikkeling, algehele O-GlcNAsilering en seldood (apoptosis en nekrose), deur van vloeisitometrie en immunofluoresensie mikroskopie gebruik te maak. Resultate: Ons het bevind dat oleïensuur 'n betekenisvolle verlaging in ROS ontwikkeling teen 48 uur soos bepaal deur die vloeisitometer, veroorsaak. Dit wys daarop dat oleïensuur deur die selle op 'n onafhanklike wyse gemetaboliseer is. Oleïensuur het ook 'n betekenisvolle verlaging in seldood by alle tydsintervalle veroorsaak. Met betrekking tot die HBW het oleïensuur hierdie weg geaktiveer maar afstroom kardiobeskermings effekte versoorsaak wat nie noodwendig langs hierdie weg onstaan nie. Gevolgtrekking:Hierdie studie het die moontlikheid van 'n mono-onversadige vetsuur, oleïensuur, om op te tree as 'n nuwe kardiobeskermingsmiddel ondersoek. Die in vitro data ondersteun hierdie konsep en hier is aangetoon dat dit wel oksidatiewe stres en seldood onderdruk. Daar is verder bevind dat alhoewel oleïensuur die HBW aktiveer dit nie die beskermings effekte alleenlik via hierdie weg medieer nie.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: OBJECTIVE – Oxidative stress increases flux through the hexosamine biosynthetic pathway (HBP) resulting in greater O-GlcNAcylation of target proteins. Since increased oxidative stress and HBP flux are associated with insulin resistance, we hypothesized that its activation leads to greater O-GlcNAcylation of BAD (pro-apoptotic) and increased myocardial apoptosis. RESEARCH DESIGN AND METHODS – To investigate our hypothesis, we employed two experimental models: 1) H9c2 cardiomyoblasts exposed to high glucose (33 mM glucose) ± HBP modulators ± antioxidant treatment vs. matched controls (5.5 mM glucose); and 2) a rat model of high fat diet-induced insulin resistance and hyperglycemia. We evaluated apoptosis in vitro by Hoechst nuclear staining, Annexin-V staining, caspase activity measurements and immunoblotting while in vivo apoptosis was assessed by immunoblotting. In vitro reactive oxygen species (ROS) levels were quantified by H2DCFDA staining (fluorescence microscopy, flow cytometry). We determined overall and BAD O-GlcNAcylation, both by immunoblotting and immunofluorescence microscopy. As BAD-Bcl-2 dimer formation enhances apoptosis, we performed immunoprecipitation analysis and immunofluorescence microscopy (co-localization) to determine BAD-cl-2 dimerization. In vivo overall O-GlcNAcylation, BAD O-GlcNAcylation and BAD-Bcl-2 dimerization was determined by immunoprecipitation and immunoblotting. 4 RESULTS – High glucose treatment of cells significantly increased the degree of apoptosis as revealed by Hoechst nuclear staining (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V staining (43 ± 5%), caspase activity assay (26 ± 2%) and immunoblotting. In parallel, overall OGlcNAcylation (p<0.001 vs. 5.5 mM), BAD O-GlcNAcylation (p<0.05 vs. 5.5 mM) and ROS levels were increased (fluorescence microscopy – p<0.05 vs. 5.5 mM; flow cytometry – p<0.001 vs. 5.5 mM). HBP inhibition using DON and antioxidant treatment (α-OHCA) attenuated these effects while HBP activation by PUGNAc exacerbated it. Likewise, insulin resistant rat hearts exhibited significantly higher caspase-3 (p<0.05 vs. controls), overall O-GlcNAcylation (p<0.05 vs. controls) and BAD O-GlcNAcylation levels (p<0.05 vs. 5.5 mM). BAD-Bcl-2 dimer formation was increased in cells exposed to hyperglycemia [immunoprecipitation analysis and co-localization] and in insulin resistant hearts. CONCLUSIONS - Our study identified a novel pathway whereby hyperglycemia results in greater oxidative stress, resulting in increased HBP activation and increased BAD OGlcNAcylation. We also found greater BAD-Bcl-2 dimerization increasing myocardial apoptosis, suggesting that this pathway may play a crucial role in the onset of the diabetic cardiomyopathy. / AFRIKAANSE OPSOMMING: DOELWIT – Oksidatiewe stres verhoog fluks deur die heksosamien biosintetiese weg (HBW) wat in „n groter O-GlcNAsetilering van teiken proteïene resulteer. Weens die feit dat verhoogde oksidatiewe stres en HBW fluks verband hou met insulienweerstandigheid, hipotetiseer ons dat die aktivering hiervan tot groter O-GlcNAsetilering van BAD (pro-aptoptoties) en verhoogde miokardiale apoptose lei. NAVORSINGS ONTWERP EN METODES – Om die hipotese te ondersoek het ons twee modelle ontplooi: 1) H9c2 kardiomioblaste is blootgestel aan hoë glukose konsentrasie (33mM glucose) ± HBW moduleerders ± antioksidant behandeling vs. gepaarde kontrole (5.5mM glucose); en 2) „n hoë vet dieetgeïnduseerde insulienweerstandige rotmodel en hiperglukemie. Ons het apoptose in vitro deur middel van Hoescht nukleuskleuring geëvalueer, kasapase aktiwiteit bepalings en immunoblotting terwyl apoptose in vivo getoets is deur immunoblotting. Reaktiewe suurstofspesie (RSS) vlakke is deur middel van H2DCFDA verkleuring (fluoresensie mikroskopie, vloeisitometrie) bepaal. Algehele en BAD O-GlcNAsetilering is beide deur immunoblotting en immunofluoresensie mikroskopie bepaal. BAD-Bcl-2 dimeervorming bevorder apoptose, om BAD-cl-2 dimerisasie te bepaal is daar van immunopresipitering analise en immunofluoresensie mikroskopie (ko-lokalisasie) gebruik gemaak. In vivo is algehele OGlcNAsetiliering, BAD O-GlcNAsetiliering en BAD-Bcl-2 dimerisasie deur immunopresipitasie en immunoblotting bepaal. 6 RESULTE – Hoë glukose behandeling van selle het die graad van apotpose betekenisvol verhoog soos blootgelê deur Hoechst nukleuskleuring (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V kleuring (43 ± 5%), kaspase aktiviteit assay (26 ± 2%) en immunoblotting. In parallel, algehele OGlcNAsetilering (p<0.001 vs. 5.5 mM), BAD O-GlcNAsetilering (p<0.05 vs. 5.5 mM) en RSS vlakke is verhoog (fluoresensie mikroskopie– p<0.05 vs. 5.5 mM; vloeisitometrie– p<0.001 vs. 5.5 mM). HBW inhibering deur van DON en van antioksidant behandeling gebruik te maak (α- OHCA) het hierdie effekte verlaag terwyl HBW aktivering deur PUGNAc dit verhoog het. Netso, het insulienweerstandige rotharte betekenisvolle hoë kaspase -3 (p<0.05 vs. kontrole), algeheel O-GlcNAsetilering (p<0.05 vs. kontrole) en BAD O-GlcNAsetiliering vlakke (p<0.05 vs. 5.5 mM) getoon. BAD-Bcl-2 dimeervorming is verhoog in hiperglukemies blootgestelde selle [immunopresipitering analise en ko-lokalisering] en in insulienweerstandige harte. GEVOLGTREKKINGS – Ons studie het „n nuwe weg geïdenifiseer waar hiperglukemie in groter oksidatiewe stres resulteer wat weer HBW aktivering verhoog en BAD O-GlcNAsetilering verhoog het. Ons het verder bevind dat groter BAD-Bcl-2 dimerisasie miokardiale apoptose verhoog wat voorstel dat hierdie weg „n belangrike rol in diabetiese kardiomiopatie speel.