During seed development, myo-inositol (Ins) hexakisphosphate or phytic acid (PA) is stored in the form of phytin with mineral cations, and is mobilized following germination, releasing these nutrients that are required for seedling growth. Outside its role in seeds, PA and other phosphoylated Ins derivatives play critical roles in biological processes in many eukaryotes. PA also has negative influences on nutrition in both non-ruminant animals and humans due to its lack of digestibility. There have been two parallel PA biosynthetic pathways proposed, yet, the pathway is still poorly understood in terms of its regulation and enzymes involved. Here, genes encoding enzymes putatively implicated in castor bean PA biosynthetic pathways were identified in the genome and expression followed. Isolated castor bean embryos have the ability to resynthesize PA following germination if exogenous phosphate is available. It was found that the genes purported to act in PA synthesis were constitutively expressed in the embryos regardless of the availability of phosphate. Castor bean Ins 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1), catalyzing the final reaction in PA biosynthesis, regardless of pathway, was chosen for further study. Even though only one copy of the RcIPK1 gene was present in the genome, numerous RNA variants were found, most likely due to alternative splicing events. These variants encoded six closely related protein isoforms based on in silico analysis. Functional analyses using yeast mutant strains lacking the IPK1 gene revealed that only three of the mRNA variants could rescue the temperature-sensitive growth phenotype, and it was demonstrated by HPLC analysis of Ins phosphates that their ability to complement the missing yeast IPK1 enzyme was associated with enzyme activity. Only these three isoforms possessed conserved motif III and motif IV important for IPK1 catalytic activity.

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.

A esterigmatocistina (ST) é uma micotoxina policetônica produzida por diferentes espécies de Aspergillus e outros gêneros fúngicos como Bipolaris e Chaetomium. Esta toxina é caracterizada como uma bifuranoxantona com fórmula molecular C18H12O6, freqüentemente encontrada como contaminante de diversos produtos alimentícios como sementes oleaginosas e cereais. Possui propriedades carcinogênicas, toxigênicas, mutagênicas e teratogênicas, entretanto, ela é menos tóxica do que a aflatoxina. O fungo Emericella nidulans (Eidam) Vuillemin, cujo anamorfo é o Aspergillus nidulans (Eidam) Winter, foi usado como modelo genético para a investigação de genes envolvidos na via biossintética da esterigmatocistina. Linhagens estoque de Utrecht (originárias de Glasgow) foram analisadas quanto à produção de ST e, entre elas, somente a linhagem UT196 [yA2 (I); metA17 (II); piroA4 (IV)] apresentou produção de 4 ppm de ST (stc+), enquanto que as linhagens UT448 e UT184 mostraram-se não produtoras dessa micotoxina (stc). Embora a linhagem UT196 seja muito bem caracterizada geneticamente, este foi o primeiro relato da produção de ST nessa linhagem. Os índices de segregação alélica e todas as freqüências de recombinação entre os marcadores genéticos ligados e não ligados foram determinados tanto pelo cruzamento meiótico UT448 (stc) x UT196 (stc+) como pelo cruzamento mitótico UT448//UT184. 175 segregantes meióticos e 140 segregantes mitóticos foram analisados quanto à produção de ST, aos marcadores de auxotrofia e de resistência a acriflavina. Essas linhagens cruzadas apresentavam vários marcadores em heterozigose e isso permitiu o mapeamento de um gene estrutural da ST (stcZ+), localizado no braço esquerdo do cromossomo I, 4% distante do gene da riboflavina (riboA1). Como um subproduto deste trabalho, foi detectado um pigmento vermelho, de Rf = 0,90, em todos os segregantes meióticos e mitóticos, produtores ou não de ST, indicando tratar-se, provavelmente, de um pigmento policetônico produzido pelos ascosporos do fungo. A baixa expressão da produção de ST em 13 segregantes meióticos e a alta expressão dessa toxina nos diplóides UT448//UT196 e UT448//UT184 (40 ppm) permitiram concluir a existência de um fator regulador, compreendido na região w-met do cromossomo II, responsável pela expressão do gene estrutural stcZ+. A análise desses genes através do ciclo parassexual sugeriu um comportamento epigenético típico envolvendo esses genes. Com base nos dados obtidos, hipóteses para explicar o controle da expressão desses genes e suas inter-relações foram aqui apresentadas. / Sterigmatocystin (ST) is a polyketide produced by different species of Aspergillus as well as by other fungus genera such as Bipolaris and Chaetomium. This toxin is characterized as a bifuranoxanthone, whose molecular formula is C18H12O6 and which is frequently found as a contaminant in several food products such as oil-seed grains and cereals. It has carcinogenic, toxigenic, mutagenic and teratogenic properties; however, it is less toxic than aflatoxin. The fungus Emericella nidulans (Eidam) Vuillemin, whose anamorph is Aspergillus nidulans (Eidam) Winter, was used as a genetic model to investigate the genes involved in the sterigmatocystin biosynthetic pathway. Strains from Utrecht stocks (originally from Glasgow) were analyzed in order to detect ST production and, among them, only the UT196 strain [yA2 (I); metA17 (II); piroA4 (IV)] showed the production of 4 ppm of ST (stc+), whereas the UT448 and UT184 strains showed to be nonproducers of such toxin (stc). Although the UT196 strain is very well characterized genetically, this has been the first report on its production of ST. The allelic segregation rates and all the recombination frequencies between linked and non-linked genetic markers were determined by both the meiotic crossing UT448 (stc) x UT196 (stc+) and mitotic crossing UT448//UT184. 175 meiotic segregants and 140 mitotic segregants were analyzed as to ST production, auxotrophy markers and resistance to acriflavine. These crossed strains presented various markers in heterozygous configuration, which allowed to map a structural gene of ST (stcZ+) located on the left arm of chromosome I, 4% distant from the riboflavin gene (riboA1). As a byproduct of this work, a red pigment of 0.90 Rf was detected in all meiotic and mitotic segregants, whether they were ST producers or not, which indicated that was probably a polyketide produced by the fungus ascopores. The low expression of ST production in 13 meiotic segregants and the high expression of such toxin in the UT448//UT196 and UT448//UT184 diploids (40 ppm) allowed to conclude that a regulating factor existed in the w-met region of chromosome II, which is responsible for the expression of the structural gene stcZ+. The analyses of those genes through the parasexual cycle suggested a typical epigenetic behavior which involved them. Based on the data obtained, hypotheses to explain the expression control of these genes as well as their inter-relationships were here presented.

A esterigmatocistina (ST) é uma micotoxina policetônica produzida por diferentes espécies de Aspergillus e outros gêneros fúngicos como Bipolaris e Chaetomium. Esta toxina é caracterizada como uma bifuranoxantona com fórmula molecular C18H12O6, freqüentemente encontrada como contaminante de diversos produtos alimentícios como sementes oleaginosas e cereais. Possui propriedades carcinogênicas, toxigênicas, mutagênicas e teratogênicas, entretanto, ela é menos tóxica do que a aflatoxina. O fungo Emericella nidulans (Eidam) Vuillemin, cujo anamorfo é o Aspergillus nidulans (Eidam) Winter, foi usado como modelo genético para a investigação de genes envolvidos na via biossintética da esterigmatocistina. Linhagens estoque de Utrecht (originárias de Glasgow) foram analisadas quanto à produção de ST e, entre elas, somente a linhagem UT196 [yA2 (I); metA17 (II); piroA4 (IV)] apresentou produção de 4 ppm de ST (stc+), enquanto que as linhagens UT448 e UT184 mostraram-se não produtoras dessa micotoxina (stc). Embora a linhagem UT196 seja muito bem caracterizada geneticamente, este foi o primeiro relato da produção de ST nessa linhagem. Os índices de segregação alélica e todas as freqüências de recombinação entre os marcadores genéticos ligados e não ligados foram determinados tanto pelo cruzamento meiótico UT448 (stc) x UT196 (stc+) como pelo cruzamento mitótico UT448//UT184. 175 segregantes meióticos e 140 segregantes mitóticos foram analisados quanto à produção de ST, aos marcadores de auxotrofia e de resistência a acriflavina. Essas linhagens cruzadas apresentavam vários marcadores em heterozigose e isso permitiu o mapeamento de um gene estrutural da ST (stcZ+), localizado no braço esquerdo do cromossomo I, 4% distante do gene da riboflavina (riboA1). Como um subproduto deste trabalho, foi detectado um pigmento vermelho, de Rf = 0,90, em todos os segregantes meióticos e mitóticos, produtores ou não de ST, indicando tratar-se, provavelmente, de um pigmento policetônico produzido pelos ascosporos do fungo. A baixa expressão da produção de ST em 13 segregantes meióticos e a alta expressão dessa toxina nos diplóides UT448//UT196 e UT448//UT184 (40 ppm) permitiram concluir a existência de um fator regulador, compreendido na região w-met do cromossomo II, responsável pela expressão do gene estrutural stcZ+. A análise desses genes através do ciclo parassexual sugeriu um comportamento epigenético típico envolvendo esses genes. Com base nos dados obtidos, hipóteses para explicar o controle da expressão desses genes e suas inter-relações foram aqui apresentadas. / Sterigmatocystin (ST) is a polyketide produced by different species of Aspergillus as well as by other fungus genera such as Bipolaris and Chaetomium. This toxin is characterized as a bifuranoxanthone, whose molecular formula is C18H12O6 and which is frequently found as a contaminant in several food products such as oil-seed grains and cereals. It has carcinogenic, toxigenic, mutagenic and teratogenic properties; however, it is less toxic than aflatoxin. The fungus Emericella nidulans (Eidam) Vuillemin, whose anamorph is Aspergillus nidulans (Eidam) Winter, was used as a genetic model to investigate the genes involved in the sterigmatocystin biosynthetic pathway. Strains from Utrecht stocks (originally from Glasgow) were analyzed in order to detect ST production and, among them, only the UT196 strain [yA2 (I); metA17 (II); piroA4 (IV)] showed the production of 4 ppm of ST (stc+), whereas the UT448 and UT184 strains showed to be nonproducers of such toxin (stc). Although the UT196 strain is very well characterized genetically, this has been the first report on its production of ST. The allelic segregation rates and all the recombination frequencies between linked and non-linked genetic markers were determined by both the meiotic crossing UT448 (stc) x UT196 (stc+) and mitotic crossing UT448//UT184. 175 meiotic segregants and 140 mitotic segregants were analyzed as to ST production, auxotrophy markers and resistance to acriflavine. These crossed strains presented various markers in heterozygous configuration, which allowed to map a structural gene of ST (stcZ+) located on the left arm of chromosome I, 4% distant from the riboflavin gene (riboA1). As a byproduct of this work, a red pigment of 0.90 Rf was detected in all meiotic and mitotic segregants, whether they were ST producers or not, which indicated that was probably a polyketide produced by the fungus ascopores. The low expression of ST production in 13 meiotic segregants and the high expression of such toxin in the UT448//UT196 and UT448//UT184 diploids (40 ppm) allowed to conclude that a regulating factor existed in the w-met region of chromosome II, which is responsible for the expression of the structural gene stcZ+. The analyses of those genes through the parasexual cycle suggested a typical epigenetic behavior which involved them. Based on the data obtained, hypotheses to explain the expression control of these genes as well as their inter-relationships were here presented.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical (HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter protein function and contribute to the onset of myocardial insulin resistance and impaired contractile function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation, and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4 translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose. Although our results were not conclusive, the data suggest a potential novel link between dexamethasone exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and related metabolic derangements and diseases), these results provide new targets for potential therapeutic agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier (HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie. Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak, die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê. Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4) translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4 translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie. Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.

Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Gene expression of the cardiac isoform of acetyl-CoA carboxylase (ACCb) is induced in a glucose-dependent manner. ACCb produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake. Previous studies show that increased flux through the hexosamine biosynthetic pathway (HBP) under hyperglycaemic conditions may contribute to the development of insulin resistance. In light of this, we hypothesised that increased HBP flux induces cardiac ACCb gene expression thereby contributing to the onset of insulin resistance. We tested our hypothesis by transiently transfecting cardiac-derived rat H9c2 myoblasts with a 1,317 bp human ACCb promoter-luciferase construct (pPIIb-1317) and an expression construct encoding the rate-limiting step of the HBP i.e. glutamine: fructose 6-phosphate amidotransferase (GFAT). Overexpression of GFAT increased ACCb gene promoter activity by 75 ± 23% versus controls (n=6, p<0.001). When cotransfection experiments were repeated in the presence of varying concentrations of L-glutamine (0 mM, 4 mM, 8 mM), a substrate for the HBP, ACCb promoter activity was dose-dependently increased. To further corroborate these findings, we employed two inhibitors of GFAT, i.e. 40 μM azaserine and 40 μM 6-diazo-5-oxo-Lnorleucine were administered to transfected cells for a period of 24 hours. Here both azaserine and 6-diazo-5-oxonorleucine attenuated ACCb gene promoter activity. In agreement, co-transfections with two dominant negative GFAT constructs also diminished ACCb gene promoter activity. We next inhibited two enzymes of the HBP acting downstream of GFAT, i.e. O-GlcNAc transferase and O-GlcNAcase using alloxan (0.1 mM, 1 mM and 2 mM) and streptozotocin (5 mM and 10 mM), respectively, for a period of 24 hours. Addition of alloxan attenuated ACCb gene promoter activity by 35.6 ± 1.9% (n=16, p<0.001) and streptozotocin increased activity by 32 ± 12% (n=12, p<0.001). We also investigated USF1 and USF2 as transcriptional regulatory candidates for HBP-induced ACCβ promoter regulation. Our data implicates USF2 as an important transcriptional regulator of HBP-induced ACCβ promoter regulation. In summary, this study demonstrates that increased flux through the hexosamine biosynthetic pathway induces ACCb gene promoter activity. We further propose that such an induction would reduce cardiac fatty acid oxidation, thereby leading to intracellular lipid accumulation due to a mismatch between sarcolemmal FA uptake and mitochondrial FA oxidation in the insulin resistant setting (i.e. hyperlipidaemia). / AFRIKAANSE OPSOMMING: Geen uitdrukking van die kardiale isoform asetiel-KoA karboksilase (ACCb) word in ‘n glukose afhanklike wyse geïnduseer. ACCb produseer maloniel-KoA, ‘n kragtige inhibeerder van mitochondriale vetsuuropname. Vorige studies toon aan dat verhoogde fluks deur die heksosamien biosintestiese weg (HBW) onder hiperglukemiese toestande bydra tot die ontwikkeling van insulienweerstand. In die lig hiervan, word daar gehipotetiseer dat verhoogde HBP fluks kardiale ACCb geenuitdrukking induseer en so bydra tot die ontstaan van insulienweerstand. Ons hipotese is getoets deur die kardiale afkomstige rot H9c2 mioblaste met ‘n 1.317 bp mens ACCb-lusiferase promotor konstruk (pPII-1317) te transfekteer en ‘n uitdrukking te konstrueer wat die tempo bepalende stap van HBP i.e. glutamien: fruktose-6-fosfaat amidotransferase (GFAT) kodeer. Ooruitdrukking van GFAT verhoog ACCb geenpromotor aktiviteit deur 75 ± 23% teenoor kontrole (n=6, p<0.001). Die herhaling van ko-transfeksie eksperimente is herhaal in die teenwoordigheid van variëerbare L-glutamienkonsentrasies (0 mM, 4 mM, 8 mM), ’n substraat vir die HBP, ACCb promotor aktiwiteit is dosisafhanglik verhoog. Om die bevindinge verder te staaf, is twee inhibeerders van GFAT, i.e. 40 μM azaserien en 40 μM 6-diazo-5-oxo-L-norleusien aan transfeksie selle toegedien vir ’n tydperk van 24 uur. Beide azaserien en 6-diazo-5-oxo-L-norleusien verlaag ACCb geenpromotor aktiwiteit. In ooreenstemming met die bogenoemde het ko-transfeksies met twee dominante negatiewe GFAT konstrukte ook ACCb geenpromoter aktiwiteit verminder. Die volgende stap is om twee ensieme van die HBP wat stroomaf van GFAT aktief is, vir ‘n periode van 24 uur te inhibeer i.e. O-GlcNAc transferase en O-GlcNAcase deur alloxan (0.1 mM, 1 mM en 2 mM) and streptozotosien (5 mM en 10 mM) onderskeidelik vir ‘n 24 uur periode te gebruik. Toevoeging van alloxan het die ACCb geenpromotor aktiwiteit by 35.6 ± 1.9% (n=16, p<0.001) verlaag en streptozotosien aktiwiteit verhoog by 32 ± 12% (n=12, p<0.001). Ons het ook die USF1 en USF2 as transkripsie regulerings kandidate vir HBP-geïnduseerde ACCβ promotor regulering ondersoek. Ons data impliseer dat USF2 as ‘n belangrike transkripsie reguleerder van HBP-geïndiseerde ACCβ promotor regulering is. Samevattend het hierdie studie demonstreer dat verhoogde fluks deur die hexosamien biosintetiese weg ACCb geenpromotor aktiwiteit induseer. Ons stel verder voor dat hierdie induksie die kardiale vetsuuroksidasie verlaag wat daartoe lei dat intrasellulêre lipied akkumulasie as gevolg van onparing tussen sarkolemma vetsuuropname en mitochondriale vetsuuroksidasie in ’n insulien weerstandige situasie (i.e. hiperlipidaemia).

La graine de soja contient de grandes quantités d'isoflavones (génistéine, daidzéine et glycitéine). En raison de leurs propriétés phytoestrogéniques, ces composés peuvent avoir des effets bénéfiques sur la santé humaine, mais ils peuvent aussi être perçus comme perturbateurs endocriniens, en particulier dans les laits pour nourrissons. La teneur et la composition en isoflavones de la graine diffèrent selon la fraction considérée. Les cotylédons contiennent de la génistéine et de la daidzéine, tandis que les germes, avec une teneur quatre à dix fois supérieure, contiennent majoritairement de la daidzéine et de la glycitéine. Le génotype influence fortement la teneur en isoflavones totales. Le déterminisme du pourcentage des isoflavones est essentiellement génétique. Ce travail porte sur l'étude du déterminisme génétique à l'origine des variations de teneurs et de compositions en isoflavones dans les germes et les cotylédons de la graine, en tenant compte également du net décalage de l'accumulation entre ces deux compartiments, au cours du développement de la graine. Dans un premier temps, les gènes des isoflavone synthases (IFS) de variétés très différenciées pour leurs teneurs et profils d'isoflavones ont été séquencés, puis les expressions des gènes clefs de la biosynthèse (neuf chalcone synthases (CHS), une chalcone réductase (CHR), quatre chalcone isomérases (CHI) et les deux isoflavone synthases (IFS) ont été suivies par RT-PCR quantitative dans les cotylédons et dans les germes, à trois stades critiques du développement de la graine (25, 40 et 60 jours après floraison). La seconde partie de ce travail a été consacrée à l'étude de l'expression de différents gènes candidats de la flavonoïde 6-hydroxylase (F6H) catalysant la première étape de la synthèse de la glycitéine. Le polymorphisme des séquences génomiques IFS1 et IFS2 des isoflavone synthases n'a pas montré de lien avec les différences de teneurs en isoflavones entre les variétés. L'activité transcriptionnelle des gènes de biosynthèse des isoflavones souligne l'existence d'une régulation bien distincte de cette synthèse dans ces deux compartiments. Les taux d'expression des gènes cibles ne sont pas toujours reliés avec les différences de teneurs ou de profils dans les germes et les cotylédons, suggérant ainsi l'effet prépondérant des régulations post-traductionnelles, notamment dans la formation du complexe multienzymatique de biosynthèse de ces composés. Nous avons aussi mis en évidence une forte expression du gène CHS9 codant pour la chalcone synthase 9, avec un profil correspondant plus à l'accumulation des isoflavones dans le germe que dans les cotylédons. Les gènes CHS7 et CHS8 codant pour les chalcone synthases 7 et 8, déjà signalés comme fortement corrélés à la synthèse des isoflavones, sont plus liés à l'accumulation dans les cotylédons que dans les germes. Ces travaux montrent aussi que le gène F6H signalé dans la littérature ne s'exprime pas dans les germes. En revanche, deux candidats dont la séquence est similaire à 79% ont été étudiés. Le gène F6H3 est le seul à s'exprimer dans la graine, uniquement dans le germe. Son expression n'a pas été détectée dans les germes d'une lignée mutante qui ne produit pas de glycitéine. Ce gène est donc un candidat potentiel clef pour la synthèse de la glycitéine dans le germe. La structure particulière de l'enzyme correspondante pourrait indiquer une forte implication de l'architecture du complexe enzymatique et des contraintes qui en découlent dans l'utilisation préférentielle d'une voie ou d'une autre dans ce schéma de biosynthèse. / The soybean seed contains large amounts of isoflavones (genistein, daidzein, and glycitein). Owing to their phytoestrogenic properties, these compounds can have beneficial effects on human health, but they can also be considered as endocrine disruptors, for example in infant formulas. The isoflavone content and composition in the seed depend on the considered fraction. The cotyledons contain only genistein and daidzein, while the hypocotyls are four to ten times more concentrated and contain three isoflavones, mostly daidzein and glycitein. The genotype has a strong influence on total isoflavone content, and even more on the percentage of individual isoflavones in cotyledons and hypocotyls. The objective of this work is to investigate the genetic determinism that underlies such contrasted contents and compositions between the two seed fractions, and the relation between main biosynthetic steps and genotypic differences. First, the genes of isoflavone synthases (IFS) were sequenced in varieties with highly contrasted content and composition. The expression of different keys genes of the biosynthesis (nine chalcone synthases (CHS), a chalcone reductase (CHR), four chalcone isomerases (CHI) and the two isoflavone synthases (IFS) have then been followed by quantitative RT - PCR in the cotyledons and hypocotyls, at three critical stages of seed development (25, 40 and 60 days after flowering). Second, the expression of different candidate genes for the flavonoid 6-hydroxylase (F6H) which catalyzes the first step in the synthesis of the glycitein has been investigated. The polymorphism of the genomic sequences IFS1 and IFS2 of isoflavone synthases was not correlated with differences in isoflavone contents. The transcriptional activity of key genes of the biosynthesis of isoflavones pointed out the existence of a distinct regulation of isoflavone biosynthesis between the two seed fractions. The expression levels of target genes were not always related to differences in isoflavone content or compositions in the hypocotyls and cotyledons. This suggests the overriding effect of post-translational regulation, especially in the formation of multienzyme complex of biosynthesis of these compounds. The chalcone synthase gene CHS9 was highly expressed, with a profile similar to the accumulation of isoflavones in hypocotyls. The chalcone synthase genes CHS7 and CHS8 expressions, already reported as highly correlated to the biosynthesis of isoflavones were more related to accumulation in the cotyledons than in hypocotyls. This work has also shown that the F6H gene, reported in the literature was not expressed in the hypocotyls. However, two candidates with as highly similar coding sequence (79%) have been studied. The F6H3 gene is the only one expressed in the seed, more precisely in the hypocotyls but it was not expressed in the cotyledons. Moreover, it was not expressed in a mutant line which did not accumulate glycitein. This gene is therefore a key potential candidate for the synthesis of the glycitein in hypocotyls. The particular structure of the corresponding enzyme may indicate a strong involvement of the architecture of the multienzyme complex of isoflavones biosynthesis and the constraints arising in the preferential use of a track or another in this scheme of biosynthesis.

<p>The Mediator (Med) complex comprises about 30 subunits and is a transcriptional co-regulator in eukaryotic systems. The core Mediator complex, consisting of the head, middle and tail modules, functions as a bridge between transcription factors and basal transcription machinery, whereas the CDK8 kinase module can attenuate Mediator’s ability to function as either a co-activator or co-repressor. Many Arabidopsis Mediator subunit has been functionally characterized, which reveals critical roles of Mediator in many aspects of plant growth and development, responses to biotic and abiotic stimuli, and metabolic homeostasis. Traditional genetic and biochemical approaches laid the foundation for our understanding of Mediator function, but recent transcriptomic and metabolomic studies have provided deeper insights into how specific subunits cooperate in the regulation of plant metabolism. In Chapter 1, we highlight recent developments in the investigation of Mediator and plant metabolism, with emphasis on the large-scale biology studies of <i>med</i> mutants.</p> <p>We previously found that MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi-dominant mutation (<i>reduced epidermal fluorescence 4-3</i>, <i>ref4-3</i>) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to <a>decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism</a>. In contrast, loss of MED5a and MED5b (<i>med5</i>) results in increased levels of phenylpropanoids. In Chapter 2, we present our finding that <i>ref4-3</i> requires CDK8, a Mediator kinase module subunit, to repress plant growth even though the repression of phenylpropanoid metabolism in <i>ref4-3 </i>is CDK8-independent. Transcriptome profiling revealed that salicylic acid (SA) biosynthesis genes are up-regulated in a CDK8-dependent manner in <i>ref4-3,</i> resulting in hyper-accumulation of SA and up-regulation of SA response genes. Both growth repression and hyper-accumulation of SA in <i>ref4-3</i> require CDK8 with intact kinase activity, but these SA phenotypes are not connected with dwarfing. In contrast, mRNA-sequencing (RNA-seq) analysis revealed the up-regulation of a DNA J protein-encoding gene in <i>ref4-3</i>, the elimination of which partially suppresses dwarfing. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.</p> <p>In Chapter 3, we characterize other phenotypes of <i>med5</i> and <i>ref4-3</i>, and find that in addition to the up-regulated phenylpropanoid metabolism, <i>med5</i> show other interesting phenotypes including hypocotyl and petiole elongation as well as accelerated flowering, all of which are known collectively as the shade avoidance syndrome (SAS), suggesting that MED5 antagonize shade avoidance in wild-type plants. In contrast, the constitutive <i>ref4-3 </i>mutant protein inhibits the process, and the stunted growth of <i>ref4-3 </i>mutants is substantially alleviated by the light treatment that triggers SAS. Moreover, <i>ref4-3</i> mimics the loss-of-function <i>med5</i> mutants in maintaining abscisic acid (ABA) levels under both normal and drought growth conditions. The phenotypic characterization of <i>med5</i> mutants extend our understanding of the role of Mediator in SAS and ABA signaling, providing further insight into the physiological and metabolic responses that require MED5.</p> <p>In Chapter 4, we explore the function of MED5 and CDK8 in gene expression regulation by investigating the effect of mutations in Mediator including <i>med5</i>, <i>ref4-3</i>, <i>cdk8-1</i> and <i>ref4-3 cdk8-1</i> on genome-wide Pol II distribution. We find that loss of MED5 results in loss of Pol II occupancy at many target genes. In contrast, many genes show enriched Pol II levels in <i>ref4-3</i>, some of which overlap with those showing reduced Pol II occupancy in <i>med5</i>. In addition, Pol II occupancy is significantly reduced when CDK8 is disrupted in <i>ref4-3</i>. Our results help to narrow down the direct gene targets of MED5 and identify genes that may be closely related to the growth deficiency observed in <i>ref4-3</i> plants, providing a critical foundation to elucidate the molecular function of Mediator in transcription regulation.</p>