Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour which secretes ACTH and other related peptides. Contrary to normal production by the pituitary, ACTH production is not inhibited by glucocorticoids (Gcs) in SCLC. This insensitivity to Gc action can be attributed to impaired Gc receptor (GR) expression in these cells. Over-expression of the GR induces apoptosis both in vitro and in vivo. Evasion of GR signalling thus confers a significant survival advantage to SCLC cells. Re-expression of endogenous GR in SCLC cells may provoke the same effect. Many tumours silence the expression of tumour suppresser genes by epigenetic mechanisms. Recent evidence suggests that the GR in SCLC cells is epigenetically silenced by hypermethylation of its promoter. The overall aim of this study was to determine whether endogenous GR re-expression induces apoptosis of SCLC cells. The DMS 79 SCLC cell line, and the control HEK and non-SCLC A549 cell lines were treated with the DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (5-aza), to determine whether treatment with 5-aza results in re-expression of endogenous GR. Conflicting results were thought to result from the use of possibly degraded 5-aza. However, a quantitative real-time PCR analysis using newly purchased, freshly prepared 5-aza indicated that 5-aza treatment up-regulated GR mRNA expression in the DMS 79 cells (p<0.0005). No significant changes in GR expression were seen in the HEK and/or A549 cells, suggesting that the GR in these cell lines is not methylated. Contrary to expectations and possibly due to the use of degraded stock, Western blot analysis revealed that 5-aza had no effect on GR protein expression in DMS 79 cells, yet affected GR protein expression in HEK and A549 cells (p=0.003 and p=0.042, respectively). Cell viability assays indicated that treatment with varying concentrations of 5-aza had no effect on the viability of DMS 79 and A549 cells, but had a minimal effect on HEK cell (p<0.0005) viability. These data reinforce the hypothesis that stock 5-aza had degraded as 5-aza is known to exert cytotoxic effects at higher concentrations. Using newly purchased, freshly prepared 5-aza, flow cytometry and/or microscopy were performed to establish whether endogenous GR re-expression was sufficient to kill the SCLC cells by apoptosis. FITC Annexin V staining and nuclear morphology showed that significant proportions of the 1 μM (p=0.010 and p=0.027) and 5 μM (p=0.002 and p=0.018) 5-aza treated DMS 79 cells were apoptosing, with little apoptosis seen in HEK cells. 5-Aza induced negligible HEK cell death, as determined by microscopic analyses. The effect of dexamethasone (Dex; a synthetic Gc) on HEK and DMS 79 cells was examined to determine whether Gc treatment could enhance apoptosis. Treatment with Dex alone, and in combination with 5-aza, resulted in significant HEK cell death (p=0.046 and p=0.005 respectively), but not apoptosis. This was unexpected as HEK cells express very little unmethylated GR, and may be due to excessive drug exposure or combined drug toxicity. The same effect was observed with DMS 79 cells (p=0.003 and p<0.0005 respectively), with 5-aza appearing to enhance cell death induced by Dex. No effects on apoptosis were seen confirming earlier reports that GR-mediated apoptosis is ligand-independent. As 5-aza does not selectively demethylate the GR, cells were exposed to the GR antagonist, RU486, to establish whether apoptosis associated with 5-aza treatment is specifically due to demethylation and subsequent expression of the GR. Treatment with RU486 in conjunction with 5-aza induced cell death (p=0.014), but not apoptosis, of HEK cells. Again, this may have been due to excessive drug exposure or combined drug toxicity. Flow cytometric data showed that DMS 79 cell death was induced by both RU486 (p=0.004), and RU486 in combination with 5-aza (p=0.003). Furthermore, although not significant, RU486 treatment appeared to inhibit apoptosis induced by 5-aza in the DMS 79 cells. The data suggest that re-expression of the GR may be responsible for apoptotic induction. Our findings, although not significant, hint that endogenous re-expression of the GR leads to apoptosis. Unlike mutations, epigenetic marks are reversible and clinical trials with DNMTis have shown promising results. The identification of a novel endogenous mechanism that specifically induces apoptosis of SCLC cells offers great promise for the development of targeted therapeutics for the treatment of this deadly disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

The indiscriminate use of fossil fuels has led to global problems of greenhouse gas emissions, environmental degradation and energy security. Developments of alternative and sustainable energy resources have assumed paramount importance over the past decades to curb these challenges. Biohydrogen is emerging as an alternative renewable source of energy and has received considerable attention in recent years due to its social, economic and environmental benefits. It can be generated by dark fermentation on Organic Fraction of Solid Municipal Waste (OFSMW). These OFSMW exist abundantly and poses disposal challenges. This study models and optimizes the production of biohydrogen on a mixture of agro-municipal wastes; it examines a semi-pilot scale production on these substrates and the feasibility of generating bioelectricity from the process effluents and reviews the prospect of enhancing fermentative biohydrogen development using miniaturized parallel bioreactors. The fermentation process of biohydrogen production on agro-municipal wastes was modelled and optimized using a two-stage design. A mixture design was used for determination of optimum proportions of co-substrates of Bean Husk (BH), Corn Stalk (CS) and OFSMW for biohydrogen production. The effects of operational setpoint parameters of substrate concentration, pH, temperature and Hydraulic Retention Time (HRT) on hydrogen response using the mixed substrates were modelled and optimized using box-behnken design. The optimized mixtures were in the ratio of OFSMW: BH: CS = 30:0:0 and OFSMW: BH: CS = 15:15:0 with yields of 56.47 ml H2/g TVS and 41.16 ml H2/g TVS respectively. Optimization on physico-chemical parameters using the improved substrate suggested optimal setpoints of 40.45 g/l, 7.9, 30.29 oC and 86.28 h for substrate concentration, pH, temperature and HRT respectively and hydrogen yield of 57.73 ml H2/g TVS. The quadratic polynomial models from the mixture and box-behnken design had a coefficient of determination (R2) of 0.94 and 0.79 respectively, suggesting that the models were adequate to navigate the optimization space. The feasibility of a large-scale biohydrogen fermentation process was studied using the optimized operational setpoints. A semi-pilot scale biohydrogen fermentation process was carried out in 10 L bioreactor and the potential of generating bioelectricity from the process effluents was further assessed using a two-chambered Microbial Fuel Cell (MFC) process. The maximum hydrogen fraction of 46.7% and hydrogen yield of 246.93 ml H2/g TVS were obtained from the semi-pilot process. The maximum electrical power and current densities of 0.21 W/m2 and 0.74 A/m2 respectively were recorded at 500 Ω and the chemical oxygen demand (COD) removal efficiency of 50.1% was achieved from the MFC process. This study has highlighted the feasibility of applying agricultural and municipal wastes for large-scale microbial production of hydrogen, with a simultaneous generation of bioelectricity from the process effluents. Furthermore, the potential of generating an economical feasible biohydrogen production process from these waste materials was demonstrated in this work. Keywords: Biohydrogen production, Organic Fraction of Solid Municipal Waste (OFSMW), Modelling and optimization, Fermentation process, Renewable energy, Bioenergy / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Aeromonas spp. are important biofilm-forming fish pathogens causing great economic loss in aquaculture. Bacterial cells within biofilms communicate with each other via the production of quorum sensing (QS) signalling molecules called acyl-homoserine lactones (AHLs), which influence biofilm development and production of virulence factors. QS together with efflux pumps, extracellular polymeric substances (EPS) and eDNA are associated with resistance of bacteria to antimicrobial agents. These mechanisms provide a target for different control strategies. The objectives of this study were to: (i) determine effective antimicrobial agents and exposure concentrations against aeromonad biofilms; (ii) ascertain whether Aeromonas spp. produce QS molecules or display efflux pump phenotypes, and (iii) investigate the effect of antimicrobial agents, lytic enzymes, efflux pump inhibitors and QS inhibitors on biofilm formation by Aeromonas spp. isolates.signalling MICs of azithromycin, ciprofloxacin, ceftazidime, and tetracycline ranged between 0.064-64 μg/ml. Gentamicin had the lowest MICs which ranged between 0.0048-32 μg/ml.The highest MBIC at which antimicrobial agents exhibited inhibition was 4096 μg/ml. Majority of the isolates displayed MIC levels ranging from 2-32 μg/ml, and thus a ≥ 128-fold increase was observed for MBICs. Of the sub-MIC, MIC and supra-MIC exposures tested, MIC exposure of biofilms was the most effective. Gentamicin MIC exposures inhibited initial attachment of 100% (28/28) of isolates tested, while azithromycin MIC exposure detached 82.1% (23/28) of isolates. Carbonyl cyanide 3-chlorophenylhydrazone completely inhibited efflux of cefpodoximeby 14.8% of isolates. However, 1-(1-naphthylmethyl)-piperazinewas more effective, decreasing adherence of 98.1% (53/54) of isolates and increasing detachment of 100% (54/54) of isolates. DNase I was more effective against the mature biofilm,where it increased biofilm detachment of 64.8% of isolates. Of the 48 Aeromonas spp. and six Plesiomonas spp. isolates used, only a single isolate induced the production of violacein by the C. violaceum CV026 biosensor, while all isolates induced the utilization of X-gal to produce a visible blue colour with the A.tumefaciens A136 biosensor. Based on the reaction to the two biosensors, aeromonads appeared to produce long-chain acylhomoserine lactones. By blocking QS, S-adenosyl homoserinewas more effective in inhibiting both initial attachment (72.2% of isolates) and pre-formed biofilms (detached 74.1% of isolates). The investigated strategies are promising for Aeromonas spp. biofilm inhibition. Thesecould be explored aspotential therapeutic measures in aquaculture systems to limit aeromonad pathogenicity and overcome antimicrobial resistance. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1975.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Complete degradation of cellulose produces mainly glucose, which can be fermented to ethanol. Therefore cellulose presents an abundant renewable energy resource for the production of an alternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanases and -glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol. Therefore, a need has arisen to develop a recombinant microorganism with the ability to produce cellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single process step, referred to as “Consolidated Bioprocessing” (CBP). This would provide a cost-effective, economically feasible strategy for the production of bioethanol. The naturally fermentative yeast, Saccharomyces cerevisiae, is often used as host for the expression of recombinant proteins due to several characteristics, including its robustness in industrial processes, the well developed genetic tools available for manipulation and its proven safety status. A number of cellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. In this study, all three components of the cellulase system were co-expressed in S. cerevisiae to test the ability of the yeast to effectively produce the heterologous proteins, and consequently produce enough glucose for growth on an amorphous cellulosic substrate. The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by a S. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1 towards CMC and barley -glucan, respectively. The major endoglucanase gene, egI (Cel7B) from T. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. coli mismatch repair deficient strain. Screening of S. cerevisiae transformants revealed a strain, S. cerevisiae Y294[pLEM1], with improved levels of endoglucanase activity (21.8 nkat.ml-1), compared to S. cerevisiae Y294[pAZ40], expressing the wild type gene (10.3 nkat.ml-1). Through subcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it was established that the mutations located in both the promoter- and gene sequences were responsible for the improved levels of activity displayed by S. cerevisiae Y294[pLEM1]. The egII gene and the altered egI gene were co-expressed with a codon optimised T. reesei cellobiohydrolase (sCBHI) and a -glucosidase from Saccharomycopsis fibuligera. This resulted in a reduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast by co-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressing strains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enable growth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likely that a bottleneck exists at its conversion to glucose, suggesting inadequate -glucosidase activity. This study has provided insight into co-expression of cellulase enzymes by the yeast S. cerevisiae. The knowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulose degradation and eventual production of ethanol by recombinant yeast. It has also demonstrated the applicability of random mutagenesis for improving the activity of cellulases.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2005. / Plasmid pTC-F14 is a 14.2kb promiscuous, broad-host range IncQ-like mobilizable plasmid isolated from Acidithiobacillus caldus f. At. caldus is a member of a consortium of bacteria (along with Acidithiobacillus ferrooxidans and Leptospirilum ferrooxidans) that is used industrially for decomposing metal sulphide ores and concentrates at temperatures of 40ºC or below which is now a well-established industrial process to recover metals from certain copper, uranium and gold-bearing minerals or mineral concentrates. These biomining microbes are usually obligately acidophilic, autotrophic, usually aerobic iron- or sulphur-oxidizing chemolithotrophic bacteria. Their remarkable physiology allows them to inhabit an ecological niche that is largely inorganic and differs from those environments populated by the more commonly studied non-acidophilic heterotrophic bacteria. At. caldus, is a moderately thermophilic (45 to 50ºC), highly acidophilic (pH1.5 to 2.5) sulphur-oxidizing bacterium, and its role as one of the major players in the industrial decomposition of metal sulphide ores has become evident in recent years. At. caldus f from which pTC-F14 was isolated was found to be one of two dominant organisms in a bacterial consortium undergoing pilot-scale testing for the commercial extraction of nickel from ores.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalis FAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivo and in vitro study. During initial tests peptide AS-48 showed no significant activity towards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity was obtained only after purification with Triton X-114 phase partitioning, followed by cation exchange chromatography. Titers for the purified peptide varied between 3200 and 12800 AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae and Streptococcus dysgalactiae, but not against Escherichia coli. The size of peptide AS-48 was determined at 7150 Da, based on electronspray mass spectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained by adding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the lag growth phase. When the same concentration of peptide AS-48 was added to a culture of S. aureus in mid-exponential growth, a slight decrease in viable cell numbers was recorded, which lasted for only 30 min. Cell growth commenced thereafter. In situ experiments in cows were done with purified peptide AS-48, encapsulated in liposomes. These in vivo studies were conducted by administering peptide AS-48 (6400 AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreated with peptide AS-48, a reduction close to 90% in the viable cell numbers of S. aureus was recorded relative to the control quarters, which were not treated with the peptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatment trial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viable cell numbers of S. aureus was recorded. In the same quarters, a reduction in SCC amounted to almost 80%. A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptide AS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which produces enterocins 1071A and 1071B. Southern blot hybridization experiments revealed thepresence of plasmid p92 in the recipient strain without the loss of plasmid pEF1071, which encodes enterocins 1071A and 1071B. All three antimicrobial peptides, i.e. enterocin 1071A, enterocin 1071B and peptide AS-48, were produced in transconjugant FA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greater than that recorded for strains FA2/Ent and FAIRE 92, respectively and included E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus and S. aureus. These organisms are not inhibited by strain FA2/Ent. However, low levels of peptide AS-48 was produced by strain FA2/Ent/AS-48. Further research in fermentation and gene expression will be needed before the transconjugant E. faecalis FA2/Ent/AS-48 may be used in the treatment of mastitis. / AFRIKAANSE OPSOMMING: Die effek van die bakteriosien-agtige, peptied AS-48, geproduseer deur Enterococcus faecalis FAIRE 92, is gedurende ‘n in vivo en in vitro studie teen ‘n mastitiese Staphylococcus aureus-isolaat getoets. Aanvanklike toetse met peptied AS-48, selfs tienvoudig gekonsentreerde selvrye supernatant, het geen beduidende aktiwiteit teen S. aureus getoon nie. Aktiwiteit is eers verkry na suiwering met Triton X-114 fase-skeiding gevolg deur katioon uitruilingschromatografie. Titers vir die gesuiwerde peptied het tussen 3200 en 12800 AE/ml gewissel. Die gesuiwerde peptied het ook aktiwiteit teen Streptococcus agalactiae en Streptococcus dysgalctiae getoon, maar nie teen Escherichia coli nie. Peptied AS-48 het ‘n molekulêre massa van 7150 Da, soos bepaal met elektronsproeimassa spektrometrie en SDS-PAGE. Totale inhibisie van selgroei is verkry deur 1 ml gesuiwerde peptied AS-48 (3200 AE/ml) by ‘n 100 ml kultuur van S. aureus in die sloerfase te voeg. Dieselfe konsentrasie peptied AS-48, toegevoeg tydens die mideksponensiële groeifase, het egter slegs ‘n klein vermindering in die aantal lewende selle teweeg gebring en het ook vir slegs ‘n 30 min geduur. Selgroei het hierna weer normaal voort gegaan. In situ eksperimente op koeie is uitgevoer met gesuiwerde peptied AS-48, geenkapsuleerd in liposome. Hierdie In vivo studies is onderneem deur peptied AS-48 (6400 AE/ml) in verskillende kwarte van die uier, kunsmatig of reeds geïnfekteerd met S. aureus, toe te dien. In ‘n voorkomings-eksperiment waar kwarte vooraf met peptied AS- 48 behandel is, is ‘n verlaging van byna 90% in die lewende seltelling van S. aureus relatief tot die kontrole kwarte, sonder behandeling met peptied AS-48, verkry. ‘n 50% verlaging in die somatiese seltelling (SST) is verkry. In die behandelings-eksperiment, waar geïnfekteerde kwarte met peptied AS-48 behandel is, is ‘n verlaging van byna 90% in lewende S. aureus selle gevind. In dieselfde kwarte is ‘n verlaging van byna 80% in die SST genoteer.‘n Rekombinante ras is gekonstrueer deur plasmied 92 (p92), wat kodeer vir peptied AS- 48, vanaf Enterococcus faecalis FAIRE 92 na E. faecalis FA2/Ent, wat enterosien 1071A en 1071B produseer, te konjugeer. Southern-klad hibridisasie het die teenwoordigheid van plasmied p92 in die ontvanger ras, sonder die verlies van plasmied pEF1071 wat enterosien 1071A en 1071B kodeer, getoon. Al drie antimikrobiese peptiede, nl. enterosien 1071A, enterosien 1071B en peptied AS-48, is deur die transkonjugant FA2/Ent/AS-48 geproduseer. Die spektum van antimikrobiese aktiwiteit van die transkonjugant vand die transkonjugant is breër as dié van rasse FA2/Ent en FAIRE 92, onderskeidelik en het ook E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus en S. aureus ingesluit. Hierdie organismes word nie deur ras FA2/Ent geïnhibeer nie. Lae vlakke van peptied AS-48 is egter deur ras FA2/Ent/AS-48 geproduseer. Verdere navorsing in fermentasie en geenuitdrukking is nodig voordat E. faecalis FA2/Ent/AS-48 in die behandeling van mastitis gebruik kan word.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the brewing industry yeast cells are re-used in successive fermentations. Consequently, the state of the cells at the end of each successive fermentation could impact on the quality of the subsequent fermentations. The use of markers to evaluate the fermentative ability of yeast to resist stress enables brewers to select populations of yeast for brewing. Yeasts are typically exposed to osmotic-, ethanol- and cold-stress during the high-gravity brewing process. In this study the vitality of the yeast cells was monitored during and after each successive high-gravity brewing fermentation. This was done by measuring the cell metabolites, which included glycerol, trehalose and glycogen. Others markers that were evaluated for yeast viability were the number of budding scars, the levels of activity of the enzymes neutral trehalase and esterase and the expression level of the heat shock protein Hsp12p. Coupled to these evaluations, the growth of the yeast and the utilisation of the sugars glucose, fructose, maltose and maltotriose were monitored during the fermentations. The experiments were conducted in 2-litre E.B.C. tubes at either 14 oC or at 18oC using standard techniques. Comparable growth patterns were obtained for different re-pitching fermentations, with fermentation 1 at 18ºC and 5 and 6 at 14°C being the most active fermentations. The higher temperature encouraged more rapid growth and a greater numbers of cells. The wort attenuation was more rapid at 18°C than at 14°C. Glucose and fructose in wort were utilised prior to maltose and maltotriose. At 18°C the yeast consumed the sugars faster, with mean utilisation values of 97.3% glucose, 100% fructose, 59.9% maltose and 65.6% maltotriose. At the lower temperature of 14°C high concentrations of residual sugars remained at the end of the fermentation. All re-pitching fermentations revealed lower viabilities at 18°C in comparison to the 14°C fermentations. Simultaneously, a number of other markers were evaluated. The intracellular trehalose concentration per cell varied considerably with each fermentation. Trehalose levels at 18°C gradually increased in concentration from 48h until the end of the stationary phase. Much lower trehalose concentrations were observed in fermentations conducted at 14°C. Higher and more consistent glycerol concentrations were found in fermentations at 14°C with mean concentrations of 12 mg/g dry weight at pitching. The expression of the heat shock protein Hsp12p level increased during the fermentation but no sharp increase was detected in any particular fermentation. No increase in yeast budding scar number was observed during re-pitching fermentations. Neutral trehalase and esterase activities in fermentations at 18°C were especially high at pitching. Neutral trehalase activities at 14°C were all generally lower than in the case of fermentations at 18°C. The fermentation ability of flocculated yeast in slurry and yeast suspended in beer was investigated after exposure to various stresses. The aged yeast present in the slurry was generally found to be more resistant to stress, in particularly to osmotic stress, throughout the serial re-pitching process. The fermentation rates of both yeast types were especially sensitive to prior exposure to ethanol stress. / AFRIKAANSE OPSOMMING: In die broubedryf word gisselle herhaaldelik gebruik vir agtereenvolgende fermentasies. Derhalwe kan die toestand van die gisselle teen die einde van elke agtereenvolgende fermentasie ‘n invloed hê op die kwaliteit van die daaropvolgende fermentasies. Deur gebruik te maak van merkers om die fermentasievermoë van gis om stres te weerstaan te evalueer, stel dit bierbrouers in staat om gispopulasies te selekteer. Gedurende die hoëdigtheid brouproses word giste tipies aan osmotiese-, etanol- en koue-stres blootgestel. In hierdie studie, gedurende hoë-digtheid fermentasies, is die lewensvatbaarheid van die gisselle gedurende en na elke agtereenvolgende fermentasie gemonitor deur die volgende selmetaboliete te bepaal: gliserol, trehalose en glikogeen. Bykomende merkers vir gis lewensvatbaarheidsbepalings was: die aantal botselletsels, die vlakke van aktiwiteit van die neutrale trehalose en esterase ensieme, en die uitdrukkingsvlak van die hitteskokprotein Hsp12p. As aanvullende evaluasies is die groei van die gis en die gebruik van die suikers glukose, fruktose, maltose en maltotriose gedurende fermentasies gemonitor:. Die proewe is in 2-liter E.B.C. buise uitgevoer, by ‘n temperatuur van 14oC of 18oC, deur van standaard tegnieke gebruik te maak. Die groeipatrone van die verskillende herhaaldelike-inokulasie gistings was ongeveer dieselfde. Fermentasie 1 by 18ºC en fermentasies 5 en 6 by 14°C was die mees aktiewe fermentasies. Die hoër temperatuur het vinniger groei en ‘n groter aantal selle begunstig. Die wortattenuasie was vinniger by 18°C as by 14°C. Glukose en fruktose in mout is voor die maltose and maltotriose opgebruik. By 18°C het die gis die suikers vinniger opgebruik. Gemiddelde gebruikswaardes vir die sewe reeksgewyse fermentasies was die volgende: 97.3% glukose, 100% fruktose, 59.9% maltose en 65.6% maltotriose. Teen die einde van fermentasie by 14°C was daar hoë konsentrasies van die oorblywende suikers, hoofsaaklik na fermentasie 1. Alle herhaaldelike inokulasie fermentasies het lae lewensvatbaarheid by 18°C in vergelyking met 14°C fermentasies getoon. Ander merkers is ook gelyktydig gebruik. In die verskillende fermentasies was daar ‘n groot verskil in die intrasellulêre trehalose konsentrasie per sel. Trehalose konsentrasies by 18°C het geleidelik toegeneem, vanaf 48 uur tot aan die einde van die stationêre fase. Baie laer trehalose konsentrasies is gemeet vir fermentasies by 14°C. In fermentasies by 14°C was die gliserolkonsentrasies hoër en meer konstant. Gemiddelde konsentrasies was 12mg/g 14°droë gewig by inokulasie. Die uitdrukking van die hitteskokproteien Hsp12p vlak het gedurende fermentasie toegeneem, maar daar was geen skerp toename vir die afsonderlike fermentasies nie. Die bepaling van die aantal botselletsels per sel het daarop gewys dat die gemiddelde aantal nie toegeneem het met die veroudering van die gis gedurende reeksgewyse herhaaldelike inokulasie nie. Neutrale trehalase aktiwiteite in fermentasies by 18°C was besonders hoog, veral by inokulasie. Die neutrale trehalase aktiwiteite in die fermentasies by 14°C was in die algemeen laer as die by 18°C. Die fermentasievermoë van die geflokkuleerde gis in die sediment en gesuspendeerde gis in die bier is ondersoek na blootstelling aan verskeie tipes stres. Die verouderde gis teenwoordig in die sediment was in die algemeen meer bestand teen stres, veral aan osmotiese stres, dwarsdeur die reeksgewyse herhaaldelike inokulasie proses. Etanolstres het die gistingstempo van beide giste dieselfde geaffekteer.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Mountain Fynbos biome, a division of the Cape Floristic Region (CFR), is home to round-leafed Buchu [Agathosma betulina (Berg.) Pillans], one of South Africa’s best-known endangered herbal medicinal plants. Agathosma betulina is renowned as a traditional additive to brandy or tea, which is used for the treatment of a myriad of ailments. In its natural habitat, A. betulina thrives on mountain slopes in acid and highly leached gravelly soils, with a low base saturation and low concentrations of organic matter. To adapt to such adverse conditions, these plants have formed mutualistic symbioses with arbuscular mycorrhizal (AM) fungi. In this study, the effect of indigenous AM taxa on the physiology of A. betulina is investigated. In addition, the AM taxa responsible for these physiological responses in the plant were identified using morphological and molecular techniques. Agathosma betulina was grown under glasshouse conditions in its native rhizosphere soil containing a mixed population of AM fungi. Control plants, grown in the absence of AM fungi, were included in the experimentation. In a time-course study, relative growth rate (RGR), phosphorus (P)-uptake, P utilization cost, and carbon (C)-economy of the AM symbiosis were calculated. The data showed that the initial stages of growth were characterized by a progressive increase in AM colonization. This resulted in an enhanced P-uptake in relation to non-AM plants once the symbiosis was established. Consequently, the lower P utilization cost in AM plants indicated that these plants were more efficient in acquiring P than non-AM plants. When colonization levels peaked, AM plants had consistently higher growth respiration. This indicated that the symbiosis was resulting in a C-cost to the host plant, characterized by a lower RGR in AM plants compared to non-AM plants. Arbuscular mycorrhizal colonization decreased with increasing plant age that coincided with a decline in P-uptake and growth respiration, along with increases in RGR to a level equal to non-AM plants. Consequently, the AM benefit was only observed during the initial stages of growth. In order to identify the AM fungi in planta, morphological and molecular techniques were employed, which indicated colonization by AM fungi belonging to the genera Acaulospora and Glomus. Phylogenetic analyses of a dataset containing aligned 5.8S ribosomal RNA gene sequences from all families within the Glomeromycota, including sequences obtained during the study, supported the above mentioned identification. / AFRIKAANSE OPSOMMING: Die Fynbos bergbioom, ‘n onderafdeling van die Kaapse Floristiese Streek, huisves rondeblaar Boegoe [Agathosma betulina (Berg.) Pillans], een van Suid Afrika se bekendste bedreigde medisinale plante. Agathosma betulina is bekend vir sy gebruik as tinktuur vir die behandeling van verskeie kwale. Die plant kom voor in bergagtige streke, in suur en mineraal-arm grond, met ‘n lae organiese inhoud. Gevolglik, om aan te pas by hierdie ongunstige kondisies, vorm die plante simbiotiese assosiasies met blaasagtige, struikvormige mikorrisa (BSM). In die huidige studie is die effek van hierdie BSM op die fisiologie van A. betulina ondersoek. Die identiteit van die BSM is ook gevolglik met morfologiese en molekulêre identifikasie tegnieke bepaal. Agathosma betulina plante is onder glashuis kondisies in hul natuurlike grond gekweek, wat ‘n natuurlike populasie van BSM bevat het. Kontroles is ook in die eksperiment ingesluit en hierdie stel plante is met geen BSM geïnokuleer nie. Gevolglik is die relatiewe groeitempo, fosfor opname, fosfor verbuikerskoste asook die koolstof ekonomie van die plante bereken. Die data het getoon dat die eerste groeifase gekarakteriseer is deur toenames in BSM kolonisasie vlakke. Dit het tot ‘n hoër fosfor opname in BSM geïnokuleerde plante gelei. Die laer fosfor verbuikerskoste gedurende hierdie fase het aangedui dat die plante wat geïnokuleer is met BSM oor beter meganismes beskik het om fosfor uit die grond te bekom. Toe BSM kolonisasie vlakke gepiek het, was groei respirasie hoër in BSM geïnokuleerde plante as in die kontroles. Dit het aangedui dat die BSM kolonisasie van plante tot hoër koolstof kostes vir hierdie plante gelei het, wat weerspieël is in die laer groeitempo van die BSM geïnokuleerde plante. Die BSM kolonisasie vlakke het gedaal met toenemende ouderdom van hul gasheer plante, wat gekarakteriseer is deur ‘n laer opname van fosfor en laer groei respirasie, tesame met ‘n toename in relatiewe groeitempo tot vlakke soortgelyk aan die van die kontrole plante. Die BSM voordele vir die plant is dus net gedurende die eerste groeifase waargeneem. Die BSM wat verantwoordelik is vir hierdie fisiologiese veranderinge is gevolglik geïdentifiseer met behulp van morfologiese en molekulêre tegnieke en dit is gevind dat BSM wat behoort tot die genera Acaulospora en Glomus binne hierdie plante voorkom. Filogenetiese analise gegrond op opgelynde 5.8S ribosomale RNA geen volgordes afkomstig van al die families binne Glomeromycota asook volgordes gevind in die studie, het die bogenoemde identifikasie gestaaf.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Thirty lactic acid bacteria were isolated from the intestinal tract of Drosophila simulans Stuvervant and nine lactic acid bacteria from Merlot grapes collected from the same winery in the Stellenbosch region, South Africa. The isolates were grouped according to morphological, biochemical and physiological characteristics. Isolates selected from each group were identified to species level by PCR with species-specific primers, PCR-based DGGE and 16S rDNA sequencing. The majority of isolates from the intestinal tract of Drosophila simulans Stuvervant belonged to the species Lactobacillus plantarum, but Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis and Pediococcus pentosaceus were also identified. As far as we could determine, this is the first report on the isolation of L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis and P. pentosaceus from vinegar flies. Lactobacillus plantarum has previously been isolated from Merlot grapes. The genotypic relatedness among isolates of L. plantarum isolated from the intestinal tract of vinegar flies and from Merlot grapes were determined by RAPD-PCR. The isolates were grouped into four genotypically well-separated clusters. Thirteen isolates from grape must and five from flies yielded identical RAPD-PCR banding patterns and grouped into one cluster, suggesting that they are descendants from the same strain. This suggests that L. plantarum has the ability to use vinegar flies as a vector. / AFRIKAANSE OPSOMMING: Dertig melksuurbakterieë is vanuit die dermkanaal van Drosophila simulans Stuvervant geïsoleer en nege melksuurbakterieë vanuit Merlot-druiwe. Die druiwe is afkomstig van dieselfde wynkelder in die Stellenbosch-area van Suid-Afrika. Die isolate is volgens morfologiese, biochemiese en fisiologiese eienskappe gegroepeer. Verteenwoordigende isolate vanuit die fenotipiese groepe is tot spesievlak met behulp van lukraak ge-amplifiseerde polimorfe-DNA (RAPD) polimerase ketting-reaksie (PKR), PKR met spesie-spesifieke inleiers, PKR-gebaseerde denaturerende gradient-jel elektroforese (DGGE) en 16S rDNA sekwensering geïdentifiseer. Die meerderheid isolate uit die ingewande van Drosophila simulans Stuvervant is as Lactobacillus plantarum geklassifiseer. Stamme van Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis en Pediococcus pentosaceus is ook geïdentifiseer. Sover bekend, is dit die eerste keer dat L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis en P. pentosaceus uit asynvlieë geïsoleer is. Lactobacillus plantarum is voorheen uit Merlot-druiwe geïsoleer. Die genotipiese ooreenkoms tussen die stamme van L. plantarum wat uit die asynvlieë en Merlot-druiwe geïsoleer is, is deur middel van RAPD-PKR bepaal. Hiervolgens is die stamme in vier genotipies goed-gedefinieerde groepe geplaas. Dertien isolate vanuit druiwemos en vyf vanuit asynvlieë het identiese RAPD-PKR bandpatrone vertoon en het in een groep gesorteer. Hierdie resultate dui daarop dat die stamme heel moontlik uit een voorouer ontstaan het en dat asynvlieë heel moontlik as vektor vir L. plantarum dien.