Quercetin-3-O-glucoside (Q3G), a glycosylated derivative of quercetin, is a polyphenolic compound known to possess diverse biological activities. Its moderately hydrophilic nature is a critical factor governing the accessibility to the active sites of oxidative damages in vivo. It was hypothesized that biological activities of Q3G can be further enhanced by regioselective acylation with fatty acids which gives more lipophilicity. Q3G was acylated with six selected long chain fatty acids: stearic acid, oleic acid, linoleic acid, ?-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), using Candida antactica lipase. The derivatives were evaluated for their potential in inhibiting lipid oxidation in food systems and human low density lipoprotein (LDL), and cytoprotection and anti-inflammatory effect in cell culture model systems. The fatty acid derivatives of Q3G possessed greater effectiveness in inhibiting lipid oxidation in oil-in-water emulsions, and better cytoprotective effect against H2O2- and cigarette smoke toxicant-induced cytotoxicity when compared to Q3G.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.

The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving . Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5.0% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1.0% activated charcoal, added before autoclaving . In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolysed The adsorption of 2, 4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal from methanol and aqueous solutions was determinated using HPLC. The amount of the added 2,4-D decreased in both methanol and aqueous solutions in the presence of activated charcoal, compared with those in the absence of activated charcoal. In methanol and aqueous solutions, activated charcoal used at the level of 0.1% significantly reduced 2,4-D. About 68.4% and 60.9% respectively of the added 2,4-D was adsorbed by activated charcoal (1.0%) from these solutions. The changes of inorganic elements in MS-salt solutions, in the presence of activated charcoal, were analysed by SEM-EDX. The concentrations of magnesium (Mg), calcium (Ca), iron (Fe) and zinc (Zn) deceased in the presence of activated charcoal, while the concentrations of potassium (K), copper (Cu), manganese (Mn), phosphorus (P), and sulphur (S) increased in the MS salt solution in the presence of activated charcoal compared with no activated charcoal in the medium. This suggests that activated charcoal adsorbed calcium, magnesium, iron and zinc and released copper, manganese, phosphorus and sulphur. Rooting occurred when 7-day-old seedling hypocotyls of Daucus carota L. Cape Market were placed on MS medium supplemented with 2,4-D, and IAN/NAA in the presence of activated charcoal. Hypocotyls did not produce roots on the 2,4-D containing media in the absence of activated charcoal. The roots were produced polarly on the NAA/IAA-containing media in the presence of activated charcoal. No-polarity of root formation was observed on media supplemented with NAA/IAA without activated charcoal. Different responses of hypocotyls to a series of 2,4-D concentrations (0.5, 1.0, 3.05.0, 8.0, and 10.0 mg l ¯¹) were observed on media supplemented with 0.02, 0.1 and 0.5% activated charcoal. In the NAA/IAA containing media in the presence of activated charcoal, root number per hypocotyl decreased. Root number perhypocotyl, on the media supplemented with NAA and IAA, increased when hypocotyls were pre-cultured on MS medium supplemented with 2,4-D (1.0 mg l ¯¹) for 2-3 days. When hypocotyls were pre-cultured on a 2,4-D containing MS medium for 5 days, embryos emerged from the hypocotyls directly on the medium supplemented with 2,4-D in the presence of activated charcoal. Addition of activated charcoal to MS medium supplemented with 2,4-D resulted in somatic embryogenesis of Daucus carota. Somatic embryos were not formed on the medium in the absence of activated charcoal. In suspension culture, the incorporation of 0.01 to 1.0% concentrations of activated charcoal to the MS medium, irrespective of 2,4-D, increased the number of somatic embryos produced. The maximum number of somatic embryos were produced with 1.0% activated charcoal. Further development of embryos of Daucus carota occurred on the media in the presence of activated charcoal, and the embryos subsequently regenerated normal plantlets. Abnormal somatic embryos followed the addition of 3.0% activated charcoal to the medium. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

Hybrid clones of the fast-growing Eucalyptus grandis and cold-tolerant E. nitens (GN clones) have been identified by the South African Forestry Industry as being highly suitable for plantations in cold-dry marginal areas. However, one of the main problems regarding their propagation is the difficulty in rooting of cuttings, both in vitro and ex vitro. The aims of this investigation, therefore, were (1) to develop widely applicable and efficient in vitro rooting system(s) for these commercially important clones, and (2) to assess some physiological characteristics of the roots produced. Adventitious shoots (15-20 mm in length) were obtained (l0 shoots/explant) from axillary buds on Murashige and Skoog's (MS) medium containing 0.01 mg.l-1 NAA, 0.01 mg.l-1 IBA and 0.2 g.l-1 FAP. The effect of various medium components, as well as modification of culture environment on in vitro rooting, were investigated. The highest rooting frequencies in clones GN121 (75%) and GN107 (65%) were achieved on l/4 MS with additional 0.22 g.l-1 CaCl2..2H2O and 0.18 g.1-1 MgS04.7H2O, 0.1 mg.l-1 IBA, 0.1 mg.l-1 biotin, 0.1 mg.l-1 calcium pantothenate, 15 g.1-1 sucrose and 4 g.l-1 Gelrite. Best culture conditions were an initial 72-hours dark incubation followed by a 16-hours day/8-hours night photoperiod at a PPFD of 37 µmol.m-2.s-1 and 23°C day/21°C night for seven days, after which the PPFD was increased to 66 µmol.m-2.s-1 at 27°C day/21°C night for 18 days. Towards the development of a more widely applicable in vitro rooting protocol for GN clones, the use of Agrobacterium rhizogenes strains was investigated. Production of transgenic roots was observed on carrot discs and shoots from seedlings of Eucalyptus grandis and E. nitens, but not on shoots of GN clones. Therefore, a method needs to be established for the successful transfer and integration of the Ri plasmid of Agrobacterium into the hybrid plant genome for induction of transgenic roots. The quality of roots produced in vitro and from cuttings was assessed by examination of root anatomy and hydraulic characteristics. Adventitious roots were prepared for measurement of hydraulic conductivity by detopping explants, then filtered, acidified distilled water was drawn through undisturbed potted root systems under partial vacuum, causing no damage to the roots. Initial studies showed that tissue culture-derived roots exhibited a higher specific root mass hydraulic conductivity than those derived from cuttings (6.46 x 10-6 vs. 3.06 X 10-6 g.kPa-1.s-1.g-1 dry root), probably due to root architecture. Curves relating vulnerability to water potential were constructed and both types of roots showed vulnerability to cavitation at high water potentials. Differences were also observed in staining reactions (safranin and fastgreen) which might suggest differences in presence and level of secondary metabolites in these roots at the juvenile stage. Applications of the developed protocols and future research strategies are discussed. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.

In vertebrate nervous system, formation of myelin sheaths around axons is essential for rapid nerve impulse conduction. However, the signals that regulate myelination in CNS remain largely unknown partially due to the lack of suitable in vitro models for studying localized cellular and molecular basis of axon-glia signals. We utilize microfabrication technologies to develop series of CNS neuron culture microsystems capable of providing localized physical and biochemical manipulation for studying neuron-glia interaction and neural progenitor development. First, a circular neuron-glia co-culture platform with one soma-compartment and one axon/glia compartment has been developed. The device allows physical and fluidic isolation of axons from neuronal somata for studying localized axon-glia interactions under tightly controlled biochemical environment. Oligodendrocyte (OL) progenitor cells co-cultured on isolated axons developed into mature-OLs, demonstrating the capability of the platform. The device has been further developed into higher-throughput devices that contain six or 24 axon/glia compartments while maintaining axon isolation. Increased number of compartments allowed multiple experimental conditions to be performed simultaneously on a single device. The six-compartment device was further developed to guide axonal growth. The guiding feature greatly facilitated the measurement of axon growth/lengths and enabled quantitative analyses of the effects of localized biomolecular treatment on axonal growth and/or regeneration. We found that laminin, collagen and Matri-gel promoted greater axonal growth when applied to somata than to the isolated axons. In contrast, chondroitin sulfate proteoglycan was found to negatively regulate axon growth only when it was applied to isolated axons. Second, a microsystem for culturing neural progenitor cell aggregates under spatially controlled three-dimensional environment was developed for studies into CNS neural development/myelination. Dense axonal layer was formed and differentiated OLs formed myelin sheaths around axons. To the best to our knowledge, this was the first time to have CNS myelin expressed inside a microfluidic device. In addition, promotion of myelin formation by retinoic acid treatment was confirmed using the device. In conclusion, we have developed series of neuron culture platforms capable of providing physical and biochemical manipulation. We expect they will serve as powerful tools for future mechanistic understanding of CNS axon-glia signaling as well as myelination.

Sinapic acid derivatives and tocopherols in refining byproducts of commercially produced expeller-pressed canola oils were characterized and isolated. Additionally, the antioxidant activities of the phenolics were examined by three systems including an in vitro non-biological related assay, a cellular assay and a deep-fat frying model. Sinapic acid (SA： 42.9 µg/g), Sinapine (SP: 199 µg/g), and Canolol (CAN: 344 µg/g) were found in different byproducts of canola oil refining, namely, soapstock, spent bleaching clay, and wash-water, respectively. Tocopherols (3.75 mg/g) and other non-identified phenolic compounds (2.7 mg /g) were found in deodistillates (DDL). CAN and DDL revealed significant protection effect (p<0.05) against hydrogen peroxide induced oxidation in two mammalian cell lines. The results of deep-fat frying studies indicated positive effects of CAN and DDL in preventing lipid oxidation. The canola oils fortified with DDL and CAN showed a considerable reduction (p < 0.05) in oxidation products of lipid after frying.

The garlic extract ajoene is considered to have antimicrobial and antitumor effects against a variety of cell types, and it is suggested to have the potential to be used as an antifungal or antitumor drug clinically. The underlying mechanism of its inhibitory effects is still uncertain. In this project, the effects of ajoene on the growth of fungal and oomycete cells were studied on Candida albicans, Neurospora crassa and Achlya bisexualis. Endometrial cancer is the most common gynecologic cancer. A 3D spheroid model of endometrial cancer cells were for the first time used to investigate the antitumor effects of ajoene and selected antitumor agents. Ajoene was extracted from fresh garlic by chromatographic methods and the outcome of the extractions was verified with Mass spectrometry and NMR spectroscopy. Ajoene was then tested on the yeast form or germ tubes of C. albicans, and the cell division and germ tube formation was analyzed. N. crassa and A. bisexualis were treated with ajoene on plates or on glass slides to measure the hyphae radial extension or individual hyphal extension. 3D endometrial adenocarcinoma cell (Ishikawa) spheroids were treated with ajoene, paclitaxel, targeted drugs everolimus, sorafenib, gefitinib and canertinib alone or in combinations. The growth activity, metabolic activity, cell proliferation, apoptotic activity and the cytoskeletons were analyzed after the treatments. Cell division of C.albicans was inhibited by ajoene at 5µg/ml or higher concentrations. The length of C.albicans germ tubes was significantly shorter in ajoene treated groups than the untreated ones. Radial extension and individual hyphal extension of N. crassa and A. bisexualis were both inhibited by ajoene. Ajoene did not show any antitumor effects on the 3D cell model of Ishikawa cells. No synergistic effect was detected between ajoene and paclitaxel or ajoene and everolimus. The targeted drugs Canertinib and everolimus showed an inhibitory effect on growth activity of the spheroids, but no synergy with paclitaxel. In conclusion, ajoene was able to inhibit various forms of fungal and oomycete growth, but any antitumor activity of ajoene did not show on 3D culture of endometrial cancer cells.

Content of free- (FPC) and bound- phenolics (BPC) significantly (p<0.05) increased during mixing, fermenting and baking. Bread crust and crumb contained the highest FPC and BPC, respectively. Antioxidant activities (AOA) followed the trends of their respective phenolic contents. HPLC analysis demonstrated that different phenolic acids showed various responses to the bread-making process. Total anthocyanin content (TAC) was significantly (p<0.05) reduced through mixing and baking, but fermentation elevated the levels. Anthocyanin extract of purple wheat exerted higher AOA than those of common wheat. Digested purple wheat extracts after in-vitro digestion demonstrated significantly (p<0.05) higher AOA than common wheat. During in-vitro testing, extracts exhibited concentration-dependent effects, while the use of different cell lines exhibited varying levels of cellular antioxidant and pro-oxidant properties. Purple wheat demonstrated higher cytoprotectivity and cellular AOA than those of common wheat. Our findings suggest that purple wheat has the potential to act as functional food in bakery products.

In order to optimize productivity of a cell culture it is necessary to understand growth and productivity and couple these features of the culture to extracellular nutrients whose profiles can be manipulated. Also, since growth and productivity are directly affected by cell death mechanisms such as apoptosis, it is imperative to understand these mechanisms. This work describes the development of a differential equation based population balance model of apoptosis in a Chinese Hamster Ovary cell culture producing Anti-RhD monoclonal antibody (mAb). The model was verified in isolation and was then coupled to a metabolic flux model. The model distinguishes between various subpopulations at normal healthy states and at various stages of apoptosis. After finding that glucose and glutamine are not limiting nutrients for this culture, different hypotheses were explored to explain growth arrest. Initially, it was hypothesized that there is some unknown nutrient in either media or serum which is depleted, thus causing growth arrest. Accordingly a first model was developed assuming depletion of this nutrient. Subsequent experiments with different additions of media and serum showed that there is no such nutrient limitation for the media and serum conditions used in most of the experiments. Additional experiments with different culture volumes showed that cell growth was actually controlled by a compound that accumulates and causes pH deviation from its optimal range of operation. Since strong correlations were found between culture volume and growth, it was hypothesized that the compound may be carbon dioxide (CO2), which is inhibitory for growth and may accumulate due to mass transfer limitations. Following this finding, a second model was proposed to take into account the accumulation of this inhibitor, although the specific inhibiting compound could not be exactly identified. This second mathematical model of cell growth was then integrated with a metabolic flux model to provide for a link between intracellular and extracellular species balances, since the latter are the ones to be manipulated for increasing productivity. This final model formulation was then used to describe mAb productivity. The model was also able to reasonably predict all cell subpopulations, nutrients, metabolites and mAb. In an attempt to mitigate the effect of CO2 accumulation and renew the cell growth, culture perfusions were performed. Although this approach resulted in some renewal of growth, the cell concentration progressively decreased after each successive perfusion event. This suggests that irreversible cell damage occurs because of CO2 accumulation. The model was used to describe the perfusion experiments. Agreement between data and model predictions were reasonable. In addition, it was shown that operation with successive perfusions results in a significant increase in productivity and therefore it can be used for further process optimization.