Magister Scientiae - MSc / This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996). / South Africa

Un concept qui a fait son apparition ces dernières années est l’utilisation de micronutriments comme agents thérapeutiques connus pour leurs propriétés anti-inflammatoires, antioxydantes, cardio-protectrices ou anticancéreuses. Dans notre travail, nous nous sommes intéressés à l’étude des mécanismes d’actions in vivo et in vitro du resvératrol et de deux formulations naturelles, l’une impliquée dans les effets antioxydants (produit « X ») et l’autre (produit « Y ») destinée à corriger les désordres de nature lipidique de sujets présentant un syndrome métabolique. Nous avons caractérisé les effets pro-différenciateurs d’un polyphénol naturel, le resvératrol, sur cellules squelettiques musculaires C2C12 en culture. L’étude a montré que ce polyphénol permet d’induire l’expression de facteurs de la différenciation musculaire (Myogénine, Scrp3) et d’augmenter le taux de la myosine, protéine impliquée dans la contraction musculaire. Enfin, le resvératrol permet de moduler l’expression de microRNA, comme par exemple miR-133b impliqué dans les mécanismes de différenciation musculaire. Les résultats sur le stress oxydant montrent que le produit « X » est capable, à la fois, de moduler de façon modérée l’expression des gènes des défenses anti-radicalaires au niveau du muscle gastrocnémiens (Sirt1, Sod1, Ucp2, Nrf1) mais aussi au niveau des cellules musculaires squelettiques murines C2C12 (Prdx1, Nrf2, Pgc1α) en culture. Les résultats au niveau des gastrocnémiens ont montré également une élévation du potentiel global des défenses anti-radicalaires par dosage KRL. Une diminution du taux de ROS intracellulaires a été observée avec sonde DHE dans les cellules C2C12 en culture. L’étude des mécanismes d’action de « Y » a montré qu’il est capable de moduler de façon modérée l’expression des gènes du métabolisme énergétique (Gs1, Srebp1c, Fabp3, Vlcad, Pparβ) dans les cellules C2C12. Nos résultats montrent également que « Y » induit un effet anti-inflammatoire par la diminution du nombre de lymphocytes sous un régime riche en calorie. Toutefois, « Y » n’est pas un agoniste de PPARα et de PPARγ dans nos conditions. L’ensemble des résultats obtenus au cours de ce travail confirme que les micronutriments sont capables d’induire des effets au niveau génique quoique de façon modérée. Des études à long terme sont nécessaires pour comprendre et confirmer réellement leur impact sur l’organisme / A concept that has emerged in recent years is the use of micronutrients as therapeutic agents known for their anti-inflammatory, antioxidant, cardio-protective or anticarcinogenic proprieties. In this work, we studied the in vivo and in vitro mechanism of action of resveratrol and two natural formulations, one involved in the antioxidant effects (product "X") and the other (product "Y") involved in the correction of lipid disorders in people affected by metabolic syndrome. We characterized the pro-differentiating effect of a natural polyphenol, resveratrol, on C2C12 skeletal muscle cell line. This study showed that this polyphenol can induce the expression of muscle differentiation factors (myogenin, Scrp3) and increases the level of myosin, a protein involved in muscular contraction. Finally, resveratrol can modulate the expression of microRNA, such as miR-133b involved in the muscular differentiation process. Concerning oxidative stress, the results shows that "X" is capable both, to slightly modulate expression of anti-radicalar defense genes in gastrocnemius muscle (SIRT1, SOD1, UCP2, NRF1) but also, in C2C12 murine skeletal muscle cells (PRDX1, NRF2, PGC1α). The results in the gastrocnemius showed a rise of the overall radicalar defense potential by using KRL assay. A decrease of intracellular ROS was observed with the DHE probe in C2C12 cells. The study of "Y" has shown that it is able to slightly modulate the expression of genes involved in energetic metabolism (Gs1, Srebp1c, Fabp3, VLCAD, Pparβ) in C2C12 cells. Our results also shown that "Y" induces an anti-inflammatory effect by reducing the number of lymphocytes in mice fed by a High Fat/ High Sucrose diet. However, "Y" is not an agonist of PPARα and PPARγ in our conditions. The overall results obtained in this work confirms that micronutrients can induce slightly effects on gene expression. Long-term studies are needed to understand and confirm their true impact on organism

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-β-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC50 ∼14 μM) followed by quercetin (IC50 ∼33 μM), quercetin-3-β-d glucoside (IC50 ∼71 μM), resveratrol (IC50 ∼94 μM), quercitrin (IC50 ∼120 μM). Inhibition was identical in both F1Fo membrane preparations as well as in isolated purified F1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β-d glucoside inhibited only ATPase activity and not ATP synthesis.

We have studied the inhibitory effect of natural and structurally modified polyphenols on Escherichia coli ATP synthase to test (I) if the beneficial dietary effects of polyphenols are related to their inhibitory actions on ATP synthase, (II) if inhibitory effects of polyphenolic compound could be augmented through structural modifications, and (III) if they can act as antimicrobial agent through their actions on ATP synthesis. X-ray crystal structures of polyphenol binding sites suggested that polyphenols bind at a distinct polyphenol binding pocket, at the interface of α,β,γ-subunits. We found that both natural and modified polyphenols inhibit E. coli ATP synthase to varying degrees and structural modifications resulted in augmented inhibition. Inhibition was reversible in all cases. Both natural and modulated compounds inhibited E. coli cell growth to varying degrees. We conclude that dietary benefits of polyphenols may be in part due to the inhibition of ATP synthase.

Peanut skins are a low-value byproduct of peanut blanching operations. They have been shown to contain significant levels of phenolic compounds with demonstrated antioxidant properties. The effects of two types of extraction methods: solid-liquid extraction (SLE) and microwave-assisted extraction (MAE) on the recovery of phenolic compounds from peanut skins were investigated. Response surface methodology was used to optimize extraction conditions based on total phenolic content (TPC), ORAC (oxygen radical absorbance capacity) activity and <i>trans</i>-resveratrol content. The protective effect of peanut skin extracts (PSE) against hydrogen peroxide (H₂O₂)-induced oxidative stress in human brain microvascular endothelial cells (HBMEC) and the effect of PSE on lipid oxidation in commercial peanut butter were also evaluated. In the SLE method, EtOH was found to be the most efficient extraction solvent followed by MeOH, water and EA. Despite EtOH extracts having a higher TPC, samples extracted with MeOH demonstrated slightly higher ORAC activity. Resveratrol was identified in MeOH extracts but was not found in EtOH, water or EA extracts. In the MAE procedure, the maximum predicted TPC under the optimized conditions was 144 mg phenols/g skins compared to 118 mg/g with SLE. The maximum predicted ORAC activity was 2789 μmol TE/g as opposed to 2149 μmol TE/g with the SLE method. MAE was able to extract more phenolic compounds (with higher antioxidant activity) in a faster time than the SLE procedure. In addition, resveratrol was identified in PSE derived from MAE although at relatively low levels. PSE were found to have some protective effects against oxidative stress in HBMEC. Higher doses of PSE appeared to have a slightly cytotoxic effect. However, the data were highly variable which made it difficult to arrive at any definitive conclusions regarding the potential benefits of PSE in preventing oxidative damage to cells. In the PB experiment, hexanal levels over the storage period were not high enough for the samples to be considered oxidized. However, hexanal values of PB samples treated with PSE were lower than the control throughout storage, which suggests that PSE may provide some protection against oxidation of PB. / Ph. D.

Combining chemotherapeutics to treat malignant tumors has been shown to be effectivein preventing drug resistance, tumor recurrence, and reducing tumor size. We modeledcombination drug therapy in PC-3 human prostate cancer cells using mixture design responsesurface methodology (MDRSM), a statistical technique designed to optimize compositions thatwe applied in a novel manner to design combinations of chemotherapeutics. Conventionalchemotherapeutics (mitoxantrone, cabazitaxel, and docetaxel) and natural bioactive compounds(resveratrol, piperlongumine, and flavopiridol) were used in twelve different combinationscontaining three drugs at varying concentrations. Cell viability and cell cycle data werecollected and used to plot response surfaces in MDRSM that identified the most effectiveconcentrations of each drug in combination. MDRSM allows for extrapolation of data fromthree or more compounds in variable ratio combinations, unlike the Chou-Talalay method.MDRSM combinations were compared with combination index data from the Chou-Talalaymethod and were found to coincide. We propose MDRSM as an effective tool in devisingcombination treatments that can improve treatment effectiveness, and increase treatmentpersonalization because MDRSM measures effectiveness rather than synergism, potentiation orantagonism.

Le resvératrol (RV/RSV) est un composé chimique organique connu pour ses effets anticancéreux mais aussi pour son effet rajeunissant sur les cellules et les organismes. Afin d’étudier le ou les multiples mécanismes à l’origine de ces effets, Y. Benslimane du laboratoire Harrington a étudié l’effet du resvératrol in vitro, à des concentrations de l’ordre de 12,5 μM à 25 μM, sur plusieurs lignées de cellules cancéreuses humaines, notamment NALM6 et JURKAT. Ses recherches ont démontré que le traitement par resvératrol conduit à l’activation de la voie de signalisation ATR/CHK1, qui maintient les cellules mitotiques en phase S afin d'induire la réparation de l'ADN. Dans ces cellules, le resvératrol présente spécifiquement une signature similaire à celle de l’hydroxyurée, un composé anticancéreux connu pour l’induction de stress réplicatif. Cette induction de stress réplicatif semble complémentaire aux mécanismes de la littérature existante, qui présente le resvératrol comme un activateur de sirtuines, une famille d’histones désacétylases hautement conservées entre les espèces et impliquées dans la réparation de l'ADN et la réponse métabolique. Cependant, alors que plusieurs articles démontrent une activation de la sirtuine 1 (SIRT1) comme conséquence notable du traitement par le resvératrol, les études de Benslimane et al. ont démontré quant à elles que le knock-out de la sirtuine 1 dans les cellules NALM6 et Jurkat n’affectait pas le phénotype de stress réplicatif observé. Or les concentrations de resvératrol employées dans l’ensemble de la littérature scientifique varient de 0,5 μM à 100 μM in vitro, laissant supposer une variance dans les conditions expérimentales qui pourrait expliquer la variance de résultats observés. Dans ce contexte, nous avons émis l’hypothèse que l’effet du resvératrol était dépendant de la concentration utilisée, autrement dit, que son effet était biphasé. Plus spécifiquement, dans les cellules NALM6 cancéreuses, nous avons supposé que les stress réplicatif causé par des concentrations élevées (>12,5 μM) de resvératrol pouvait masquer l’activation des sirtuines et spécifiquement celle de la sirtuine 1, tandis que des concentrations plus faibles (<5 μM) 4 permettraient l’observation potentielle de mécanismes parallèles à l’activation de la voie ATR/CHK1. Nous avons également émis l’hypothèse que l’échelle temporelle d’observation jouait un rôle dans la mesure des résultats et leur observabilité. Afin d’examiner nos hypothèses, nous avons dans un premier temps fixé le temps d’observation à 72h après traitement, et évalué l'effet du resvératrol et de l'inhibiteur de l'ATR VE-821 sur la viabilité cellulaire relative dans les cellules NALM6 WT, p53 KO et SIRT1 KO. La lignée p53 KO fut choisie, car p53 est en aval des voies de signalisation ATR/CHK1 et SIRT1. Cette expérience a montré une augmentation légère et non significative de la viabilité relative dans les trois lignées à des concentrations de resvératrol d'environ 2,5 μM, et une réduction significative de la viabilité à des concentrations supérieures à 10 μM. En outre, le score de synergie Bliss a révélé un effet additif entre le resvératrol et VE-821. Dans un second temps, nous avons quantifié l’effet de différentes concentrations de resvératrol sur le cycle cellulaire. Dans les trois lignées cellulaires, on trouve un pourcentage significatif de cellules arrêtées en phase S après un traitement de 24 heures avec 12,5 μM de resvératrol, mais aucune différence significative entre les conditions DMSO et 2,5 μM de resvératrol. On n’observe pas de différence significative entre les lignées. Enfin, une expérience temporelle révèle que les effets du resvératrol pourraient s'estomper après 16h, et montre une légère augmentation des cellules arrêtées en phase S après un traitement à 2,5 μM de resvératrol à 16h, par rapport au contrôle DMSO, suggérant un temps d’observation intéressant en deçà de 16h et un potentiel effet du resvératrol à faible dose. Ces résultats semblent confirmer l'action du resvératrol sur la voie ATR/CHK1, avec un arrêt du cycle cellulaire en phase S après traitement par 12,5 μM de resvératrol, dans les trois lignées WT, SIRT1 KO et p53 KO. Ils suggèrent également un effet potentiellement bénéfique du resvératrol à faibles doses par le biais d'un stress réplicatif de faible intensité, sans démontrer de dépendance à SIRT1 ou p53. Enfin, ils soulignent la nécessité d’une observation à différentes échelles temporelles. / Resveratrol (RV/RSV) is an organic chemical compound known for its anti-cancer and rejuvenating effects on cells and organisms. To investigate the multiple mechanisms behind these effects, Y. Benslimane of the Harrington laboratory studied the effect of resveratrol in vitro, at concentrations ranging from 12.5 μM to 25 μM, on several human cancer cell lines, including NALM6 and JURKAT. His research has shown that treatment with resveratrol leads to activation of the ATR/CHK1 signaling pathway, which keeps mitotic cells in S phase to induce DNA repair. Resveratrol specifically displays a signature similar to that of hydroxyurea, an anti-cancer compound known to induce replicative stress. This induction of replicative stress seems complementary to the mechanisms exposed in the literature, which presents resveratrol as an activator of sirtuins, a family of histone deacetylases highly conserved across species and involved in DNA repair and metabolic response. However, while several articles demonstrate activation of sirtuin 1 (SIRT1) as a notable consequence of resveratrol treatment, studies by Benslimane et al. showed that sirtuin 1 knockout in NALM6 and Jurkat cells did not affect the observed phenotype of replicative stress. Because concentrations of resveratrol used in the literature vary from 0.5 μM to 100 μM in vitro, we hypothesized that this variance in experimental conditions might explain the variety of results observed. More specifically, in NALM6 cancer cells, we hypothesized that replicative stress caused by high concentrations (>12.5 μM) of resveratrol could mask activation of sirtuins and specifically sirtuin 1, while lower concentrations (<5 μM) would allow the potential observation of mechanisms parallel to activation of the ATR/CHK1 pathway. We also hypothesized that the time scale of observation played a role in the measurement of results and their observability. To examine our hypotheses, we first set the observation time at 72h post-treatment, and assessed the effect of resveratrol and the ATR inhibitor VE-821 on relative cell viability in NALM6 WT, p53 KO and SIRT1 KO cells. The p53 KO line was chosen, as p53 is downstream of the 6 ATR/CHK1 and SIRT1 signaling pathways. This experiment showed a slight, non-significant increase in relative viability in all three lines at resveratrol concentrations of around 2.5 μM, and a significant reduction in viability at concentrations above 10 μM. In addition, the Bliss synergy score revealed an additive effect between resveratrol and VE-821. In a second step, we quantified the effect of different resveratrol concentrations on the cell cycle. In all three cell lines, we found a significant percentage of cells arrested in S phase after 24h treatment with 12.5 μM resveratrol, but no significant difference between DMSO and 2.5 μM resveratrol conditions. No significant difference was observed between lines. Finally, a time-course experiment revealed that the effects of resveratrol might fade after 16h, and showed a slight increase in S-phase arrested cells after treatment with 2.5 μM RSV at 16h, compared with the DMSO control, suggesting an interesting observation time below 16h and a potential effect of resveratrol at low doses. These results seem to confirm the action of resveratrol on the ATR/CHK1 pathway, with cell cycle arrest in S phase after treatment with 12.5 μM resveratrol, in the three lines WT, SIRT1 KO and p53 KO. They also suggest a potentially beneficial effect of low-dose resveratrol via low-intensity replicative stress, without demonstrating dependence on SIRT1 or p53. Finally, they emphasize the need for observation on different time scales.

Made available in DSpace on 2015-04-17T14:49:32Z (GMT). No. of bitstreams: 1 parte1.pdf: 3805618 bytes, checksum: c5283752b1a8775c6dd8cef062f7c370 (MD5) Previous issue date: 2009-11-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this research was to evaluate the physical, physical-chemical, bioactive, and sensorial attributes of the first Brazilian seedless grapes (Vitis vinifera L.) varieties 'BRS Clara', 'BRS Linda', 'BRS Morena', and Advanced Selection 8, compared with the North American 'Crimson Seedless'. Three experiments had been carried out. For the first one, taking into account the Normative Brazilian/2002 and the requirements for exportations, it was determined that Advanced Selection 8 develops large cluster, inserted in class 2, with dark reddish berry, caliber 20, and ellipsoid format; 'BRS Morena' presents cluster of medium size, in class 2, with berry colored of black nuance, caliber 20, and format between ellipsoid and globoid; 'BRS Linda' with large cluster, in class 2, light-green color, and 'Crimson Seedless' with pink colored and small cluster inserted in class 1, offer berries in caliber 18 with ellipsoid formats; 'BRS Clara' develops medium size cluster in class 2, berry with caliber, green yellowish green color and a format elongated ellipsoid. All of these grapes meet the standards for national market that demands a minimum of 12 millimeters of diameter. On the other hand, 'BRS Clara' does not meet the minimum diameter demanded for exportations. The red grapes ' BRS Morena' e 'Crimson Seedless' develop the firmest berries. The hydrogenionic potential, titratable acidity and soluble solids content meet the official standard values. The largest clusters are significantly less acid and present higher soluble solid/titratable acidity ratio, inferring to be the sweetest when mature. For the second experiment, it was determined and correlated the content of acid Lascorbic, total phenolic composites, trans-resveratrol, and the total antioxidative capacity of the studied grapes. All grape samples presented low acid L-ascorbic contents. The white 'BRS Clara' presents marked content of total phenolic compounds, followed by the white 'BRS Linda', in third position are the red ones: Advanced Selection 8, 'BRS Morena', and 'Crimson Seedless'. 5 Grapes presented distinct contents of trans-resveratrol in this following order of superiority: 'BRS Morena', 'Crimson', Advanced Selection 8, 'BRS Linda', and 'BRS Clara'. Trans-resveratrol seems to provide varietal identity. Red grapes presented higher contents of trans-resveratrol. 'BRS Clara', ' Crimson Seedless', and 'BRS Linda', similarly, presented the highest antioxidative capacity and the color of the grape did not influence on that. The contents of total extractable phenolic compounds influenced positively on the antioxidative capacity. Grapes presenting larger contents of total extractable phenolic compounds presented lower content of transresveratrol. For the third experiment it was used the Quantitative Descriptive Sensorial Analysis, by means of selected and trained judges. On the global quality, 'BRS Morena' was superior than others grapes evaluated, while Advanced Selection 8 was in second position, and 'BRS Linda' and 'Crimson Seedless' occupied the third position, while 'BRS Clara' showed the most inferior sensorial quality. The general characteristics flavor, with large superiority, followed, in this order, by the strongest tonality, the red color, the most spherical format, the highest firmness, succulence, crispness, and sweetness contributed positively with the global quality. The bitter taste and the sensorial acidity, in this sequence of intensity, were the attributes of larger negative influence. The results for physical and physical-chemical evaluations, together, reflect significantly the sensorial perceptions. Therefore, once inserted in the official standards of identity and quality, beside the varietal characteristic flavor, producers must prefer to produce grapes of stronger tone, larger sizes, and more spherical or globular formats. / O objetivo desta pesquisa foi avaliar os atributos físicos, físico-químicos, bioativos e sensoriais das primeiras variedades de uvas (Vitis vinifera L.) sem sementes brasileiras, 'BRS Clara', 'BRS Linda' e 'BRS Morena', mais a Seleção Avançada 8, comparadas com a norte-americana 'Crimson Seedless'. Foram conduzidos três experimentos. Assim, com o primeiro considerando as Normativas brasileiras/2002 e as exigências para exportações, determinou-se que a Seleção Avançada 8 possui cacho grande na classe 2 com baga avermelhada escura, calibre 20 e formato elipsóide; A 'BRS Morena' apresenta cacho médio na classe 2, com baga colorida de nuança preta, calibre 20 e de formato elipsóide a quase globoso; a 'BRS Linda' com cacho grande, classe 2, de cor verde clara e a 'Crimson' com cacho róseo, pequeno na classe 1 oferecem bagas de calibre 18 e com formatos elipsóides; a 'BRS Clara' possui cacho médio e de classe 2, com baga de calibre 14, tom verde amarelado e de formato elipsóide alongado. Todas atendem o mercado nacional quanto aos diâmetros mínimos de 12 milímetros. A 'BRS Clara' não atende os diâmetros exigidos para exportações. As uvas tintas 'BRS Morena' e 'Crimson Seedless' dispõem das bagas mais firmes. O potencial hidrogeniônico, a acidez titulável e os teores de açúcares solúveis atendem aos padrões oficiais. Os cachos maiores são os menos ácidos e apresentam a melhor relação de sólidos solúveis/acidez titulável, inferindo serem os mais doces, quando maduros. O segundo experimento determinou e correlacionou os teores de ácido L-ascórbico, compostos fenólicos totais extraíveis, trans-resveratrol e a capacidade antioxidante total das uvas estudadas. Todas as amostras apresentaram baixos conteúdos de ácido L-ascórbico. A branca 'BRS Clara' apresenta destacado teor de compostos fenólicos totais extraíveis, seguida pela branca 'BRS Linda', em terceiro lugar encontram-se as tintas Seleção 8, 'BRS Morena' e 3 'Crimson'. As uvas apresentaram conteúdos distintos do trans-resveratrol e nessa ordem de superioridade: 'BRS Morena', 'Crimson', Seleção Avançada 8, 'BRS Linda' e 'BRS Clara'. O trans-resveratrol parece conferir identidade varietal. As tintas apresentaram maiores conteúdos de trans-resveratrol. A 'BRS Clara', 'Crimson' e 'BRS Linda', igualmente, apresentaram a maior capacidade antioxidante. A cor não interferiu na capacidade antioxidante. Os compostos fenólicos totais influenciaram positivamente na capacidade antioxidante. Uvas com maiores quantidades de compostos fenólicos totais extraíveis, apresentam menores teores de transresveratrol. No terceiro experimento utilizou-se a Análise Sensorial Descritiva Quantitativa, mediante julgadores selecionados e treinados. Quanto à qualidade global, a 'BRS Morena' apresentou-se superior, a Seleção Avançada 8 destacou-se em segundo lugar, enquanto, a 'BRS Linda' e 'Crimson Seedless' ocuparam o terceiro lugar e a BRS Clara' foi de qualidade inferior, sensorialmente. O sabor geral característico, com ampla superioridade, seguido na ordem, pela tonalidade mais forte, a cor tinta, o formato mais esférico, a maior firmeza, suculência, crocância e doçura contribuíram positivamente com a qualidade global. O amargor e a acidez sensorial, nesta seqüência de intensidade, foram os atributos de maior influência negativa. Os resultados das avaliações físicas e físico-químicas, em conjunto, refletem com significância as percepções sensoriais. Uma vez, dentro dos padrões de identidade e qualidade oficiais, além do sabor varietal característico, os produtores devem preferir produzir uvas de tons mais fortes, tamanhos maiores e de formatos mais esféricos ou globosos.

Thesis (MSc (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2002. / Pathogen devastation of food products has been the topic of extensive research efforts worldwide. Fungal infections are foremost amongst these pests, contributing not only to losses in product yield, but also significantly affecting the quality thereof. It is not surprising then that producers of these foodstuffs and their derived products continually strive towards the highest possible product quality. Therefore, it remains imperative that satisfactory methods are implemented to control these fungal pathogens. The current strategies are all hampered by drawbacks, and severe crop losses are still experienced. New technologies are being explored; one such technology is the genetic transformation of plant species. This method has enabled scientists to introduce foreign genes, with known functions and predictable outcomes, into plants. Genes identified to be involved in disease resistance have become the focus of numerous research efforts concerned with the improvement of the plant's innate defence response. This study aimed to enhance disease resistance to fungal pathogens by means of the genetic transformation of two genes previously shown to be involved in disease resistance. These genes encode polygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrol synthase from Vitis vinifera. PGIPs specifically inhibit the action of fungal polygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown of plant cell walls. These enzymes were also found to be the first enzymes that are secreted by fungal pathogens during infection of the host plant. Additionally, PGIP-PG interaction results in the existence of molecules involved in the activation of plant defence responses. Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destruction of the microbial cellular membranes. These mentioned genes were transformed alone, and in combination, into Nicotiana tabacum and the resultant transgenic lines were evaluated for enhanced disease resistance and for possible synergistic effects between the transgenes. Several independent transgenic lines were regenerated with genes integrated into the tobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressed these genes at a high frequency. Some non-expressing lines were identified from the transgenic plants that had integrated both genes, but several lines were obtained expressing both transgenes. Good correlations were observed between transgene product activity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in planta assay. Lines showing the highest PGIP activity and resveratrollevels were more resistant to the pathogen, leading to disease resistance of up to 80% seven days after inoculation in comparison to an untransformed control. These lines maintained their strong inhibition, even three weeks post-inoculation, showing a complete halt in disease development and fungal growth. These results provide good indications of the efficacy of these transgenes in the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of these genes. In an additional pilot study, genes encoding for precursors and for the formation of resveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultant recombinant yeast strains were evaluated for their ability to produce the phenolic substance, resveratrol. This compound has been implicated in beneficial aspects relating to human health, including positive effects on atherosclerosis and platelet aggregation as a direct result of its antioxidant and anti-inflammatory activities. Recombinant yeast strains were constructed that expressed genes coding for coenzyme A ligase and resveratrol synthase. These strains were shown to be able to produce the phenolic compound resveratrol from the precursors present in the yeast as well as from the products introduced with the transformation. The resveratrol was complexed with an added glucose moiety. These results are extremely positive, considering the possibility of manipulating wine yeasts to produce resveratrol during the wine fermentation, thereby adding to the health aspects of both red and white wine. This is the first report of the production of this compound by the introduction of genes necessary for its biosynthesis in a foreign host. This study has confirmed the importance of PGIPs and resveratrol in the effort to enhance disease resistance in plants through genetic transformation technology. It has also shown that the health benefits of resveratrol could be exploited more optimally in the wine industry, by producing wine yeasts with the ability to synthesise this important antioxidant.

The lens of the eye needs to be transparent with a high refractive index to focus images on the retina. In cataracts the lens becomes opaque, eventually leading to blindness. There are many possible causes of cataract but a lot of evidence implicates oxidative damage as contributing to opacification. This includes epidemiological studies showing that diets rich in antioxidants lowered the prevalence of cataract. This research tested the hypothesis that if cataracts were at least partially caused by oxidative damage then their progression would be slowed by application of antioxidants. The antioxidants used were two plant compounds found in the diet, resveratrol and quercetin. The system used was sheep lenses cultured in Eagles Minimal Essential Medium (EMEM). Lenses remained transparent for up to 7 days in EMEM but became opaque within 24 h when exposed to 1 mM hydrogen peroxide (H₂O₂). The lens is exposed to H₂O₂ in vivo as it is found in the aqueous humor. Prior Lenses pre-treated with quercetin reduced but did not prevent opacification. Lens cell death, as determined by measurement of leakage of lactate dehydrogenase, was found to increase with H₂O₂ and the increase was prevented by pre-treatment with antioxidants. The role of the endogenous antioxidant glutathione was also investigated. It was found that H₂O₂ decreased the amount of reduced glutathione in the lens cortex and increased the levels of oxidised glutathione but only at levels of 2 mM and above. Thus the results of this research indicate that H₂O₂ at low concentration (1 mM) is able to damage lens cells and cause opacification without affecting the reduced glutathione levels and that the exogenous antioxidants have some ability to protect the lens.