Philosophiae Doctor - PhD / This study explores the roles of exogenously applied nitric oxide, exogenously applied caffeic acid and salt stress on the ontioxidant system in cereal (exemplified by maize) and legume (using soybean as an example) plants together with their influence on membrane integrity and cell death. This study investigates changes in H₂O₂ content, root lipid peroxidation, root cell death and antioxidant enzymatic activity in maize roots in response to exogenously applied nitric oxide (NO) and salt stress. This part of the study is based on the partially understood interaction between NO and reactive oxygen species (ROS) such as H₂O₂ and the role of antioxidant enzymes in plant salt stress responses. The results show that application of salt (NaCl) results in elevated levels of H₂O₂ and an increase in lipid peroxidation, consequently leading to increased cell death. The study also shows that by regulating the production and detoxification of ROS through modulation of antioxidant enzymatic activities, NO plays a pivotal role in maize responses to salt stress. The study argues for NO as a regulator of redox homeostasis that prevents excessive ROS accumulation during exposure of maize to salinity stress that would otherwise be deleterious to maize. This study extends the role of exogenously applied NO to improve salt stress tolerance in cereals crops (maize) further to its role in enhancing salt stress tolerance in legumes. The effect of long-term exposure of soybean to NO and salt stress on root nodule antioxidant activity was investigated to demonstrate the role of NO in salt stress tolerance. The results show that ROS scavenging antioxidative enzymes like SOD, GPX and GR are differentially regulated in response to exogenous application of NO and salt stress. It remains to be determined if the NO induced changes in antioxidant enzyme activity under salt stress are sufficient to efficiently reduce ROS accumulation in soybean root nodules to levels close to those of unstressed soybean root nodules. Furthermore, this study investigates the effect of long-term exposure of soybean to exogenous caffeic acid (CA) and salt stress, on the basis of the established role of CA as an antioxidant and the involvement of antioxidant enzymes in plant salt stress responses. The effect of CA on soybean nodule number, biomass (determined on the basis of nodule dry weight, root dry weight and shoot dry weight), nodule NO content, and nodule cyclic guanosine monophosphate (cGMP) content in response to salt stress was investigated. Additionally, CA-induced changes in nodule ROS content, cell viability, lipid peroxidation and antioxidant enzyme activity as well as some genes that encode antioxidant enzymes were investigated in the presence or absence of salt stress. The study shows that long-term exposure of soybean to salt stress results in reduced biomass associated with accumulation of ROS, elevated levels of lipid peroxidation and elevated levels of cell death. However, exogenously applied CA reversed the negative effects of salt stress on soybean biomass, lipid peroxidation and cell death. CA reduced the salt stress-induced accumulation of ROS by mediating changes in root nodule antioxidant enzyme activity and gene expression. These CA-responsive antioxidant enzymes were found to be superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR), which contributed to the scavenging of ROS in soybean nodules under salt stress. The work reported in Chapter 2 has been published in a peer-reviewed journal [Keyster M, Klein A, Ludidi N (2012) Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide. Plant Signaling and Behavior 7: 349-360]. My contribution to the published paper was all the work that is presented in Chapter 2, whereas the rest of the work in the paper (which is not included in Chapter 2) was contributed by Dr Marshall Keyster.

Philosophiae Doctor - PhD

Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites, the total antioxidant capacity, cytokines, biochemical and haematological parameters in the plasma of patients with CP and control subjects. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescence determination. Nitrites were determined using Griess reaction. Cytokines - TNF-alfa; TNF RI; PDGF-AB; TGF-beta, together with myeloperoxidase and hyaluronan were determined using ELISA Kits. Biochemical and haematological parameters were measured by standard methods.

Magister Scientiae (Biodiversity and Conservation Biology) / Wheat is a key global commodity in terms of acreage and tradeable value and as a staple in household diets. Many factors including biotic stress conditions have detrimental effects on global wheat production and yield. The increasing prevalence of biotic stress inflicted by fungal species such as Fusarium has significantly reduced yield and quality of cereal crops thus, threatening sustainable agriculture and food security. Interactions between wheat and Fusarium spp. such as Fusarium proliferatum triggers the accumulation of reactive oxygen species (ROS) to levels toxic to the plant thus leading to oxidative damage and ultimate cellular death. In order to maintain redox homeostasis, plants rely on ROS-scavenging antioxidants (enzymatic and non-enzymatic) to control ROS molecules to levels less toxic to plants. This study investigated the impact of F. proliferatum on the physio-biochemical responses of two wheat cultivars (SST 015 and SST 088). Changes in seed germination, growth, biomass, chlorophyll and mineral contents were monitored. Furthermore, changes in ROS accumulation and antioxidant enzyme activity was measured in the shoots of both wheat cultivars.

>Magister Scientiae - MSc / Biotic stress is one of the main causes for agricultural loss of economically important cereal crops. The increasing prevalence of biotic stress inflicted by fungal species such as Fusarium has significantly reduced yields and quality of cereals, threatening sustainable agriculture and food security worldwide. Interactions between wheat and Fusarium spp. such as Fusarium oxysporum promotes the accumulation of reactive oxygen species (ROS). Overproduction of ROS can become toxic to plants depending on the scavenging ability of antioxidant systems to maintain redox homeostasis. This study investigated the effects of F. oxysporum on the physiological and biochemical response of three wheat cultivars namely, SST 056, SST 088 and SST 015. Physiological responses were monitored by measuring changes observed in plant growth parameters including shoot and root growth and biomass, relative water content as well as photosynthetic metabolism and osmolyte content in all three wheat cultivars. Downstream biochemical analysis involved monitoring the accumulation of ROS biomarkers (superoxide and hydrogen peroxide) as well as the detection of enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD). These biochemical responses were only monitored on the two wheat cultivars which presented contrasting responses to F. oxysporum infection. Results showed that F. oxysporum significantly reduced plant growth, biomass, chlorophyll pigments and relative water content of all three cultivars, with the highest reduction observed for SST 088 relative to SST 015 and SST056. On the other hand, proline content was significantly enhanced in all three wheat cultivars, with the highest increase observed for SST 015 relative to SST 056 and SST 088. Based on the contrasting physiological results observed for these three cultivars, downstream biochemical analysis was focused on SST 015 and SST 088. F. oxysporum trigged an increased in superoxide and hydrogen peroxide contents in both cultivars, with the highest increase observed for SST 088. A similar trend was observed for the extent of lipid peroxidation, manifested as enhanced MDA levels. Furthermore, F. oxysporum differentially altered antioxidant enzyme activity relative to the control of both wheat cultivars. A Significant increase in SOD activity was observed for both cultivars in response to F. oxysporum. However, contrasting responses in APX and POD activity (as seen for the band intensities of individual isoforms) was observed in these wheat cultivars in response to F. oxysporum. Based on the results obtained in this study we suggest that F. oxysporum infection has varying degrees of severity in different wheat cultivars. In light of the significant reduction of plant development coupled with enhanced ROS accumulation and differential antioxidant capacity for SST 015 relative SST 088, we suggest that SST 015 is more resilient to F. oxysporum. We thus conclude that a direct relationship exists between ROS accumulation and antioxidant scavenging in regulating plant tolerance against F. oxysporum pathogens.

Masters of Science / In this study, we observed that phosphate (P) deficiency stunted plant growth and produced plants with poor morphological characteristics (yellow and small leaves). Furthermore, we treated plants with 0.8 mM (control) and 0.02 mM P (deficient) in addition to 6 μM methylglyoxal (MG) and we observed that the plants treated with MG had a higher germination, and better morphological characteristics (the leaves were more dark green and bigger in size) compared to the P deficient plants. However, we also observed that the P deficient plants treated with MG had low levels of both O2- and H2O2 and this could be a possible reason for the improved growth and morphological characteristics. In contrast, the P deficient plants not treated with MG had high levels of O2- and H2O2 which could be the possible reason for the observed cell death. We also performed biochemical assays including superoxide dismutase, ascorbate peroxidase, malondialdehyde content, ascorbic acid content, catalase, and most of the assays showed high levels of reactive oxygen species (ROS) and low levels of antioxidant activities in plants not treated with MG while high levels of antioxidant activities and low levels of ROS were observed in plants treated with exogenous MG. Since nitric oxide (NO) is also known to be a signalling molecule, we did a NO assay and observed that NO content increased under low exogenous doses of MG. From our findings we came to a hypothesis that MG modulates P deficiency stress in P. vulgaris through NO signalling or it might be that NO and MG work in tandem to modulate signalling pathways under P deficiency. Finally, we looked at the nutrient profile and the results showed that while there was a poor nutrient profile generally under P deficiency, there was an improvement in nutrient profile when MG was administered at low doses.

<p><strong>Apstrakt je obrađen tehnologijama za optičko prepoznavanje teksta (OCR).</strong></p><p>Ispitan je uticaj heksanskih, etanolnih i etil-acetanih ekstrakata zrna soje i punomasnih hraniva od zrna soje, ekstrudiranog sojinog griza i hidrotermički tretiranog zrna soje, na termičku i katalitičku oksidaciju metil-linoleata primenom ESR spektroskopije i &quot;spin- traping&quot; tehnike. Utvrđeno je da ispitivani ekstrakti inhibiraju termičku i katalitičku oksidaciju metil-linoleata različitim mehanizmima antioksidativnog delovanja. Ustanovljeni antioksidativni efekti dovedeni su u vezu sa vrstama i sadržajem liposolubilnih (a-tokoferol, P-karotin, ukupni ksantofili) i neliposolubilnih antioksidanata (izoflavoni i fitinska kiselina) soje, odnosno u vezu sa termičkim tretmanima primenjenim u proizvodnji ovih hraniva. Ispitani su i uticaji primene različitih rastvarača (etil-acetat, etanol, acetonitril) na antioksidativne efekte ekstrakata neliposolubilnih antioksidanata punomasnih hraniva od zrna soje tokom termičke i katalitičke oksidacije sojinog ulja primenom ESR.</p> / <p><strong>Abstract was processed by technology for Optical character recognition (OCR).</strong></p><p>The influence of hexane, ethanol and ethyl acetate extracts of soybean grain and full-fat feeds of soybean grain, fullfat extruded soybean grits and hydrothermal treated soybean grain on the thermal and catalytic oxidation of methyl linoleate has been studied by ESR spectroscopy and spin-trapping method. It has been found that the investigated extracts inhibit the thermal and catalytic oxidation of methyl linoleate through different mechanisms of antioxidation action. The antioxidative effects found were related to the kinds and contents of soybean liposolubile (a-tocopherol, P-carotene, total xanthophyls) and nonliposolibile antioxidants (isoflavones, phytic acid), that is, they were put in relation to thermal treatments used in production of these feeds. The effects of the application of different solvents on the antioxidative actions of the extracts of nonliposolubile antioxidants of full-fat soybean feeds during thermal and catalytic oxidation of soybean oil have been studied by ESR.</p>

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Recent evidence suggests that oxidatively modified forms of low-density lipoprotein (LDL) may be particularly atherogenic. In this investigation, the catabolism of human LDL modified by lipid peroxidation in vitro was studied with a recirculating rat liver perfusion system. A dual-labelling technique was used that permitted native LDL and modified LDL to be studied simultaneously in the liver perfusion system. Native human LDL was found to have a fractional catabolic rate (FCR) of 1.00 +/- 0.21%/h, in agreement with other investigators. Subjecting LDL to oxidation for 12 h in the presence of 30 microM FeEDTA did not significantly affect its FCR. LDL treated with a superoxide-generating system (xanthine oxidase, hypoxanthine, O2) in the presence of 30 microM FeEDTA did, however, show a significant increase in FCR (3.23 +/- 0.19%/h). The hepatic uptakes of native LDL and LDL oxidized with FeEDTA+O2 were similar, but both were significantly lower than the hepatic uptake of LDL treated with the superoxide-radical-generating system. The proteolysis of LDL with pancreatin did not influence either its susceptibility to oxidation or its FCR. LDL oxidation resulted in the preferential loss of alpha-tocopherol rather than gamma-tocopherol. These data indicate that the rat liver effectively catabolizes LDL oxidatively modified by treatment with the superoxide-generating system. Furthermore, our results suggest that only very low plasma levels of highly oxidized LDL could be found under conditions in vivo. The liver may therefore play a major role in protecting the arterial vasculature from highly atherogenic forms of LDL.