Bibliography: leaves 161-204 / xv, 204 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine and Pathology, 1985

L'étude de l'impact des champignons sur les processus géologiques qui altèrent les mineraux s'appelle la “géo-mycologie”. <br />Les serpentinites sont des roches de la famille des ophiolites et peuvent contenir du chrysotile (amiante serpentine).<br />La réactivité des fibres d'amiante est due à la composition chimique de surface et principalement à la présence d'ions métalliques qui peuvent catalyser des réactions chimiques dangereuses. L'amiante est un problème ambiantale à cause de la présence de roches contenant ce minéral mais aussi à cause des anciennes mines d'amiante. La revalorisation (remediation) de ces sites naturellement contaminés passe par la modification de la toxicité des fibres. Les champignons sont des bons candidats pour la bioremediation de l'amiante.<br />L'objectif de cette thèse est l'isolement de souches fongiques à partir de sols serpentiniques afin de savoir quelles sont les espèces fongiques les plus abondantes dans ces sols et de sélectionner les souches les plus efficaces pour leur interaction avec les fibres d'amiantes. Les modifications des fibres et l'altération du métabolisme fongique ont été considérés.<br />Verticillium leptobactrum semble être l'espèce fongique dominante dans tous les sols serpentiniques examinés : cette espèce n'avait jusque là été que rarement isolée, ce qui rend ces résultats intéressants. <br />Trois espèces fongique peuvent extraire Fe et Mg des fibres de amiante (chrysotile et crocidolite), en modifiant la composition et la réactivité chimique de la surface de fibres et la génotoxicité mesuré dans un système acellulaire.<br />Les champignons expriment, en présence des fibres, des enzymes liés à la réponse aux stress oxydants.

During repetitive stimulation of motor nerve terminals, mitochondrial Ca2+ uptake limits increases in free cytosolic [Ca2+] and helps ensure faithful neuromuscular transmission. Changes in cytosolic [Ca2+] and in mitochondrial [Ca2+] as well as changes in mitochondrial membrane potential (Psi m) were studied in mouse motor nerve terminals using Ca2+ sensitive indicator and potentiometric dyes, respectively. Trains of action potentials (APs) at 50 to 100 Hz produced a rapid increase in mitochondrial [Ca2+] followed by a plateau which usually continued beyond the end of stimulation. After stimulation, mitochondrial [Ca2+] decayed back to baseline over the course of tens of seconds to minutes. Increasing the Ca2+ load delivered to the terminal by increasing the number of stimuli (500-2000), increasing bath [Ca2+], or prolonging the AP with 3,4-diaminopyridine (3-4, DAP, 100 micromolar), prolonged the post-stimulation decay of mitochondrial [Ca2+] without increasing the amplitude of the plateau. Inhibiting openings of the mitochondrial permeability transition pore with cyclosporin A (5 micromolar) had no significant effect on the decay of mitochondrial [Ca2+]. Inhibition of the mitochondrial Na+-Ca2+ exchanger with CGP-37157 (50 micromolar) dramatically prolonged the post-stimulation decay of mitochondrial [Ca2+], reduced post-stimulation residual cytosolic [Ca2+], and reduced the amplitude of end-plate potentials evoked after the end of stimulation. Stimulation-induced mitochondrial Ca2+ uptake resulted in Psi m depolarizations that were small or undetectable at near-physiological temperatures (~30 degrees C). Their amplitude became larger at lower temperatures (~20 degrees C), or when AP duration was increased with 3,4-DAP (20 micromolar). Psi m depolarizations were inhibited by lowering bath [Ca2+] or by blocking P/Q-type Ca2+ channels with omega-agatoxin (0.3 micromolar). Partial inhibition of complex I of the electron transport chain (ETC) with rotenone (50 nM) increased the amplitude of stimulation-induced Psi m depolarizations. These findings suggest that: (1) Ca2+ extrusion from motor terminal mitochondria occurs primarily via the Na+-Ca2+ exchanger and helps sustain post-tetanic transmitter release, and (2) that the depolarization of Psi m that accompanies Ca2+ uptake is limited by accelerated proton extrusion via the ETC.

Das Superoxidradikal kann mit fast allen Bestandteilen von Zellen reagieren und diese schädigen. Die medizinische Forschung stellte eine Beteiligung des Radikals an Krebs, Herzinfarkten und neuraler Degeneration fest. Ein empfindlicher Superoxidnachweis ist daher zum besseren Verständnis von Krankheitsverläufen wichtig. Dabei stellen die geringen typischen Konzentrationen und seine kurze Lebensdauer große Anforderungen. Ziel dieser Arbeit war es zum einen, zwei neuartige Proteinarchitekturen auf Metallelektroden zu entwickeln und deren elektrochemisches Ansprechverhalten zu charakterisieren. Zum anderen waren diese Elektroden zur empfindlichen quantitativen Superoxiddetektion einzusetzen. Im ersten Teil der Arbeit wurde eine Protein-Multischichtelektrode aus Cytochrom c und dem Polyelektrolyten Poly(anilinsulfonsäure) nach dem Layer-by-layer-Verfahren aufgebaut. Für zwei bis 15 Schichten an Protein wurde eine deutliche Zunahme an elektrodenaktivem Cytochrom c mit jedem zusätzlichen Aufbringungsschritt nachgewiesen. Die Zunahme verlief linear und ergab bei 15 Schichten eine Zunahme der redoxaktiven Proteinmenge um deutlich mehr als eine Größenordnung. Während das formale Potential im Multischichtsystem sich im Vergleich zur Monoschichtelektrode nicht veränderte, wurde für die Kinetik eine Abhängigkeit der Geschwindigkeit des Elektronentransfers von der Zahl der Proteinschichten beobachtet. Mit zunehmender Scangeschwindigkeit trat ein reversibler Kontaktverlust zu den äußeren Schichten auf. Die lineare Zunahme an elektroaktivem Protein mit steigender Zahl an Depositionsschritten unterscheidet sich deutlich von in der Literatur beschriebenen Protein/Polyelektrolyt-Multischichtelektroden, bei denen ab etwa 6-8 Schichten keine Zunahme an elektroaktivem Protein mehr festgestelltwurde. Auch ist bei diesen die Zunahme an kontaktierbaren Proteinmolekülen auf das Zwei- bis Fünffache limitiert. <br><br> Diese Unterschiede des neu vorgestellten Systems zu bisherigen Multischichtassemblaten erklärt sich aus einem in dieser Arbeit für derartige Systeme erstmals beschriebenen Elektronentransfermechanismus. Der Transport von Elektronen zwischen der Elektrodenoberfläche und den Proteinmolekülen in den Schichten verläuft über einen Protein-Protein-Elektronenaustausch. Dieser Mechanismus beruht auf dem schnellen Selbstaustausch von Cytochrom c-Molekülen und einer verbleibenden Rotationsflexibilität des Proteins im Multischichtsystem. Die Reduzierung des Proteins durch das Superoxidradikal und eine anschließende Reoxidation durch die Elektrode konnten nachgewiesen werden. In einem amperometrischen Messansatz wurde das durch Superoxidradikale hervorgerufene elektrochemische Signal in Abhängigkeit von der Zahl an Proteinschichten gemessen. Ein maximales Ansprechverhalten auf das Radikal wurde mit 6-Schichtelektroden erzielt. Die Empfindlichkeit der 6-Schichtelektroden wurde im Vergleich zum Literaturwert der Monoschichtelektrode um Faktor 14, also mehr als eine Größenordnung, verbessert. <br><br> Somit konnte eine Elektrode mit 6 Schichten aus Cytochrom c und Poly(anilinsulfonsäure) als neuartiger Superoxidsensor mit einer 14-fachen Verbesserung der Empfindlichkeit im Vergleich zum bislang benutzten System entwickelt werden. <br><br> Der zweite Teil dieser Arbeit beschreibt die Auswahl, Gewinnung und Charakterisierung von Mutanten des Proteins Cu,Zn-Superoxiddismutase zur elektrochemischen Quantifizierung von Superoxidradikalen. Monomere Mutanten des humanen dimeren Enzyms wurden entworfen, die durch Austausch von Aminosäuren ein oder zwei zusätzliche Cysteinreste besaßen, mit welchem sie direkt auf der Goldelektrodenoberfläche chemisorbieren sollten. 6 derartige Mutanten konnten in ausreichender Menge und Reinheit in aktiver Form gewonnen werden. Die Bindung der Superoxiddismutase-Mutanten an Goldoberflächen konnte durch Oberflächen-plasmonresonanz und Impedanzspektroskopie nachgewiesen werden. Alle Mutanten wiesen einen quasi-reversiblen Elektronentransfer zwischen SOD und Elektrode auf. Durch Untersuchung von kupferfreien SOD-Mutanten sowie des Wildtyps konnte nachgewiesen werden, das die Mutanten über die eingefügten Cysteinreste auf der Elektrode chemisorptiv gebunden wurden und der Elektronentransfer zwischen der Elektrode und dem Kupfer im aktiven Zentrum der SOD erfolgte. <br><br> Die Superoxiddismutase katalysiert die Zersetzung von Superoxidmolekülen durch Oxidation und durch Reduktion der Radikale. Somit sind beide Teilreaktionen von analytischem Interesse. Zyklovoltammetrisch konnte sowohl die Oxidation als auch die Reduktion des Radikals durch die immobilisierten Superoxiddismutase-Mutanten nachgewiesen werden. In amperometrischen Messanordnungen konnten beide Teilreaktionen zur analytischen Quantifizierung von Superoxidradikalen genutzt werden. Im positiven Potentialfenster wurde die Empfindlichkeit um einen Faktor von etwa 10 gegenüber der Cytochrom c–Monoschichtelektrode verbessert. / The superoxide radical can react with almost all components of a cell and thus damage them. Enzymatic and non-enzymatic scavengers remove it from the body. An implication of the radical in cancer, heart disease, and neuronal degredation has been found in medical research. Therefore, a sensitive quantification of superoxide is necessary for a better understanding of diseases as well as for the study of biological degradation processes. <br><br> The aim of this work was to develop two new protein architectures on metal electrodes and to characterize their electrochemical behavior. Secondly, both electrodes were to be applied as superoxide biosensors. <br><br> In the first part of the work, a protein multilayer electrode consisting of cytochrome c and the polyelectrolyte poly(aniline sulfonated acid) was built up by the layer-by-layer procedure. SPR experiments proved the formation of multilayers. For 2 to 15 protein layers, a significant increase in electroactive protein was found with every deposition step in a linear fashion. For 15 layers, this increase was found to be more than one order of magnitude. While the formal potential did not change for the proteins in the layers, the rate of electron transfer was found to be dependent on the number of layers deposited. With increased scanning speed, a reversible loss of contact to the outer layers was noted. The linear increase in electroactive protein loading differed significantly from protein/polyelectrolyte electrodes described in the literature, where after 6-8 layers no further increase was found. Additionally, these systems increase the number of electroactive protein molecules only by a factor of 2 to 5. <br><br> These differences can be explained by an electron transfer mechanism which was demonstrated in this work for the first time. The transport of electrons between the electrode surface and the proteins in the layers takes place by a protein-protein electron transfer. This mechanism relies on the fast self-exchange of cytochrome c and a residual rotational flexibility of the protein molecules inside the structure. The reduction of the protein by the radical and its subsequent reoxidation by the electrode could be shown. In the amperometric mode, the sensor signal was determined for 2 to 15 layer electrodes. A maximum signal was found for 6 layers, where the sensitivity was improved by a factor of 14, compared to monolayer sensors. <br><br> The second part of this work describes the selection, production and characterization of mutants of the protein Cu,Zn-superoxide dismutase and their application as superoxide sensors. Monomeric mutants of the human dimeric enzyme were designed, which contained one ore two additional cysteines in order to chemisorb directly onto gold surfaces. 6 such mutants were gained in sufficient amount and purity. The binding to gold was characterized by surface plasmon resonance studies. All mutants showed quasi-reversible electrochemistry on gold electrodes. Experiments with copper-free mutants and the wildtype enzyme proved that the mutants bind to gold via the additional cysteines, while the electron transfer takes place between the electrode and the active site copper. Superoxide dismutases catalyze the removal of superoxide by both oxidation and reduction. Thus, both partial reactions are of analytical interest. In cyclic voltammetry, both oxidation and reduction of the radical could be proved. In amperometric experiments, both reactions were used for a quantification of superoxide concentrations. In the positive potential window, the sensitivity was found to be increased by about one order of magnitude, as compared to the cytochrome c monolayer electrode.<br> -----------<br><br> <b>Hinweis zum Copyright:</b><br>Einige Abbildungen dieser Arbeit sind in Artikeln des Verfassers in den Zeitschriften <i>Angewandte Chemie, Angewandte Chemie International Edition, Analytical Chemisty</i> und <i>Elektroanalysis</i> erschienen.<br> Ihre Darstellung im Rahmen dieser Arbeit erfolgt auch online mit ausdrücklicher Genehmigung der Verlage.

Light and oxygen generate harmful reactive oxygen species (ROS) in the lens, causing biochemical changes that gradually disarrange the lens fibres resulting in light scattering and loss of transparency. In the healthy eye, this chronic exposure to oxidative stress may lead to age-related cataract. However, there are also some conditions that accelerate cataract formation, such as diabetes mellitus, in which increased glucose levels may contribute to increased generation of ROS. The superoxide dismutases (SOD) participate in the defence against ROS by catalysing the dismutation of superoxide radicals. The main SOD isoenzyme in the lens is copper-zinc superoxide dismutase (SOD1). The aim of this thesis was to explore if this antioxidant enzyme is important for the protection against age-related and diabetes-induced cataract development. Lenses from wild-type mice and mice lacking SOD1 were incubated in high levels of glucose in vitro and their transparency and damage evaluated daily. Also, the impact of nitric oxide was studied by adding a nitric oxide synthase inhibitor. Furthermore, in vivo cataract formation in relation to the oxidative status of the lens was evaluated in streptozotocin-induced diabetic mice as well as in non-diabetic mice of both genotypes. Finally, the spontaneous age-related cataract development was studied in both genotypes. In vitro, the SOD1 null lenses showed increased levels of superoxide radicals and developed dense nuclear lens opacities upon exposure to high levels of glucose. They also showed increased lens leakage of lactate dehydrogenase, reduced transport function across cell membranes, and increased water contents. However, the lens damage and cataract formation were eliminated when the synthesis of nitric oxide was inhibited. This indicates that both superoxide and nitric oxide have important roles in glucose-induced cataract development possibly through their reaction with each other which generates the highly reactive peroxynitrite. In vivo, both the SOD1 null and the wild-type mice showed cortical cataract changes after 8 weeks of diabetes, although the SOD1 null mice showed a more pronounced cataract formation than the wild-type mice in relation to the level of hyperglycaemia. As cataract formation was accentuated the lenses showed diminishing levels of glutathione but increasing amounts of protein carbonyls, suggesting a reduced lens antioxidant capacity as well as increased lens protein oxidation. Non-diabetic young (18 weeks of age) SOD1 null mice did not show any signs of cataract. At 1 year of age they had developed some cortical lens obscurity as compared to the wild-type mice which did not show equivalent changes until 2 years of age. The results presented in this thesis show that SOD1 null mice are more prone to develop diabetes-induced and age-related cataract than wild-type mice. The findings thus further endorse the importance of oxidative stress as a contributor to cataract development and indicate that both superoxide and nitric oxide may be damaging to the lens. I therefore conclude that the antioxidant enzyme SOD1 is important for the protection against cataract.

Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation.

Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation.

Hepatocellular carcinoma (HCC) is one of the most common and devastating malignant tumors in Taiwan. Due to an imbalanced between reactive oxygen species (ROS) production and detoxification, oxidative stress, has been implicated in liver carcinogenesis. Superoxide dismutases (SODS) play a key role in the detoxification of superoxide radicals and thus protect cells from damage induced by free radicals. Manganese superoxide dismutase (MnSOD or SOD2) is a member of the superoxide dismutase family located in mitochondria. SOD2 transforms toxic superoxide, a byproduct of the mitochondrial electron transport chain, into hydrogen peroxide and diatomic oxygen. Though reduced SOD2 protein level and activities have been reported in hepatoma tissues, it remains unclear how SOD2 expression affected the tumorigenic processes of hepatoma cells. Expression analysis of an array of human HCC cell lines revealed that SOD2 were down-regulated in poorly differentiated SK-Hep-1 hepatoma cells. Moreover, SOD2 is downregulated in 68.8% of resected HCC samples (97 out of 141 cases). Adenovirus-mediated SOD2 gene delivery increased the cellular SOD2 protein level and H2O2 production, but reduced the superoxide anion level in SK-Hep-1 cells. Furthermore, SOD2 restoration significantly reduced the proliferation, motility, and colony formation of SK-Hep-1 cells. In vivo animal model, the finding of SOD2 overexpression inhibited the proliferation of Sk-Hep-1 hepatoma cells while reduced the tumor growth in mice. Flow cytometry analysis showed that SOD2 gene transfer inhibited the growth of hepatoma cells through induction of cell cycle arrest at G2/M phase. This was associated with declined cdc2/cdk1 and cyclin B1 expression and upregulation p21Cip1 by SOD2 gene delivery. However, SOD2 overexpression had no effect on the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9.In conclusion, SOD2 overexpression suppresses the tumorigenicity of hepatoma cells and may hold promise for HCC treatment.

In this study, effect of cold stress and recovery on the superoxide dismutase (SOD) activities and the expression levels of Cu/ZnSOD gene were investigated in two barley cultivars (Tarm-92-winter type, Zafer-160-spring type). Eight days old barley seedlings were subjected to two different cold stresses / chilling stress at 4&deg / C for 1, 3, 7 days and freezing stress at -3&deg / C and -7&deg / C. Analyses were performed both on leaf and root tissues. The SOD activities and isozyme patterns were determined by Native PAGE activity staining technique. Relative RT-PCR was used for the transcript levels of Cu/ZnSOD gene. The SOD activities and expression levels of control and cold stressed plants were compared by densitometric analysis. Under chilling stress, the activities of Cu/ZnSODx and Cu/ZnSOD2 did not have any significant change in leaf and root tissues of both cultivars. However, the expression levels of Cu/ZnSOD gene were more variable than activity results. After -3&deg / C freezing stress, the activities of SOD enzyme in leaf tissues of Tarm-92 increased significantly, however, these activities significantly decreased in leaves of Zafer-160. These results suggested that both cultivars were not affected by chilling stress in terms of SOD enzyme activities and expression levels. Furthermore, under freezing stress conditions, the increment of SOD activities and expression levels in Zafer-160 was higher than Tarm-92. In conclusion, the changes in SOD isozyme activities and expression levels may not be enough for understanding of the cold stress mechanism. Therefore, further studies have to be carried on other antioxidant enzyme systems.

Life as we know it would be impossible in the absence of oxygen. However, too much oxygen can be toxic to the aerobic organisms which depend on it for their very existence. This apparent paradox arises as a result of oxygen's ability to accept electrons, forming highly reactive (reduced) oxygen species such as superoxide, hydrogen peroxide and the hydroxyl radical. The toxicity of oxygen is greatly enhanced in illuminated plants, due to the photosynthetic reactions which produce both oxygen and highly energetic electrons in close proximity to one another. These problems are further exacerbated when plants are exposed to a variety of stress conditions, since these conditions reduce the ability of plants to utilise excess electrons. As a result of the danger posed by· these reactive oxygen species, plants have· evolved a complex antioxidant system for their scavenging. Research has shown that plants with naturally elevated levels of the components of the antioxidant system are better equipped to deal with stress conditions which enhance the production of reactive oxygen species. A considerable amount of research has thus been dedicated to the elucidation of the antioxidant system. Almost as much research has been dedicated to enhancing the antioxidant system, with the aim of improving plant productivity under stress conditions. This study sought to evaluate plants carrying elevated levels of two of the enzymes of the antioxidant system. For these purposes, tobacco was transformed with the gene for E. coli glutathione reductase (GR), an enzyme believed to catalyse the rate limiting reaction in the scavenging of hydrogen peroxide. This gene was fused to the gene for the RUBISCO small subunit transit peptide - a peptide capable of targeting proteins to the chloroplast. Due to the presence of this peptide the transformed plants exhibited high chloroplastic levels of GR activity. These plants were crossed with a second tobacco transformant carrying high levels of chloroplastic tomato superoxide dismutase (SOD) - an enzyme responsible for the scavenging of superoxide. These hybrid plants were shown to exhibit high GR and SOD activities in the chloroplast .- the subcellular compartment most susceptible to damage caused by reactive oxygen species. The transgenic hybrids were evaluated for their ability to tolerate oxidative stress by treating them with paraquat - a herbicide whose mode of action involves the production of large quantities of activated oxygen. Under stress conditions, plants carrying just E. coli GR showed a slight improvement in their ability to deal with oxidative stress. In contrast to this, the SOD transformants showed more cellular damage than untransformed control plants. This was attributed to the inability of other enzymes in the antioxidant pathway to deal with the increased flow of metabolites through the pathway. The hybrid transformants showed enhanced stress tolerance in the initial stages of oxidative stress, but this declined with ongoing exposure to stress conditions. As with the SOD transformants, this decline in protection was . ascribed to the relatively low activities of the other enzymes in the antioxidant pathway. It was concluded that elevated levels of the two enzymes conferred greater stress tolerance than just one of the enzymes, but for true stress tolerance it will be necessary to evaluate the antioxidant system and enhance the activity of further enzymes in the pathway. It may also be necessary to improve the regulation of transgene expression, ensuring that none of the enzymes are overwhelmed by the increased flow of metabolites through the system. / Thesis (M.Sc.)-University of Natal, 1996