5-Lipoxygenase (5LO) is a lipid-peroxidizing enzyme which inserts molecular oxygen into fatty acids leading to the biosynthesis of leukotrienes. This protein is widely expressed in the brain including the cortex and hippocampus regions, where its levels and activity increase in an age-dependent manner. Previous work has shown that 5LO modulates both amyloid beta (A) and tau pathology in Alzheimer's disease (AD) models. However, whether the effect of 5LO on tau is direct or indirect still remains unclear. Tau is a microtubule-associated protein usually found in the axons of neurons where it promotes assembly and stabilization of microtubules. In post-mortem brains of AD patients, tau is hyperphosphorylated and altered conformationally, followed by the formation of intracellular aggregates known as neurofibrillary tangles (NFTs), which are also the major pathological hallmark of another group of neurodegenerative diseases collectively referred to as tauopathies such as Pick's Disease, Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD) and Parkinsonism linked to chromosome 17. The central hypothesis of the thesis is that 5LO directly influences tau metabolism, the development of related neuropathology and behavioral phenotype. To prove this hypothesis, a comprehensive genetic and pharmacologic experimental approach, combining both in vivo and in vitro experiments, was implemented. We initially showed that human brains from patients with a confirmed diagnosis of PSP, had significantly higher levels of 5LO when compared with brains form healthy controls. Next, we assayed the levels of 5LO in brains from htau (transgenic tau mice) mice at 4 different age time-points and two regions (cortex and cerebellum). Interestingly, compared with wild type controls, cortices from htau mice had a non-significant increase in 5LO protein levels as early as 6 months of age, which became significant by 10 months of age in the cortex only. Taken together, the age-dependent and region-specificity of the 5LO up-regulation supports the hypothesis that this pathway may have a functional role in the development of the tauopathy phenotype. To prove it, we treated tau mice with a selective 5LO inhibitor, zileuton, and explored the effect on learning and memory. Treatment of the htau mice with zileuton restored their short term working memory and spatial memory deficits. Shortly after completion of the behavioral tests, mice were euthanized and brains harvested for biochemistry and immunohistochemistry analyses. In association with the changes in behavior, we observed that pharmacologic inhibition of 5LO had an influence on tau metabolism, more specifically a significant decrease in tau phosphorylation. In search for the molecular mechanism involved in this biological effect, we assayed different kinases and phosphatase which have been implicated in tau metabolism and showed the specific involvement of the cdk5 pathway. This observation was further confirmed by in vitro studies, in which by using primary neuronal cells we showed that zileuton decreased tau phosphorylation via a cdk-5-dependent mechanism. Since the development of tau pathology results in biochemical and functional manifestation of synaptic deficits, next we assessed levels of pre- and post-synaptic protein markers. Compared with wild type, htau mice had significant reduction in the levels of three distinct markers of synaptic integrity (that is synaptophysin, post-synaptic density protein-95 and microtubule associated protein-2). By contrast, the decrease was completely restored to wild type levels by zileuton treatment. To further support the involvement of this pathway in the improvement of the behavioral and cognitive deficits, we explored the effects of its pharmacological blockade on synaptic function by performing electrophysiological studies. As reported previously, there was a significant difference in Long Term Potentiation (LTP) between the wild type and htau mice, with the latter showing significant deficits. However, pharmacologic blockade of 5LO in the htau mice was adequate to restore the LTP responses to a level comparable to those measured in the wild type mice. In the genetic portion of the study, WT and htau pups were intracranially injected with both AAV2/1 control vector and AAV2/1 5LO vector. Compared with the htau control group, the htau mice injected with AAV2/1 5LO displayed a significant deficits in cognition and memory associated with a decline in their synaptic integrity. Also, genetic upregulation of 5LO yielded significant increases in tau phosphorylation associated with an increase in cdk-5 kinase activation both in vivo and in vitro. Taken together these results describe a pluripotent role for 5LO in the context of tauopathy by representing its direct functional role in modulating behavior along with tau phosphorylation, neuroinflammation and synaptic function in a relevant mouse model of the human disease. The demonstration of the pleiotropic role 5LO in tauopathy pathogenesis makes it not only a valid pharmacological target, as 5LO inhibitors are already FDA approved but, more importantly represents a unique therapeutic opportunity with true disease modifying potential for the treatment of these dementing disorders for which there is no cure. / Pharmacology

Cardiovascular disease (CVD) including atherosclerosis is the leading cause of illness and death in the United States. The American Heart Association indicated that an estimated 81,100,000 American adults have one or more types of CVD and the estimated direct and indirect cost of CVD for 2010 is $503.2 billion, which is $27.9 billion more than last year. Although the exact cause of this disease remains unsolved, previous studies have demonstrated that pro-oxidative and pro-inflammatory pathways in vascular endothelium play a critical role in early pathological events of atherosclerosis. However, the detailed molecular signaling mechanisms underlying this process are not yet completely understood. Recently, the role of interleukin-4 (IL-4) in atherogenesis became controversial and gained attention. IL-4 is a pleiotropic immunomodulatory cytokine secreted by T-helper 2 (Th2) lymphocytes, eosinophils, and mast cells. It was traditionally believed to be an anti-inflammatory cytokine. Increasing evidence, however, has suggested that IL-4 contributes to the initiation and progression of atherosclerosis by oxidative stress-mediated up-regulation of pro-inflammatory mediators such as vascular cell adhesion moledule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in vascular endothelium. 5-Lipoxygenase (5-LOX) is one of the key sources that generate reactive oxygen species (ROS) via metabolic pathways of arachidonic acid. Although 5-LOX has been implicated in the development of atherosclerosis, it remains unclear whether 5-LOX-mediated ROS generation is associated with IL-4-induced MCP-1 expression in vascular endothelium. The present study was focused on the oxidative mechanisms by which IL-4 induces vascular inflammation as well as how 5-LOX is involved in this process. The results showed that IL-4 significantly up-regulates mRNA and protein expression of MCP-1 in vascular endothelium. In addition, DHE and DCF fluorescence staining demonstrated that IL-4 increases ROS production in human vascular endothelial cells. We have also provided the first novel evidence that 5-LOX, one of the enzymes associated with arachidonic acid metabolism, is responsible for the IL-4-induced ROS generation and MCP-1 expression in human vascular endothelial cells. / Master of Science

Lipoxygenasen sind nichthämeisenhaltige Dioxygenasen, die die Bildung von Hydroperoxiden aus molekularem Sauerstoff und mehrfach ungesättigten Fettsäuren katalysieren. Im Rahmen dieser Arbeit wurden verschiedene Lipoxygenase-Ligand-Wechselwirkungen untersucht: a) Durch gezielte Substratveränderungen und ortsgerichtete Mutagenese konnte gezeigt werden, dass Fettsäuren normalerweise mit dem Methylende in der Bindungstasche tierischer Lipoxygenasen gebunden werden. Unterschiedliche Positionsspezifitäten basieren demzufolge auf dem Volumen der Substratbindungstasche (Volumenhypothese). Darüber hinaus konnte bei der 15-Lipoxygenase eine inverse Substratbindung (Carboxylgruppe im aktiven Zentrum) durch Modifikation beider Enden der Fettsäure erzwungen werden, wodurch ausschließlich 5-Lipoxygenierung katalysiert wurde. b) Untersuchungen mit dem Hemmstoff Ebselen ergaben unterschiedliche Hemmmechanismen für verschiedene Enzymzustände. Die Hemmung der Lipoxygenase im Grundzustand (Fe[II]) erfolgt durch kovalente Bindung und Veränderung der Eisenligandensphäre irreversibel nach einem nicht-kompetitiven Mechanismus. Dagegen wird die aktive Lipoxygenase (Fe[III]) nur noch kompetitiv durch Ebselen gehemmt. c) Die Membranbindung der tierischen 15-Lipoxygenase erfolgt über hydrophobe Wechselwirkungen, vermittelt durch oberflächenexponierte, hydrophobe Aminosäuren aus beiden Enzymdomänen. Die Expression einer enzymatisch aktiven Trunkationsmutante, der die N-terminale Domäne fehlt, zeigte, dass diese nicht essentiell ist für die Membranbindung. / Lipoxygenases are nonheme iron-containig dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives. Here, the interaction of lipoxygenases with various ligands was investigated: a) From substrate modifications and site directed mutagenesis it was concluded that fatty acids are bound with their methyl end in the substrate binding pocket of mammalian lipoxygenases. The positional specificity is therefore related to different volumes of this binding pocket (space-based hypothesis). For the 15-lipoxygenase an inverse substrate binding (carboxy terminus in the pocket) could be forced by simultaneous modification of both ends of the fatty acid. This lead to an exclusive 5-lipoxygenation by the 15-lipoxygenase. b) The mechanism of lipoxygenase inhibition by ebselen depends on the enzyme's state. The groundstate lipoxygenase (containing Fe[II]) is irreversibly inhibited in a non-competetive manner due to covalent modification and alteration of the iron ligand sphere. The active enzyme (containing Fe[III]) on the other hand is only competetively inhibited. c) The membrane binding of the mammalian 15-lipoxygenase is based on hydrophobic interactions mediated by solvent exposed, hydrophobic amino acid residues of both enzyme domains. The expression of an enzymatically active truncation mutant, which lacks the entire N-terminal domain, showed that this domain is not essential for membrane binding.

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

Meloidogyne incognita (Kofoid and White) is a major pest of soybean in South Africa and due to its high level of pathogenicity to the crop it is quintessential that research in this regard should receive priority. Root-knot nematode control has in the past mostly included the use of nematicides, while crop rotation and inclusion of cultivars with genetic host plant resistance (henceforth referred to as resistance only) to these pests were also used. Since no synthetically-derived and/or biological agents are registered locally as nematicides on soybean, the use of resistant cultivars represents one of the most viable and environmentally-friendly strategies to protect local soybean crops against damage resulting from parasitism by M. incognita. Although numerous exotic soybean cultivars have been identified with resistance to M. incognita, only a few locally adapted ones have proved to exhibit resistance to the latter species. Moreover, at present Egret is the only cultivar still available for commercial use in South Africa. Little and fragmented information is, however, available on the use of plant enzymes, that are interrelated in biochemical pathways that are expressed in root-knot nematode resistant cultivars, for its use as an additional parameter to exploit such a trait. Therefore, the present study was undertaken to identify M. incognita resistance in selected, locally adapted soybean cultivars by quantifying and exploiting the latter trait by using enzyme activities as an additional parameter. In addition, resistance to M. incognita in selected resistant soybean cultivars was also verified by means of histopathological studies to identify cellular changes associated with the trait. In the first part of the present study, 31 locally adapted soybean cultivars of which 23 were commercially available in the 2012 growing season were evaluated for resistance to M. incognita. The latter was done by means of traditional screening protocols for which M. incognita-gall rating, egg and second-stage juvenile as well as the reproductive factor data per root system for each cultivar screened were recorded. Two greenhouse experiments were subsequently conducted concurrently, one of which the abovementioned nematode parameters were recorded 30 and the other 56 days after inoculation. Reproduction factor values were used as the main criterium to identify M. incognita resistance in local soybean cultivars since it is considered as a more reliable parameter for this specific type of evaluations. Reproduction factor values equal to and lower than one, indicating resistance to the M. incognita population used in this study, were recorded only for cultivar LS5995, as well as seven pre-released GCI cultivars. These eight cultivars also had very low egg, as well as egg and second-stage juvenile counts per root system, all of which differed significantly from the susceptible control, as well as a number of other cultivars. Root gall indices, on the other hand, did not show consistent results in terms of the identification of the host status of the 31 cultivar screened during this study. Using reproduction factor values, local farmers can thus be supplied with information on the resistance of commercially-available soybean cultivars. Eventually, such M. incognita-resistant cultivars can be used to reduce population levels of this nematode pest in fields of producers and also as valuable germplasm sources in breeding programs to introgress/stack this trait in newly-developed soybean cultivars. The second part of the study aimed to verify and exploit M. incognita-resistance in soybean either identified as resistant or susceptible during the screenings experiments, using enzymatic activity as biochemical markers. Cultivar LS5995 was included as the resistant and Dundee as the susceptible standard. The activity of three enzymes, namely guaiacol peroxidase, lipoxygenase and catalase were recorded at different time intervals in roots and leaf samples of the latter cultivars, of both nematode-inoculated and nematode-free plants of each cultivar. Significant (P ≤ 0.05) increases in guaiacol peroxidase activity in leaf and root samples of the M. incognita-resistant cultivars GCI7 and LS5995 (inoculated with J2) were recorded 24 hours (h) after onset of the experiment. Use of this enzyme thus emanated as a useful parameter to identify soybean cultivars that exhibit resistance against M. incognita, especially in leaves, which could substantially reduce the time needed to screen cultivars. In terms of lipoxygenase activity recorded, substantial variation existed between the cultivars tested. The M. incognita-susceptible cultivar Egret was the only cultivar for which a significant (P ≤ 0.05) increase in lipoxygenase activity in the roots was evident 24 h after inoculation. However, during the 48 h sampling time, significant (P ≤ 0.05) differences in lipoxygenase activity were also recorded for the two M. incognita resistant cultivars GCI7 and LS5995. Although the increase in lipoxygenase activity for the susceptible cultivar Egret was unexpected, it may indicate that some level of resistance is present in the latter cultivar, which has in previous studies been identified as resistant to M. incognita. Other factors such as a different M. incognita populations used and temperature differences in greenhouse conditions that applied in this study compared to that for an earlier study may, however, serve as explanations for the latter differences in host status identification of cultivar Egret. In terms of catalase activity recorded in leaf samples of the M. incognita-resistant cultivar LS5995, substantial reductions of as much as 35.6 % were recorded for J2-inoculated plants compared to those of the J2-free control plants. In leaf samples of the susceptible cultivars, Egret and Dundee, catalase was also reduced, but to a lesser extent and ranged from 6 to 26 %. Conversely, catalase activity in the leaves of J2-inoculated plants of the highly susceptible cultivar LS6248R was substantially increased by as much as 29.3 %. Enzyme data obtained as a result of the current study thus generally complemented those of traditional screening assays in which resistance in locally adapted cultivars were identified to a certain degree. It is, however, recommended that enzyme activity, to be used as bio-markers, still needs further refinement and more investigation to optimise their use in identification, verification and exploitation of M. incognita resistance in soybean cultivars. The third and final part of the study encompassed a comparison of cellular changes induced by M. incognita in resistant and susceptible soybean cultivars to verify the resistant reactions expressed in the enzyme data. According to light- and transmission electron microscope observations, distinct differences in the appearance and development of giant cells in roots of the M. incognita-resistant cultivars LS5995 and GCI7 existed when compared to those in roots of the susceptible cultivars Dundee and LS6248R. In the latter cultivars, giant cells that formed were characteristically large and contained a dense cytoplasm, with thick irregularly surfaced cell walls. Cell walls also displayed thick aggregations that appeared to be cell-wall ingrowths. These giant cells are optimal to facilitate M. incognita development and reproduction. In contrast, giant cells that were associated with the resistant cultivars LS5995 and GCI7 were small, irregularly shaped and contained increased amounts of deposited cell-wall material in the cytoplasm known as cell wall inclusions. Necrosis was also present in M. incognita-infected root cells of both cultivars. Such giant cells have been associated with retarded feeding, development and reproduction of the latter root-knot nematode species. However, it was evident that neither GCI7 nor LS5995 are immune to M. incognita since J2 survived and developed to third- and fourth and ultimately mature females that reproduced in their roots. Optimal giant cells that were formed in the roots of the M. incognitasusceptible cultivars Dundee and LS6248R thus supported the nutritional needs of the developing M. incognita individuals and led to significant increases in M. incognita populations 56 days after inoculation as was evident from the high reproduction factor values that were obtained for such cultivars during host status assessments that represented the first part of this study. The opposite was recorded the M. incognita-resistant cultivars LS5995 and GCI7 since sub-optimal giant cells in their roots could not sustain high offspring from such mature females. The presence of necrotic root tissue adjacent to giant cells, furthermore, indicated that hypersensitive reactions occurred in the latter resistant cultivars. Enzyme data obtained in the second part of this study supported the presence of hypersensitive reactions in root cells of the latter resistant cultivars. Guaiacol peroxidase and lipoxygenase inductions in particular in plant tissues have been reported to play integral roles in hypersensitive reactions that are exhibited by cultivars that are resistant to pests and diseases. Finally, results obtained from the different parts of this study complemented each other. It resulted in the resistance that was identified in the GCI7 pre-released cultivar being verified and exploited against that of the resistant standard LS5995. Research that was done during this study also represented the first investigations into the use of enzymes as biochemical markers of resistance against M. incognita in soybean in South Africa. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Meloidogyne incognita (Kofoid and White) is a major pest of soybean in South Africa and due to its high level of pathogenicity to the crop it is quintessential that research in this regard should receive priority. Root-knot nematode control has in the past mostly included the use of nematicides, while crop rotation and inclusion of cultivars with genetic host plant resistance (henceforth referred to as resistance only) to these pests were also used. Since no synthetically-derived and/or biological agents are registered locally as nematicides on soybean, the use of resistant cultivars represents one of the most viable and environmentally-friendly strategies to protect local soybean crops against damage resulting from parasitism by M. incognita. Although numerous exotic soybean cultivars have been identified with resistance to M. incognita, only a few locally adapted ones have proved to exhibit resistance to the latter species. Moreover, at present Egret is the only cultivar still available for commercial use in South Africa. Little and fragmented information is, however, available on the use of plant enzymes, that are interrelated in biochemical pathways that are expressed in root-knot nematode resistant cultivars, for its use as an additional parameter to exploit such a trait. Therefore, the present study was undertaken to identify M. incognita resistance in selected, locally adapted soybean cultivars by quantifying and exploiting the latter trait by using enzyme activities as an additional parameter. In addition, resistance to M. incognita in selected resistant soybean cultivars was also verified by means of histopathological studies to identify cellular changes associated with the trait. In the first part of the present study, 31 locally adapted soybean cultivars of which 23 were commercially available in the 2012 growing season were evaluated for resistance to M. incognita. The latter was done by means of traditional screening protocols for which M. incognita-gall rating, egg and second-stage juvenile as well as the reproductive factor data per root system for each cultivar screened were recorded. Two greenhouse experiments were subsequently conducted concurrently, one of which the abovementioned nematode parameters were recorded 30 and the other 56 days after inoculation. Reproduction factor values were used as the main criterium to identify M. incognita resistance in local soybean cultivars since it is considered as a more reliable parameter for this specific type of evaluations. Reproduction factor values equal to and lower than one, indicating resistance to the M. incognita population used in this study, were recorded only for cultivar LS5995, as well as seven pre-released GCI cultivars. These eight cultivars also had very low egg, as well as egg and second-stage juvenile counts per root system, all of which differed significantly from the susceptible control, as well as a number of other cultivars. Root gall indices, on the other hand, did not show consistent results in terms of the identification of the host status of the 31 cultivar screened during this study. Using reproduction factor values, local farmers can thus be supplied with information on the resistance of commercially-available soybean cultivars. Eventually, such M. incognita-resistant cultivars can be used to reduce population levels of this nematode pest in fields of producers and also as valuable germplasm sources in breeding programs to introgress/stack this trait in newly-developed soybean cultivars. The second part of the study aimed to verify and exploit M. incognita-resistance in soybean either identified as resistant or susceptible during the screenings experiments, using enzymatic activity as biochemical markers. Cultivar LS5995 was included as the resistant and Dundee as the susceptible standard. The activity of three enzymes, namely guaiacol peroxidase, lipoxygenase and catalase were recorded at different time intervals in roots and leaf samples of the latter cultivars, of both nematode-inoculated and nematode-free plants of each cultivar. Significant (P ≤ 0.05) increases in guaiacol peroxidase activity in leaf and root samples of the M. incognita-resistant cultivars GCI7 and LS5995 (inoculated with J2) were recorded 24 hours (h) after onset of the experiment. Use of this enzyme thus emanated as a useful parameter to identify soybean cultivars that exhibit resistance against M. incognita, especially in leaves, which could substantially reduce the time needed to screen cultivars. In terms of lipoxygenase activity recorded, substantial variation existed between the cultivars tested. The M. incognita-susceptible cultivar Egret was the only cultivar for which a significant (P ≤ 0.05) increase in lipoxygenase activity in the roots was evident 24 h after inoculation. However, during the 48 h sampling time, significant (P ≤ 0.05) differences in lipoxygenase activity were also recorded for the two M. incognita resistant cultivars GCI7 and LS5995. Although the increase in lipoxygenase activity for the susceptible cultivar Egret was unexpected, it may indicate that some level of resistance is present in the latter cultivar, which has in previous studies been identified as resistant to M. incognita. Other factors such as a different M. incognita populations used and temperature differences in greenhouse conditions that applied in this study compared to that for an earlier study may, however, serve as explanations for the latter differences in host status identification of cultivar Egret. In terms of catalase activity recorded in leaf samples of the M. incognita-resistant cultivar LS5995, substantial reductions of as much as 35.6 % were recorded for J2-inoculated plants compared to those of the J2-free control plants. In leaf samples of the susceptible cultivars, Egret and Dundee, catalase was also reduced, but to a lesser extent and ranged from 6 to 26 %. Conversely, catalase activity in the leaves of J2-inoculated plants of the highly susceptible cultivar LS6248R was substantially increased by as much as 29.3 %. Enzyme data obtained as a result of the current study thus generally complemented those of traditional screening assays in which resistance in locally adapted cultivars were identified to a certain degree. It is, however, recommended that enzyme activity, to be used as bio-markers, still needs further refinement and more investigation to optimise their use in identification, verification and exploitation of M. incognita resistance in soybean cultivars. The third and final part of the study encompassed a comparison of cellular changes induced by M. incognita in resistant and susceptible soybean cultivars to verify the resistant reactions expressed in the enzyme data. According to light- and transmission electron microscope observations, distinct differences in the appearance and development of giant cells in roots of the M. incognita-resistant cultivars LS5995 and GCI7 existed when compared to those in roots of the susceptible cultivars Dundee and LS6248R. In the latter cultivars, giant cells that formed were characteristically large and contained a dense cytoplasm, with thick irregularly surfaced cell walls. Cell walls also displayed thick aggregations that appeared to be cell-wall ingrowths. These giant cells are optimal to facilitate M. incognita development and reproduction. In contrast, giant cells that were associated with the resistant cultivars LS5995 and GCI7 were small, irregularly shaped and contained increased amounts of deposited cell-wall material in the cytoplasm known as cell wall inclusions. Necrosis was also present in M. incognita-infected root cells of both cultivars. Such giant cells have been associated with retarded feeding, development and reproduction of the latter root-knot nematode species. However, it was evident that neither GCI7 nor LS5995 are immune to M. incognita since J2 survived and developed to third- and fourth and ultimately mature females that reproduced in their roots. Optimal giant cells that were formed in the roots of the M. incognitasusceptible cultivars Dundee and LS6248R thus supported the nutritional needs of the developing M. incognita individuals and led to significant increases in M. incognita populations 56 days after inoculation as was evident from the high reproduction factor values that were obtained for such cultivars during host status assessments that represented the first part of this study. The opposite was recorded the M. incognita-resistant cultivars LS5995 and GCI7 since sub-optimal giant cells in their roots could not sustain high offspring from such mature females. The presence of necrotic root tissue adjacent to giant cells, furthermore, indicated that hypersensitive reactions occurred in the latter resistant cultivars. Enzyme data obtained in the second part of this study supported the presence of hypersensitive reactions in root cells of the latter resistant cultivars. Guaiacol peroxidase and lipoxygenase inductions in particular in plant tissues have been reported to play integral roles in hypersensitive reactions that are exhibited by cultivars that are resistant to pests and diseases. Finally, results obtained from the different parts of this study complemented each other. It resulted in the resistance that was identified in the GCI7 pre-released cultivar being verified and exploited against that of the resistant standard LS5995. Research that was done during this study also represented the first investigations into the use of enzymes as biochemical markers of resistance against M. incognita in soybean in South Africa. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Selection of raw materials for a consistent and high quality end product has been a challenge for brewers globally. Various different factors may influence quality and although a great number of methods for malt analysis exist today for the prediction of end product quality, some still do not accurately represent malt performance in beer. This research focussed on determining parameters in malts to predict two of the major beer quality determining factors namely, foam- and flavour stability. Specific biochemical markers in barley malt such as lipid transfer protein 1 (LTP1) lipoxygenase-1 (LOX-1), anti-radical/oxidant potential (AROP), free amino nitrogen and intact protein were determined and used in beer quality prediction from malt character. These biochemical quality predictions were then correlated with the end product beer quality as assessed in sensory analysis trials on micro-brewed beers. Being such a multi-faceted factor in beer, LTP1 have already become an attractive field of study. LTP1 is primarily associated with stable beer foam, as a foam protein in its own right, and acting as a lipid scavenger. This protein is also theorised to play a role in the stability of beer flavour by possibly acting as anti-oxidant. Lastly LTP1 is known to have anti-yeast activity, which could negatively impact fermentation. In this study LTP1 and its lipid bound isoform LTP1b were successfully purified in an economical and easy five step protocol. Both isoforms showed temperature stability at temperatures >90°C and prefer more neutral and basic pH environments. Although the reported antioxidant activity was not observed, both purified LTP1 and LTP1b inhibited lipoxygenase-1 (LOX-1) activity, which is responsible for the enzymatic breakdown of linoleic acid to form 2(E)-nonenal. This is a novel finding that links LTP1 also to flavour stability. LTP1 exhibited anti-yeast activity whereas LTP1b lost most if not all the activity. However, since most of the LTP1 is converted to LTP1b and glycosylated isoforms during the brewing process fermentation will not be greatly influenced, while foam and flavour stability could still be promoted by the presence of LTP1b. Flavour deterioration of the final packaged product is partially due to the enzymatic production of 2(E)-nonenal by LOX-1 and the presence of free oxygen radical species, limited anti-radical/oxidant potential (AROP) and LTP1. The development of two 96-well micro-assays based on the ferrous oxidation-xylenol orange (FOX) assay for the determination of LOX-1 and AROP was successfully accomplished and compared well with established assays. The LOXFOX and AROP-FOX assays were specifically developed for the on-site, high throughput comparative determination of LOX-1 and AROP in malt and other brewery samples. The AROP-FOX and LOX-FOX micro-assays and a number of established assays were used to categorise malts in different predicted quality groups, various biochemical markers were measured which included LOX activity, LTP1 content, FAN values, intact protein concentration and AROP. An excellent trend (R2=0.93) was found between FAN/LOX and LTP1/LOX which also correlated with the novel observation that LOX-1 activity is inhibited by LTP1 at various concentrations. These trends could assist brewers in optimal blending for not only high quality end products but also fermentation predictions. To determine whether these biochemical markers selected for screening in barley malt are predictive of shelf life potential of the end product, sensory trials were performed. Three barley malt cultivars were selected for LOX, AROP, LTP1, protein and FAN content and used in micro-brewery trials at 0 and 3 months and evaluated using sensory analysis. Good correlation was found between the biochemical predictors and sensory trial for the best quality malt and beer. These parameters were therefore highly relevant for predicting shelf life potential, although additional research is required to elucidate the effect of LTP1 and LOX-1 on each other during the brewing process, since it seems that high LOX-1 concentrations could be leading to LTP1 decreases. With this study it is proposed that if more detailed protein or FAN characterisation is used together with the screening of LOX-1, LTP1 and AROP, an more accurate shelf life prediction, based on malt analysis, is possible and with the help of these parameters brewers can simply blend malts accordingly. / AFRIKAANSE OPSOMMING: Die keuse van roumateriaal om 'n konstante eindproduk van goeie kwaliteit te lewer, was nog altyd 'n uitdaging vir brouers wêreldwyd aangesien verskeie faktore 'n invloed het op die kwaliteit van die produk. Alhoewel daar tans verskeie metodes vir moutanalise bestaan wat die eindproduk–kwaliteit voorspel, is daar min wat werklik die eindproduk kwaliteit soos voorspel deur moutanalise verteenwoordig. Hierdie navorsing fokus op die bepaling van mout-eienskappe om twee van die belangrikste bierkwaliteitvereistes, naamlik skuim- en geurstabiliteit te voorspel. Spesifieke biochemiese eienskappe in garsmout soos lipiedtransportproteien-1 (LTP1), lipoksigenase-1 (LOX-1), antioksidant-antiradikaal potensiaal (AROP), vry aminostikstof (FAN) is geïdentifiseer en gebruik in voorspelling van bierkwaliteit vanaf moutkarakter. Hierdie biochemiese kwaliteit voorspellings is dan gekorreleer met die eindproduk soos ge-evalueer d.m.v sensoriese analise op mikro-gebroude bier. Omdat LTP1 soveel fasette in bier beïnvloed, het dit reeds 'n aanloklike studiefokus geword. LTP1 word hoofsaaklik geassosieer met stabiele skuimkwaliteit in bier en tree op as 'n lipiedmop (“lipid scavenger”). Die proteien speel teoreties ook 'n rol in die stabiliteit van bier geur deur moontlik as 'n anti-oksidant op te tree. Laastens is LTP1 bekend vir sy antigis aktiwiteit wat moontlik 'n negatiewe uitwerking op fermentasies het. Gedurende hierdie navorsing is LTP1 en sy lipiedbinding isoform LTP1b suksesvol gesuiwer met 'n ekonomies en eenvoudige 5-stap protokol. Beide isoforme het stabiliteit by temperature >90°C en meer neutrale en basiese pH omgewings getoon. Alhoewel die voorheen gerapporteerde anti-oksidant aktiwiteit vir LTP1 nie bevestig kon word nie, is daar wel gevind dat beide LTP1 en LTP1b, LOX-1, wat verantwoordelik is vir die ensimatiese afbraak van linoleensuur na 2(E)-nonenal, se aktiwiteit inhibeer. Dit is 'n unieke bevinding wat LTP1 ook koppel aan geurstabiliteit. LTP1 het antigis aktiwiteit getoon, maar LTP1b het die meeste, indien nie alle antigis-aktiwiteit verloor. Omdat die meeste van die LTP1's omgeskakel word na LTP1b's en geglikosileerde isoforme tydens die brouproses, sal fermentasie nie beduidend beinvloed word nie, maar die skuim- en geurstabiliteit sal steeds bevorder word deur die blote teenwoordigheid van die LTP1b. Geurverval van die finale verpakte produk is gedeeltelik a.g.v die ensimatiese produksie van 2(E)-nonenal deur LOX-1 en die teenwoordigheid van vry suurstofradikaal spesies, beperkte AROP en LTP1. Die ontwikkeling van twee 96-putjie mikroessaïs, gebasseer op die yster oksidasie-xilenol oranje (FOX) essai vir die bepaling van LOX-1 en AROP, was suksesvol en het goed vergelyk met reeds gevestigde essaïs. Die LOX-FOX en AROP-FOX mikroessaïs is spesifiek ontwikkel vir die residente, hoë deurvloei vergelykende bepaling van LOX-1 en AROP in mout en ander brouery-monsters. Die AROP-FOX en LOX-FOX mikroessaïs en 'n paar gevestigde essaïs is gebruik om moute te kategoriseer in die verskillende voorspelde kwaliteitsgroepe. Die biochemiese merkers wat gemeet is het die volgende ingesluit: LOX aktiwiteit, LTP1 inhoud, FAN waardes, proteïen konsentrasie en AROP. 'n Merkwaardige korrelasie (R2=0.93) is gevind tussen FAN/LOX en LTP1/LOX wat ook ooreenstem met die waarneming dat LOX-1 aktiwiteit onderdruk word deur LTP1 by verskeie konsentrasies. Hierdie korrelasies kan brouers help met optimale versnitting van moute vir, nie net die hoogste kwaliteit eindproduk nie, maar ook vir fermentasie voorspellings. Om te bepaal of hierdie geselekteerde biochemiese merkers in mout die potensieële raklewe van die eindproduk verteenwoordig, is sensoriese evaluerings uitgevoer. Drie gars-mout kultivars is geselekteer o.g.v LOX-, AROP-, LTP1-, proteïen- en FAN-inhoud en gebruik in mikro-brouery proewe en op 0 en 3 maande en is ge-evalueer deur sensoriese analise. Goeie korrelasie is gevind tussen die biochemiese voorspellers en sensoriese evaluering vir die beste kwaliteit mout en bier. Hierdie maatstawwe is daarom uiters relevant vir voorspelling van die potensiele rakleeftyd, alhoewel addisionele navorsing nodig is om die effek van LTP1 en LOX-1 op mekaar gedurende die brouproses te bepaal. Dit blyk dat 'n hoë LOX-1 konsentrasies kan lei tot 'n afname in LTP1. Met hierdie studie word dit voorstel dat, as meer gedetaileerde proteien of FAN karakterisering saam met LOX-1, LTP1, en AROP analise uitgevoer word, 'n meer akkurate raklewe voorspelling moontlik is en met behulp van hierdie parameters kan brouers moute dienooreenkomstig versnit.