An investigation into the neuroprotective properties of melatonin
- Authors: Southgate, Garrick Steven
- Date: 1999
- Subjects: Melatonin
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3900 , http://hdl.handle.net/10962/d1003959
- Description: Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and circannual rhythms. In the last eight years, a whole new dimension to melatonin’s role in biological organisms has emerged. In 1991 it was discovered [1,2] that melatonin exhibited antioxidant properties. Since then, many researchers [3,4] have found melatonin to be a powerful free radical scavenger and antioxidant. In the present study, the ability of melatonin to offer neuroprotection against glutamate, N-methyl-D-aspartate (NMDA), quinolinic acid (QA) and kainic acid (KA) (collectively referred to as the glutamate receptor agonists) was investigated. It was first shown that stress causes an increase in circulating glucocorticoid concentrations, which resulted in an increase the number of glutamate receptors on synaptic membranes in rat brain homogenate. Melatonin acted to reduce the number of glutamate receptors present on the synaptic membranes, implying that melatonin has neuroprotective properties, as overstimulation of the glutamate receptors leads to excitotoxicity and neurodegeneration. Further investigations showed that the glutamate receptor agonists induce neurodegeneration in primary neuronal cell cultures. Both co-treatment and posttreatment with melatonin against the glutamate receptor agonists, increased neuronal cell viability in a dose dependent manner. Melatonin also appeared to offer protection against quinolinic acid-induced neurodegeneration following intrahippocampal injections of quinolinic acid. The mechanism whereby melatonin offered this protection was investigated. The glutamate receptor agonists caused an increase in intracellular calcium concentrations, which is known [5] to be responsible for initiating the excitotoxic response. Melatonin had no effect on regulating intracellular calcium concentrations Additional studies indicated that melatonin was effective at scavenging superoxide radicals. Production of superoxide radicals was induced by the glutamate receptor agonists in primary neuronal cultures. Superoxide radicals induce lipid peroxidation, which involves the destruction of lipid membranes by chain reactions. By acting as an antioxidant, melatonin was able to reduce quinolinic acid-induced lipid peroxidation in rat brain homogenate, in a dose dependent manner. Melatonin was also effective at reducing lipid peroxidation induced by the glutamate receptor agonists in primary neuronal cultures. Melatonin therefore appeared to be offering neuroprotection by removing superoxide radicals and inhibiting lipid peroxidation. It had been reported [6] that melatonin inhibits nitric oxide synthase activity. This enzyme produces the free radical, nitric oxide, and can also produce superoxide radicals. Melatonin was able to reduce nitric oxide synthase activity in a dose dependent manner. This is a novel method of neuroprotection, as melatonin was now acting as an enzyme regulator. The results obtained demonstrate that melatonin offers neuroprotection against glutamate induced excitotoxicity, by removing free radicals and preventing lipid peroxidation. The neurohormone offers further protection by decreasing the activity of enzymes that aid in the neurotoxic cascade. Melatonin is the most potent naturally occurring free radical scavenger in the body [3]. During aging, the serum concentrations of melatonin decrease [7]. During the senescence of life, free radical damage to the body is at its highest [8], while at the same time melatonin concentrations are at their lowest. Melatonin therefore shows potential for the treatment of diseases and disorders that exhibit an excitotoxic pathology.
- Full Text:
- Date Issued: 1999
- Authors: Southgate, Garrick Steven
- Date: 1999
- Subjects: Melatonin
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3900 , http://hdl.handle.net/10962/d1003959
- Description: Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and circannual rhythms. In the last eight years, a whole new dimension to melatonin’s role in biological organisms has emerged. In 1991 it was discovered [1,2] that melatonin exhibited antioxidant properties. Since then, many researchers [3,4] have found melatonin to be a powerful free radical scavenger and antioxidant. In the present study, the ability of melatonin to offer neuroprotection against glutamate, N-methyl-D-aspartate (NMDA), quinolinic acid (QA) and kainic acid (KA) (collectively referred to as the glutamate receptor agonists) was investigated. It was first shown that stress causes an increase in circulating glucocorticoid concentrations, which resulted in an increase the number of glutamate receptors on synaptic membranes in rat brain homogenate. Melatonin acted to reduce the number of glutamate receptors present on the synaptic membranes, implying that melatonin has neuroprotective properties, as overstimulation of the glutamate receptors leads to excitotoxicity and neurodegeneration. Further investigations showed that the glutamate receptor agonists induce neurodegeneration in primary neuronal cell cultures. Both co-treatment and posttreatment with melatonin against the glutamate receptor agonists, increased neuronal cell viability in a dose dependent manner. Melatonin also appeared to offer protection against quinolinic acid-induced neurodegeneration following intrahippocampal injections of quinolinic acid. The mechanism whereby melatonin offered this protection was investigated. The glutamate receptor agonists caused an increase in intracellular calcium concentrations, which is known [5] to be responsible for initiating the excitotoxic response. Melatonin had no effect on regulating intracellular calcium concentrations Additional studies indicated that melatonin was effective at scavenging superoxide radicals. Production of superoxide radicals was induced by the glutamate receptor agonists in primary neuronal cultures. Superoxide radicals induce lipid peroxidation, which involves the destruction of lipid membranes by chain reactions. By acting as an antioxidant, melatonin was able to reduce quinolinic acid-induced lipid peroxidation in rat brain homogenate, in a dose dependent manner. Melatonin was also effective at reducing lipid peroxidation induced by the glutamate receptor agonists in primary neuronal cultures. Melatonin therefore appeared to be offering neuroprotection by removing superoxide radicals and inhibiting lipid peroxidation. It had been reported [6] that melatonin inhibits nitric oxide synthase activity. This enzyme produces the free radical, nitric oxide, and can also produce superoxide radicals. Melatonin was able to reduce nitric oxide synthase activity in a dose dependent manner. This is a novel method of neuroprotection, as melatonin was now acting as an enzyme regulator. The results obtained demonstrate that melatonin offers neuroprotection against glutamate induced excitotoxicity, by removing free radicals and preventing lipid peroxidation. The neurohormone offers further protection by decreasing the activity of enzymes that aid in the neurotoxic cascade. Melatonin is the most potent naturally occurring free radical scavenger in the body [3]. During aging, the serum concentrations of melatonin decrease [7]. During the senescence of life, free radical damage to the body is at its highest [8], while at the same time melatonin concentrations are at their lowest. Melatonin therefore shows potential for the treatment of diseases and disorders that exhibit an excitotoxic pathology.
- Full Text:
- Date Issued: 1999
Development and characterisation of a membrane gradostat bioreactor for the bioremediation of aromatic pollutants using white rot fungi
- Authors: Leukes, W
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
- Full Text:
- Date Issued: 1999
- Authors: Leukes, W
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
- Full Text:
- Date Issued: 1999
Development of integrated biological processing for the biodesalination of sulphate- and metal-rich wastewaters
- Authors: Boshoff, Genevieve Ann
- Date: 1999
- Subjects: Sewage -- Purification -- Biological treatment Sulfates Mineral industries -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3899 , http://hdl.handle.net/10962/d1003958
- Description: The substantial pollution threat to the South African environment from acid mine drainage (AMD) effluents has been well documented. Due to the juvenile nature of acidity in these flows, any remediation strategies implemented will need to function effectively and at low cost for long periods of time. The widespread use of sulphate reducing biological systems for the treatment of such effluents, and in particular large volume flows, has been limited. The supply of inexpensive electron donor and carbon sources, as well as appropriate reactor designs capable of handling large volume flows, have been identified as among the principal factors limiting development of this technology. The broad aim of the research programme reported here was to undertake an evaluation of the feasibility of an algal-bacterial integrated ponding system for the treatment of AMD, and the waste stabilisation pond (WSP) as an appropriate reactor design for this application. The study attempted to demonstrate the feasibility of individual unit operations in a proposed process train using complex organic carbon serving as the electron donor source for the sulphate reducing bacteria (SRB). Studies were undertaken as laboratory and pilot-scale investigations. Tannery effluent was shown to be a functional carbon source for biological sulphate reduction, with effective removal of sulphate and organics being recorded. In turn, the use of biological sulphate reduction for the treatment of tannery effluent was demonstrated. Algal biomass was shown in laboratory studies to function as an effective carbon source for biological sulphate reduction. It is known that micro-algae produce large quantities of photosynthate which is released to the growth medium under conditions of physiological stress. The potential for the use of photosynthate production in high rate algal ponding systems and its manipulation and use as a sustainable carbon source for sulphate reduction was investigated. Growth of a mixed culture of Dunaliella under conditions of light, temperature and salinity stress demonstrated production of large quantities of organic carbon. However, growth was inhibited at high temperatures. An elevation of salinity levels led to a decrease in growth of Dunaliella, but to increased organic carbon production. Spirulina spp., on the other hand, grew well at higher temperatures but showed the highest organic carbon production, and release to the medium, under low light conditions. These results led to a proposed process for the integration of algal ponding into an integrated system for the treatment of AMD. The algal biomass may be fed into the anaerobic digester as a carbon source, or it may be passed into a High Rate Algal Pond (HRAP) where it is stressed to enhance the organic carbon content. This can then be fed into the anaerobic digester as a carbon source. The impact of high levels of sulphide in the water feeding to the algal growth compartment was investigated. Spirulina spp. isolated from a tannery waste stabilisation pond was shown to be a sulphidophilic strain of cyanobacterium, capable of being adapted to high concentrations of sulphide. Dunaliella salina on the other hand was less tolerant. These results demonstrated the practical use of algal biomass providing an oxygen-rich cap for odour control on the surface of the facultative pond as well for the secondary treatment of sulphide-rich overflow to the High Rate Algal Pond. The ability of micro-algae to elevate the pH of their surrounding environment was evaluated as a functional precipitant and neutralisation reagent for acidic metal containing wastewater. Spirulina spp. was shown to perform effectively. D. salina was less functional in this environment. Anacystis spp. was effective in elevating the pH of a defined medium as well as a zinc-rich effluent. These results indicated the practicality of a neutralising function for algal ponds in the treatment of AMD. Metal removal in the system was found to be a combined function of sulphide precipitation, removal by binding to micro-algal biomass and extracellular polymeric substances. The feasibility of waste stabilisation ponding technology use for the treatment of large volume AMD effluents was provisionally demonstrated. It was shown that complex carbon sources would be used as efficient electron donors for sulphate reduction. The integration of algal ponding into the system provides for the generation of a sustainable carbon source, odour control with the recycling of oxygen-rich water onto the top of the facultative pond, secondary treatment of the anaerobic digester overflow, and the neutralisation of the incoming acidic effluents and removal of heavy metals. Integration of the individual unit operations, the feasibility of which has been provisionally demonstrated in this study, into a continuous process train is being investigated in follow-upstudies.
- Full Text:
- Date Issued: 1999
- Authors: Boshoff, Genevieve Ann
- Date: 1999
- Subjects: Sewage -- Purification -- Biological treatment Sulfates Mineral industries -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3899 , http://hdl.handle.net/10962/d1003958
- Description: The substantial pollution threat to the South African environment from acid mine drainage (AMD) effluents has been well documented. Due to the juvenile nature of acidity in these flows, any remediation strategies implemented will need to function effectively and at low cost for long periods of time. The widespread use of sulphate reducing biological systems for the treatment of such effluents, and in particular large volume flows, has been limited. The supply of inexpensive electron donor and carbon sources, as well as appropriate reactor designs capable of handling large volume flows, have been identified as among the principal factors limiting development of this technology. The broad aim of the research programme reported here was to undertake an evaluation of the feasibility of an algal-bacterial integrated ponding system for the treatment of AMD, and the waste stabilisation pond (WSP) as an appropriate reactor design for this application. The study attempted to demonstrate the feasibility of individual unit operations in a proposed process train using complex organic carbon serving as the electron donor source for the sulphate reducing bacteria (SRB). Studies were undertaken as laboratory and pilot-scale investigations. Tannery effluent was shown to be a functional carbon source for biological sulphate reduction, with effective removal of sulphate and organics being recorded. In turn, the use of biological sulphate reduction for the treatment of tannery effluent was demonstrated. Algal biomass was shown in laboratory studies to function as an effective carbon source for biological sulphate reduction. It is known that micro-algae produce large quantities of photosynthate which is released to the growth medium under conditions of physiological stress. The potential for the use of photosynthate production in high rate algal ponding systems and its manipulation and use as a sustainable carbon source for sulphate reduction was investigated. Growth of a mixed culture of Dunaliella under conditions of light, temperature and salinity stress demonstrated production of large quantities of organic carbon. However, growth was inhibited at high temperatures. An elevation of salinity levels led to a decrease in growth of Dunaliella, but to increased organic carbon production. Spirulina spp., on the other hand, grew well at higher temperatures but showed the highest organic carbon production, and release to the medium, under low light conditions. These results led to a proposed process for the integration of algal ponding into an integrated system for the treatment of AMD. The algal biomass may be fed into the anaerobic digester as a carbon source, or it may be passed into a High Rate Algal Pond (HRAP) where it is stressed to enhance the organic carbon content. This can then be fed into the anaerobic digester as a carbon source. The impact of high levels of sulphide in the water feeding to the algal growth compartment was investigated. Spirulina spp. isolated from a tannery waste stabilisation pond was shown to be a sulphidophilic strain of cyanobacterium, capable of being adapted to high concentrations of sulphide. Dunaliella salina on the other hand was less tolerant. These results demonstrated the practical use of algal biomass providing an oxygen-rich cap for odour control on the surface of the facultative pond as well for the secondary treatment of sulphide-rich overflow to the High Rate Algal Pond. The ability of micro-algae to elevate the pH of their surrounding environment was evaluated as a functional precipitant and neutralisation reagent for acidic metal containing wastewater. Spirulina spp. was shown to perform effectively. D. salina was less functional in this environment. Anacystis spp. was effective in elevating the pH of a defined medium as well as a zinc-rich effluent. These results indicated the practicality of a neutralising function for algal ponds in the treatment of AMD. Metal removal in the system was found to be a combined function of sulphide precipitation, removal by binding to micro-algal biomass and extracellular polymeric substances. The feasibility of waste stabilisation ponding technology use for the treatment of large volume AMD effluents was provisionally demonstrated. It was shown that complex carbon sources would be used as efficient electron donors for sulphate reduction. The integration of algal ponding into the system provides for the generation of a sustainable carbon source, odour control with the recycling of oxygen-rich water onto the top of the facultative pond, secondary treatment of the anaerobic digester overflow, and the neutralisation of the incoming acidic effluents and removal of heavy metals. Integration of the individual unit operations, the feasibility of which has been provisionally demonstrated in this study, into a continuous process train is being investigated in follow-upstudies.
- Full Text:
- Date Issued: 1999
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