High-throughput modelling and structural investigation of cysteine protease complexes with protein inhibitors
- Authors: Kroon, Matthys Christoffel
- Date: 2013
- Subjects: Cysteine proteinases Cysteine proteinases -- Inhibitors Papain Cystatins Malaria -- Chemotherapy Homology (Biology) Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3885 , http://hdl.handle.net/10962/d1001619
- Description: The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb
- Full Text:
- Date Issued: 2013
- Authors: Kroon, Matthys Christoffel
- Date: 2013
- Subjects: Cysteine proteinases Cysteine proteinases -- Inhibitors Papain Cystatins Malaria -- Chemotherapy Homology (Biology) Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3885 , http://hdl.handle.net/10962/d1001619
- Description: The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb
- Full Text:
- Date Issued: 2013
The microbial production of polyphenol oxidase enzyme systems and their application in the treatment of phenolic wastewaters
- Scherman, Patricia Ann (neé Goetch)
- Authors: Scherman, Patricia Ann (neé Goetch)
- Date: 1992
- Subjects: Phenol oxidase Water -- Purification -- Biological treatment Enzymes -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4047 , http://hdl.handle.net/10962/d1004108
- Description: Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.
- Full Text:
- Date Issued: 1992
- Authors: Scherman, Patricia Ann (neé Goetch)
- Date: 1992
- Subjects: Phenol oxidase Water -- Purification -- Biological treatment Enzymes -- Regulation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4047 , http://hdl.handle.net/10962/d1004108
- Description: Phenolic compounds are a group of organic chemicals present in the wastewaters of many synthetic industrial processes. Due to their extreme toxicity to man and animals, and deleterious impact on the environment, a range of techniques exist for the effective treatment and disposal of these pollutants. Biological degradation using microbial enzymes presents a valuable alternative to conventional wastewater treatment systems. This research was therefore initiated to investigate the polyphenol oxidase enzyme system and the feasibility of its application for effluent treatment and studies in organic solvents. The enzyme system is widely distributed in nature, with Agaricus bisporus (the common mushroom) being the best known producer. Biochemical investigations of the enzyme system were therefore carried out using this extract. A screening programme was initiated to identify microbial polyphenol oxidase producers which could be cultured in liquid media, thereby enabling the production of large quantities of enzyme in fermentation systems. Extensive growth optimization and enzyme induction and optimization studies were carried out on selected cultures. A number of good producers were isolated, namely a bacterial culture designated AECI culture no. 26, Streptomyces antibioticus, Streptomyces glaucescens and a manipulated strain, Streptomyces lividans (pIJ702). Enzyme production by Agaricus bisporus mycelia was optimized in deep-liquid culture; enzyme extracts showed high phenol removal efficiencies. Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lividans (pIJ702) and AECI culture no. 26 whole cells were also investigated for phenol-removing ability in simulated phenolic effluents. The use of whole cells reduces enzyme inactivation and instability due to the protection of the enzyme system within the cell. All cultures showed improved removal efficiencies in phenolic growth media. These results strongly suggest their use for phenol removal in continuous systems.
- Full Text:
- Date Issued: 1992
The effect of combined vitamin E succinate and ascorbic acid supplementation on growth and cyclooxygenase expression in murine melanoma (BL6) cells
- Authors: Van Rooyen, Megan Lynne
- Date: 1999
- Subjects: Vitamin E , Vitamin C , Melanoma
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3982 , http://hdl.handle.net/10962/d1004041 , Vitamin E , Vitamin C , Melanoma
- Description: This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.
- Full Text:
- Date Issued: 1999
- Authors: Van Rooyen, Megan Lynne
- Date: 1999
- Subjects: Vitamin E , Vitamin C , Melanoma
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3982 , http://hdl.handle.net/10962/d1004041 , Vitamin E , Vitamin C , Melanoma
- Description: This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.
- Full Text:
- Date Issued: 1999
Isolation, purification and characterization of inulin and fructooligosaccharides from chicorium intybus and inulinase from aspergillus niger
- Mavumengwana, Vuyo Bhongelethu
- Authors: Mavumengwana, Vuyo Bhongelethu
- Date: 2005
- Subjects: Aspergillus , Inulin , Chicory -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3954 , http://hdl.handle.net/10962/d1004013 , Aspergillus , Inulin , Chicory -- South Africa
- Description: Inulin is a non-digestible carbohydrate fructan polymer consisting mainly of β (1→2) fructosyl fructose links. Enzymatic hydrolysis of inulin by inulinase results in the production of low D.P (degree of polymerization) oligosaccharides also called fructooligosaccharides(FOS). Isolation of inulin from chicory root (Chicorium intybus) was achieved by first, extraction using deionized water (600C), followed by carbonation (0.1 M Ca(OH)2 and CO2 gas). This was filtered in order to remove the non sugars, thereafter, treated successfully with polyamide 6 powder. A cation exchanger and an anion exchanger were used to further exclude other components such as tannins and pigments. The extracted inulin was quantified using the Somogyi-Nelson colourimetric assay. Chicory root (207 g, 30 % being water) yielded 30 g of the raw extract. A 100 mg of the raw extract was assayed and found to contain 11 % yield of inulin which was 80.2 % in purity and 4 % free fructose. Analysis of the crude and purified inulin extracts on the MALDI TOF spectrometry showed the samples to have a DP of 2 to 22 and 2 to 27 respectively. Maximum inulinase production from Aspergillus niger grown on inulin was observed after 60 hours. The enzyme activity was found to be 1.168 U/ml with a temperature and pH optimum of 30 °C and 7.7 respectively. The enzyme proved to be unstable as it progressively lost its total activity during attempts at purification.
- Full Text:
- Date Issued: 2005
- Authors: Mavumengwana, Vuyo Bhongelethu
- Date: 2005
- Subjects: Aspergillus , Inulin , Chicory -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3954 , http://hdl.handle.net/10962/d1004013 , Aspergillus , Inulin , Chicory -- South Africa
- Description: Inulin is a non-digestible carbohydrate fructan polymer consisting mainly of β (1→2) fructosyl fructose links. Enzymatic hydrolysis of inulin by inulinase results in the production of low D.P (degree of polymerization) oligosaccharides also called fructooligosaccharides(FOS). Isolation of inulin from chicory root (Chicorium intybus) was achieved by first, extraction using deionized water (600C), followed by carbonation (0.1 M Ca(OH)2 and CO2 gas). This was filtered in order to remove the non sugars, thereafter, treated successfully with polyamide 6 powder. A cation exchanger and an anion exchanger were used to further exclude other components such as tannins and pigments. The extracted inulin was quantified using the Somogyi-Nelson colourimetric assay. Chicory root (207 g, 30 % being water) yielded 30 g of the raw extract. A 100 mg of the raw extract was assayed and found to contain 11 % yield of inulin which was 80.2 % in purity and 4 % free fructose. Analysis of the crude and purified inulin extracts on the MALDI TOF spectrometry showed the samples to have a DP of 2 to 22 and 2 to 27 respectively. Maximum inulinase production from Aspergillus niger grown on inulin was observed after 60 hours. The enzyme activity was found to be 1.168 U/ml with a temperature and pH optimum of 30 °C and 7.7 respectively. The enzyme proved to be unstable as it progressively lost its total activity during attempts at purification.
- Full Text:
- Date Issued: 2005
Evaluation and application of electroanalysis for the determination of antioxidants
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
- Full Text:
- Date Issued: 2007
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
- Full Text:
- Date Issued: 2007
Pineal-adrenal gland interactions in search of an anti-stressogenic role for melatonin
- Authors: Van Wyk, Elizabeth Joy
- Date: 1993
- Subjects: Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4054 , http://hdl.handle.net/10962/d1004115 , Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Description: The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.
- Full Text:
- Date Issued: 1993
- Authors: Van Wyk, Elizabeth Joy
- Date: 1993
- Subjects: Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4054 , http://hdl.handle.net/10962/d1004115 , Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Description: The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.
- Full Text:
- Date Issued: 1993
The role of Hsp90 in the Wnt pathway of MCF7 breast cancer cells
- Authors: Cooper, Leanne Claire
- Date: 2011
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3985 , http://hdl.handle.net/10962/d1004044 , Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Description: Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.
- Full Text:
- Date Issued: 2011
- Authors: Cooper, Leanne Claire
- Date: 2011
- Subjects: Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3985 , http://hdl.handle.net/10962/d1004044 , Cancer -- Treatment , Heat shock proteins , Cancer cells , Molecular chaperones
- Description: Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.
- Full Text:
- Date Issued: 2011
Recombinant expression, purification and in vitro interaction analysis of HOP and RhoC
- Vaaltyn, Michaelone Chantelle
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
Bacterial degradation of the acaricide amitraz
- Authors: Baker, Penelope Bridget
- Date: 1976
- Subjects: Acaricides , Biodegradation , Gas chromatography , Bacteriology -- Cultures and culture media
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4099 , http://hdl.handle.net/10962/d1009498
- Description: This thesis describes dip tank field trials and laboratory investigations on the acaricide Amitraz. Amitraz is a triazapenta- diene compound which is relatively unstable in fouled dip washes. The field trials were conducted on the farm Sea View according to the "Total Replacement Method" and on the farm Sea Ways according to the "Lime Stabilization Method" of dipping. The results of these trials showed that Amitraz was stable in clean dip washes, and under conditions of high pH resulting from the addition of slaked lime to the dip wash. Using mixed bacterial populations optimum conditions for degradation of Amitraz in the laboratory were determined. Bacterial cultures degraded Amitraz most efficiently in media supplemented with yeast extract or with a high content of sterile cattle faeces. Amitraz concentrations were determined by gas chromatography. A culture. efficient at degrading Amitraz was enriched from a dip tank sludge inoculum. From this culture ten bacterial isolates were identified; nine of these were of the genus Pseudomonas and one was an Achromobacter sp. Experiments with both mixed and pure cultures demonstrated that bacterial degradation of Amitraz was by the process of co-metabolism. The existence of four degradation products was shown using thin layer chromatography. Tentative identification of two of the products was made.
- Full Text:
- Date Issued: 1976
- Authors: Baker, Penelope Bridget
- Date: 1976
- Subjects: Acaricides , Biodegradation , Gas chromatography , Bacteriology -- Cultures and culture media
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4099 , http://hdl.handle.net/10962/d1009498
- Description: This thesis describes dip tank field trials and laboratory investigations on the acaricide Amitraz. Amitraz is a triazapenta- diene compound which is relatively unstable in fouled dip washes. The field trials were conducted on the farm Sea View according to the "Total Replacement Method" and on the farm Sea Ways according to the "Lime Stabilization Method" of dipping. The results of these trials showed that Amitraz was stable in clean dip washes, and under conditions of high pH resulting from the addition of slaked lime to the dip wash. Using mixed bacterial populations optimum conditions for degradation of Amitraz in the laboratory were determined. Bacterial cultures degraded Amitraz most efficiently in media supplemented with yeast extract or with a high content of sterile cattle faeces. Amitraz concentrations were determined by gas chromatography. A culture. efficient at degrading Amitraz was enriched from a dip tank sludge inoculum. From this culture ten bacterial isolates were identified; nine of these were of the genus Pseudomonas and one was an Achromobacter sp. Experiments with both mixed and pure cultures demonstrated that bacterial degradation of Amitraz was by the process of co-metabolism. The existence of four degradation products was shown using thin layer chromatography. Tentative identification of two of the products was made.
- Full Text:
- Date Issued: 1976
Identification of SANCDB compounds against G2019S and I2020T variants of leucine-rich repeat Kinase 2 (LRRK2) for the development of drugs against Parkinson’s Disease
- Authors: Baye, Bertha Cinthia
- Date: 2020
- Subjects: Antiparkinsonian agents , Parkinson's disease -- Treatment , Protein kinases , Parkinson's disease -- Chemotherapy , Molecules -- Models
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/138764 , vital:37671
- Description: Parkinson’s disease is a type of movement disorder that occurs when nerve cells in the brain stop producing dopamine. It is the second neurodegenerative disease affecting 1-2% of people above the ages of 65 years old. There is a worldwide prevalence of 7 to 10 million affected people of all cultures and race. Studies have shown that mutation that causes Parkinson’s disease result in increased kinase activity. The c.6055 G > A in exon 41 is the most prevalent LRRK2 variation which causes a substitution of glycine to serine in G2019S in the highly activated loop of its MAP kinase domain. The LRRK2 G2019S variant is the most common genetic determinant of Parkinson’s disease identified to date. This work focused on building accurate 3D models of the LRRK2 kinase domain, that were used for large-scale in silico docking against South African natural compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/). Molecular docking was performed to identify compounds that formed interactions with the active site of the protein and had the lowest binding energy scores. Molecular dynamics simulations showed different movements of the protein-ligand complexes and behavioural difference of the wildtype and the variants, all three structures proved to be compact. Network analysis was done to study residue interactions, contact maps, dynamic cross correlations, average BC and average L were used to study the residue interactions and general residue contribution to the functioning of the protein..
- Full Text:
- Date Issued: 2020
- Authors: Baye, Bertha Cinthia
- Date: 2020
- Subjects: Antiparkinsonian agents , Parkinson's disease -- Treatment , Protein kinases , Parkinson's disease -- Chemotherapy , Molecules -- Models
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/138764 , vital:37671
- Description: Parkinson’s disease is a type of movement disorder that occurs when nerve cells in the brain stop producing dopamine. It is the second neurodegenerative disease affecting 1-2% of people above the ages of 65 years old. There is a worldwide prevalence of 7 to 10 million affected people of all cultures and race. Studies have shown that mutation that causes Parkinson’s disease result in increased kinase activity. The c.6055 G > A in exon 41 is the most prevalent LRRK2 variation which causes a substitution of glycine to serine in G2019S in the highly activated loop of its MAP kinase domain. The LRRK2 G2019S variant is the most common genetic determinant of Parkinson’s disease identified to date. This work focused on building accurate 3D models of the LRRK2 kinase domain, that were used for large-scale in silico docking against South African natural compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/). Molecular docking was performed to identify compounds that formed interactions with the active site of the protein and had the lowest binding energy scores. Molecular dynamics simulations showed different movements of the protein-ligand complexes and behavioural difference of the wildtype and the variants, all three structures proved to be compact. Network analysis was done to study residue interactions, contact maps, dynamic cross correlations, average BC and average L were used to study the residue interactions and general residue contribution to the functioning of the protein..
- Full Text:
- Date Issued: 2020
Integrated anaerobic/aerobic bioprocess environments and the biodegradation of complex hydrocarbon wastes
- Authors: Ehlers, George A C
- Date: 2004
- Subjects: Hydrocarbons -- Biodegradation Sewage -- Purification -- Anaerobic treatment Water -- Purification -- Biological treatment Anaerobic bacteria Aerobic bacteria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4011 , http://hdl.handle.net/10962/d1004071
- Description: An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.
- Full Text:
- Date Issued: 2004
- Authors: Ehlers, George A C
- Date: 2004
- Subjects: Hydrocarbons -- Biodegradation Sewage -- Purification -- Anaerobic treatment Water -- Purification -- Biological treatment Anaerobic bacteria Aerobic bacteria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4011 , http://hdl.handle.net/10962/d1004071
- Description: An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.
- Full Text:
- Date Issued: 2004
The effect of sewage effluent from De Beers marine diamond mining operations on the expression of cytochrome P450 (CYP1A) and vitellogenin (vtg)
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
Biological synthesis of metallic nanoparticles and their interactions with various biomedical targets
- Authors: Sennuga, Afolake Temitope
- Date: 2012
- Subjects: Nanoparticles Biosynthesis Nanotechnology Biomineralization Morphology Ceruloplasmin Ribonucleases Adenosine triphosphatase Acetylcholinesterase Platinum Gold Silver
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4009 , http://hdl.handle.net/10962/d1004069
- Description: The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.
- Full Text:
- Date Issued: 2012
- Authors: Sennuga, Afolake Temitope
- Date: 2012
- Subjects: Nanoparticles Biosynthesis Nanotechnology Biomineralization Morphology Ceruloplasmin Ribonucleases Adenosine triphosphatase Acetylcholinesterase Platinum Gold Silver
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4009 , http://hdl.handle.net/10962/d1004069
- Description: The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.
- Full Text:
- Date Issued: 2012
Establishing experimental systems for studying the replication biology of Providence virus
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Date Issued: 2009
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Date Issued: 2009
Metallophthalocyanines as photocatalysts for transformation of chlorophenols and self-assembled monolayers for electrochemical detection of thiols and cyanides
- Authors: Ozoemena, Kenneth Ikechukwu
- Date: 2003
- Subjects: Electrochemistry Cyanides Thiols Chlorophenols Photocatalysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4089 , http://hdl.handle.net/10962/d1007709
- Description: Photochemical properties of sulphonated phthalocyanine complexes of aluminium, zinc, tin and silicon, and octa-carboxyphthalocyanine complexes of aluminium and zinc have been investigated. These water-soluble metallophthalocyanine (MPc) complexes, especially the sulphonated aluminium and zinc phthalocyanines, were found to be good photosensitisers for the transformation of the toxic mono-, tri- and penta-chlorophenols in aqueous solutions. The efficiency of MPc sensitiser towards photo-transformation of chlorophenols depends on its effectiveness to generate singlet oxygen as well as its photostability. Octa-substituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were synthesized and their spectral and electrochemical properties investigated. The photochemical properties ofthe zinc phthalocyanine complexes in non-aqueous solutions were comparable to those in literature. Ultrathin films of the octasubstituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were, for the first time, immobilized onto gold electrodes using the self-assembling technique. Surface electrochemistry indicates that the ultrathin films are surface-confined self-assembled monolayer (SAM) species. Gold electrodes modified with the redox-active SAMs of cobalt and iron phthalocyanine complexes proved to be potential electrochemical sensors for the detection of thiols (L-cysteine, homocysteine and penicillamine) and thiocyanate in aqueous solutions (pH 4). The limits of detection for the thiols and thiocyanate were in the range of ∼ 10⁻⁷ and 10⁻⁶ mol dm⁻³, respectively. The modification process was reproducible and the modified electrodes showed good stability and, if stored in pH 4 buffer solutions, could be used for the analysis of thiols and thiocyanate for about a month without the need for recalibration. Etching of gold marred electrochemical detection of cyanide with the MPc-SAM-modified gold electrodes. Interestingly, however, kinetic and equilibria studies revealed strong interaction of octabutylthiophthalocyaninatoiron (II), FeOBTPc, with cyanide in both DMF and DMSO solutions.
- Full Text:
- Date Issued: 2003
- Authors: Ozoemena, Kenneth Ikechukwu
- Date: 2003
- Subjects: Electrochemistry Cyanides Thiols Chlorophenols Photocatalysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4089 , http://hdl.handle.net/10962/d1007709
- Description: Photochemical properties of sulphonated phthalocyanine complexes of aluminium, zinc, tin and silicon, and octa-carboxyphthalocyanine complexes of aluminium and zinc have been investigated. These water-soluble metallophthalocyanine (MPc) complexes, especially the sulphonated aluminium and zinc phthalocyanines, were found to be good photosensitisers for the transformation of the toxic mono-, tri- and penta-chlorophenols in aqueous solutions. The efficiency of MPc sensitiser towards photo-transformation of chlorophenols depends on its effectiveness to generate singlet oxygen as well as its photostability. Octa-substituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were synthesized and their spectral and electrochemical properties investigated. The photochemical properties ofthe zinc phthalocyanine complexes in non-aqueous solutions were comparable to those in literature. Ultrathin films of the octasubstituted thiol-derivatised phthalocyanine complexes of cobalt, iron and zinc were, for the first time, immobilized onto gold electrodes using the self-assembling technique. Surface electrochemistry indicates that the ultrathin films are surface-confined self-assembled monolayer (SAM) species. Gold electrodes modified with the redox-active SAMs of cobalt and iron phthalocyanine complexes proved to be potential electrochemical sensors for the detection of thiols (L-cysteine, homocysteine and penicillamine) and thiocyanate in aqueous solutions (pH 4). The limits of detection for the thiols and thiocyanate were in the range of ∼ 10⁻⁷ and 10⁻⁶ mol dm⁻³, respectively. The modification process was reproducible and the modified electrodes showed good stability and, if stored in pH 4 buffer solutions, could be used for the analysis of thiols and thiocyanate for about a month without the need for recalibration. Etching of gold marred electrochemical detection of cyanide with the MPc-SAM-modified gold electrodes. Interestingly, however, kinetic and equilibria studies revealed strong interaction of octabutylthiophthalocyaninatoiron (II), FeOBTPc, with cyanide in both DMF and DMSO solutions.
- Full Text:
- Date Issued: 2003
The development of the emerging technologies sustainability assessment (ETSA) and its application in the design of a bioprocess for the treatment of wine distillery effluent
- Authors: Khan, Nuraan
- Date: 2005
- Subjects: Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3953 , http://hdl.handle.net/10962/d1004012 , Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Description: Emerging Technologies Sustainability Assessment (ETSA) is a new technology assessment tool that was developed in order to compare emerging processes or technologies to existing alternatives. It utilizes infoIDlation modules, with the minimum use of resources such as time and money, in order to deteIDline if the process under development is comparatively favourable and should be developed beyond the early conceptual phase. The preliminary ETSA is vital in order to identify the gaps in the existing information and the specific methodologies to be used for data capture and analysis. The use of experimental design tools, such as Design-Expert, can facilitate rapid and efficient collection of necessary data and fits in well with the rationale for the ETSA. Wine distillery effluent (vinasse) is the residue left after alcohol has been distilled from fennented grape juice. It is an acidic, darkly coloured effluent, with a high COD and polyphenol content. The most popular method of disposal of this effluent, land application, is no longer viable due to stricter legislation and pressure on the industry to better manage its wastes. Although the ability of whiterot fungi to degrade a number of pollutants is well-known, fungal treatment of wine distillery effluent is still in the conceptual phase. The perfoIDlance of the fungal remediation system was assessed experimentally in terms of COD removal and laccase production using Design-Expert. Although Pycnoporus sanguine us was found to be most efficient at COD removal (85%) from 30% vinasse, laccase production was low (0.021 U/I). The optimum design for economically viable fungal treatment used Trametespubescens. This fungus was able to remove over 50% of the COD from undiluted vinasse while producing almost 800U/l of the valuable laccase enzyme within three days. Since the effluent from the fungal system did not meet the legal limits for wastewater disposal, a two-stage aerobicanaerobic system is suggested to improve the quality of the effluent prior to disposal. The ETSA was used to assess the fungal technology in relation to the two current methods of vinasse treatment and disposal, namely land application and anaerobic digestion. Based on the ETSA, which considered environmental, social and economic impacts, the fungal system proved to be potentially competitive and further development of the technology is suggested.
- Full Text:
- Date Issued: 2005
- Authors: Khan, Nuraan
- Date: 2005
- Subjects: Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3953 , http://hdl.handle.net/10962/d1004012 , Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Description: Emerging Technologies Sustainability Assessment (ETSA) is a new technology assessment tool that was developed in order to compare emerging processes or technologies to existing alternatives. It utilizes infoIDlation modules, with the minimum use of resources such as time and money, in order to deteIDline if the process under development is comparatively favourable and should be developed beyond the early conceptual phase. The preliminary ETSA is vital in order to identify the gaps in the existing information and the specific methodologies to be used for data capture and analysis. The use of experimental design tools, such as Design-Expert, can facilitate rapid and efficient collection of necessary data and fits in well with the rationale for the ETSA. Wine distillery effluent (vinasse) is the residue left after alcohol has been distilled from fennented grape juice. It is an acidic, darkly coloured effluent, with a high COD and polyphenol content. The most popular method of disposal of this effluent, land application, is no longer viable due to stricter legislation and pressure on the industry to better manage its wastes. Although the ability of whiterot fungi to degrade a number of pollutants is well-known, fungal treatment of wine distillery effluent is still in the conceptual phase. The perfoIDlance of the fungal remediation system was assessed experimentally in terms of COD removal and laccase production using Design-Expert. Although Pycnoporus sanguine us was found to be most efficient at COD removal (85%) from 30% vinasse, laccase production was low (0.021 U/I). The optimum design for economically viable fungal treatment used Trametespubescens. This fungus was able to remove over 50% of the COD from undiluted vinasse while producing almost 800U/l of the valuable laccase enzyme within three days. Since the effluent from the fungal system did not meet the legal limits for wastewater disposal, a two-stage aerobicanaerobic system is suggested to improve the quality of the effluent prior to disposal. The ETSA was used to assess the fungal technology in relation to the two current methods of vinasse treatment and disposal, namely land application and anaerobic digestion. Based on the ETSA, which considered environmental, social and economic impacts, the fungal system proved to be potentially competitive and further development of the technology is suggested.
- Full Text:
- Date Issued: 2005
Biological sulphide oxidation in heterotrophic environments
- Authors: Rein, Neil Berthold
- Date: 2002
- Subjects: Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3919 , http://hdl.handle.net/10962/d1003978 , Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Description: Acid mine drainage is a major environmental pollution concern associated with the mining of sulphide-containing ore bodies. Both physicochemical and biological options have been investigated for the treatment of acid mine drainage with recent interest in biological processes targeting low-cost and passive treatment applications. All acid mine drainage biological treatment processes are based to some extent on the activity of sulphate reducing bacteria, and their ability to reduce sulphate to sulphide in the presence of a range of carbon and electron donor sources. A portion of the sulphide produced may be consumed in the precipitation of heavy metals present in the mine drainage. Residual sulphide must be removed, not only due to its toxicity, but especially to prevent its reoxidation to sulphate where salinity reduction is a target of the treatment process. The partial oxidation of sulphide to elemental sulphur is an option that has received considerable attention and both physicochemical and biological options have been investigated. Biological processes have substantial potential cost advantages and run at ambient temperatures and pressures. However, the oxidation of sulphide to elemental sulphur is poised over a narrow redox range and process control to maintain optimum conditions remains a serious problem. In addition little has been reported in the literature on process control of sulphide oxidation to elemental sulphur, in the heterotrophic conditions prevailing in the reaction environment following sulphate reduction. This study undertook an investigation of biological sulphide oxidation under heterotrophic conditions in order to establish the effect of organic compounds on biological sulphide oxidation, and to determine whether the presence of organics, and associated heterotrophic oxygen consumption, may be manipulated to maintain the defined redox conditions required for the production of elemental sulphur. Biological sulphide oxidation under heterotrophic conditions was investigated in a series of flask experiments. Based on these results three different reactor configurations, a Fixed-Film Trickle Filter Reactor, Submerged Fixed-Film Reactor and a Silicone Tubular Reactor were used to investigate sulphur production. The flask studies indicated that organics, and associated heterotrophic metabolism in the presence of excess oxygen in the sulphide oxidation reaction environment, did contribute to the poising of redox conditions and thereby enabling the production of elemental sulphur. While the Fixed-Film Trickle Filter Reactor was found to be redox unstable, probably due to excess oxygen ingress to the system, a reduced oxygen challenge in the Submerged Fixed-Film Reactor configuration was found to be more successful for production of elemental sulphur. However, due to the production of a predominantly filamentous sulphur producing microbial population, recovery of sulphur from the column was intermittent and unpredictable. Extended residence times for produced sulphur on the column increased the likelihood for its eventual oxidation to sulphate. The Silicone Tubular Reactor was found to support a vigorous sulphide oxidising biofilm and produced elemental sulphur effectively. Electron microscopic studies showed that this occurred as both biologically produced sulphur and, probably mainly, as crystalline sulphur in the ortho-rhomic form. Given the linear extension of the sulphur production reaction environment it is was possible to investigate the sequence of the reaction mechanism in grater detail than is possible in mixed systems. Based on these findings a model explaining sulphur production under heterotrophic conditions has been proposed and is presented. The commercial implications of the development have also been noted.
- Full Text:
- Date Issued: 2002
- Authors: Rein, Neil Berthold
- Date: 2002
- Subjects: Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3919 , http://hdl.handle.net/10962/d1003978 , Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Description: Acid mine drainage is a major environmental pollution concern associated with the mining of sulphide-containing ore bodies. Both physicochemical and biological options have been investigated for the treatment of acid mine drainage with recent interest in biological processes targeting low-cost and passive treatment applications. All acid mine drainage biological treatment processes are based to some extent on the activity of sulphate reducing bacteria, and their ability to reduce sulphate to sulphide in the presence of a range of carbon and electron donor sources. A portion of the sulphide produced may be consumed in the precipitation of heavy metals present in the mine drainage. Residual sulphide must be removed, not only due to its toxicity, but especially to prevent its reoxidation to sulphate where salinity reduction is a target of the treatment process. The partial oxidation of sulphide to elemental sulphur is an option that has received considerable attention and both physicochemical and biological options have been investigated. Biological processes have substantial potential cost advantages and run at ambient temperatures and pressures. However, the oxidation of sulphide to elemental sulphur is poised over a narrow redox range and process control to maintain optimum conditions remains a serious problem. In addition little has been reported in the literature on process control of sulphide oxidation to elemental sulphur, in the heterotrophic conditions prevailing in the reaction environment following sulphate reduction. This study undertook an investigation of biological sulphide oxidation under heterotrophic conditions in order to establish the effect of organic compounds on biological sulphide oxidation, and to determine whether the presence of organics, and associated heterotrophic oxygen consumption, may be manipulated to maintain the defined redox conditions required for the production of elemental sulphur. Biological sulphide oxidation under heterotrophic conditions was investigated in a series of flask experiments. Based on these results three different reactor configurations, a Fixed-Film Trickle Filter Reactor, Submerged Fixed-Film Reactor and a Silicone Tubular Reactor were used to investigate sulphur production. The flask studies indicated that organics, and associated heterotrophic metabolism in the presence of excess oxygen in the sulphide oxidation reaction environment, did contribute to the poising of redox conditions and thereby enabling the production of elemental sulphur. While the Fixed-Film Trickle Filter Reactor was found to be redox unstable, probably due to excess oxygen ingress to the system, a reduced oxygen challenge in the Submerged Fixed-Film Reactor configuration was found to be more successful for production of elemental sulphur. However, due to the production of a predominantly filamentous sulphur producing microbial population, recovery of sulphur from the column was intermittent and unpredictable. Extended residence times for produced sulphur on the column increased the likelihood for its eventual oxidation to sulphate. The Silicone Tubular Reactor was found to support a vigorous sulphide oxidising biofilm and produced elemental sulphur effectively. Electron microscopic studies showed that this occurred as both biologically produced sulphur and, probably mainly, as crystalline sulphur in the ortho-rhomic form. Given the linear extension of the sulphur production reaction environment it is was possible to investigate the sequence of the reaction mechanism in grater detail than is possible in mixed systems. Based on these findings a model explaining sulphur production under heterotrophic conditions has been proposed and is presented. The commercial implications of the development have also been noted.
- Full Text:
- Date Issued: 2002
Intraspecific comparison of Phanerochaete chrysosporium strains peroxidase production, pollutant degradation and mycelial differentiation
- Authors: Fraser, Sheena Janet
- Date: 2005
- Subjects: Phanerochaete Pollutants -- Biodegradation Lignin -- Biodegradation Bioremediation Peroxidase Fungi -- Differentiation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3964 , http://hdl.handle.net/10962/d1004023
- Description: The wood-degrading basidiomycete, Phanerochaete chrysosporium, has been studied as a model organism in elucidating the mechanisms and pathways enabling this white-rot fungus to degrade recalcitrant lignin. These same mechanisms are implicated in the mineralisation of environmentally persistent, toxic phenolic chemicals. For this reason, P. chrysosporium has been exploited in a number of environmentally sound technologies, including the degradation of the indigestible lignin component in agricultural waste for the generation of digestible animal feedstocks or high sugar content raw materials for ethanol production; brightening processes in the pulp and paper industry; the detoxification and decolourisation of industrial effluents; and the bioremediation of hazardous waste sites. The improvement of these technologies is dependant on ongoing research involving strain selection, strain development using genetic engineering approaches and process development. Strain improvement using non-recombinant methods is beneficial in that it does not limit the inherent robustness observed amongst natural variants. In this research, through a breeding programme, ten P.chrysosporium sibling strains were screened for variable ligninase activities and pollutant degradation capabilities in order to further describe previously identified differences between these organisms. A conventional stationary liquid culture technique was effectively miniaturised from 10 ml flask cultures to a 96-well microtitre plate format, for the assessment of multigenic traits amongst sibling strains. Using the 96-well microtitre plate method, the relationships between P. chrysosporium growth kinetics, peroxidase production, pollutant sensitivity and pollutant degradation was explored. Significant correlations were primarily associated with P. chrysosporium growth [P < 0.05]. Percentage p-cresol removal and tannic acid tolerance were both correlated with a shorter lag phase in growth [tannic acid: r = 0.7698, P < 0.05; p-cresol: r = 0.7584, P < 0.05] and lower stationary phase biomass levels [tannic acid: r = 0.8177, P < 0.05; p-cresol: r = 0.7803, P < 0.05]. A significant correlation (linear relationship) was also detected between percentage Poly-R478 decolourisation and time of onset of MnP [r = 0.9689, P < 0.001]. No correlation was observed between dye decolourisation, p-cresol degradation, lignin degradation and lignin peroxidase (LiP) or manganese peroxidase (MnP) activities [P > 0.05]. These results imply that differences in the biosynthetic pathways for biomass accumulation in sibling strains play a significant role in the intraspecific variation observed in pollutant sensitivity, pollutant degradation, and enzyme production. Categorical analysis of intraspecific differences was assessed according to four criterions. These included growth, extracellular peroxidase activities, tolerance to toxic pollutants and the biodegradation of model pollutants. Sibling strains showing the most variable responses in three or more of the selective criterion were recommended for further studies. These strains include P. chrysosporium ME446, BS 2.52, BS 13, BS 17, BS 18, and BS 24. Interestingly, BS 2.52 (a dikaryotic strain generating from the crossing of two haploid progeny) showed significantly lower degradation capabilities than the wildtype parent strain ME446. The inherited variability observed between sibling strains is to be further explored through proteome and transcriptome analysis and genetic linkage studies aimed at describing the mechanisms or pathways conferring tolerance to or degradation of environmental pollutants. In examining fewer organisms at this next level, the number of replicates examined can be increased and thus the power of detection of experimental procedures improved, enabling the detection of multigenic traits amongst genetically related organisms. Growth was shown to play a significant role in the intraspecific differences detected in pollutant sensitivity and degradation between sibling strains. Little is known about the mechanism of growth and differentiation, or the role of differentiation in regulating the lignolytic activity in this organism. The membrane gradostat bioreactor and a unique plug-flow membrane bioreactor were evaluated as novel tools with which to further explore the relationship between secondary metabolism, pollutant degradation and biofilm development in sibling strains. High yield MnP production at levels as high as 1478.8 U.l-1 was achieved using a laboratory scale membrane gradostat bioreactor. Furthermore, extensive mycelial differentiation and tissue formation are reported for P. chrysosporium in both the membrane gradostat bioreactor and plug-flow membrane bioreactor. Intraspecific differences in the extent of this differentiation were observed in strains ME446, BS 13, BS 17 and BS 26 cultured using the membrane gradostat bioreactor, highlighting the potential of these techniques as a platform for future strain improvement strategies.
- Full Text:
- Date Issued: 2005
- Authors: Fraser, Sheena Janet
- Date: 2005
- Subjects: Phanerochaete Pollutants -- Biodegradation Lignin -- Biodegradation Bioremediation Peroxidase Fungi -- Differentiation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3964 , http://hdl.handle.net/10962/d1004023
- Description: The wood-degrading basidiomycete, Phanerochaete chrysosporium, has been studied as a model organism in elucidating the mechanisms and pathways enabling this white-rot fungus to degrade recalcitrant lignin. These same mechanisms are implicated in the mineralisation of environmentally persistent, toxic phenolic chemicals. For this reason, P. chrysosporium has been exploited in a number of environmentally sound technologies, including the degradation of the indigestible lignin component in agricultural waste for the generation of digestible animal feedstocks or high sugar content raw materials for ethanol production; brightening processes in the pulp and paper industry; the detoxification and decolourisation of industrial effluents; and the bioremediation of hazardous waste sites. The improvement of these technologies is dependant on ongoing research involving strain selection, strain development using genetic engineering approaches and process development. Strain improvement using non-recombinant methods is beneficial in that it does not limit the inherent robustness observed amongst natural variants. In this research, through a breeding programme, ten P.chrysosporium sibling strains were screened for variable ligninase activities and pollutant degradation capabilities in order to further describe previously identified differences between these organisms. A conventional stationary liquid culture technique was effectively miniaturised from 10 ml flask cultures to a 96-well microtitre plate format, for the assessment of multigenic traits amongst sibling strains. Using the 96-well microtitre plate method, the relationships between P. chrysosporium growth kinetics, peroxidase production, pollutant sensitivity and pollutant degradation was explored. Significant correlations were primarily associated with P. chrysosporium growth [P < 0.05]. Percentage p-cresol removal and tannic acid tolerance were both correlated with a shorter lag phase in growth [tannic acid: r = 0.7698, P < 0.05; p-cresol: r = 0.7584, P < 0.05] and lower stationary phase biomass levels [tannic acid: r = 0.8177, P < 0.05; p-cresol: r = 0.7803, P < 0.05]. A significant correlation (linear relationship) was also detected between percentage Poly-R478 decolourisation and time of onset of MnP [r = 0.9689, P < 0.001]. No correlation was observed between dye decolourisation, p-cresol degradation, lignin degradation and lignin peroxidase (LiP) or manganese peroxidase (MnP) activities [P > 0.05]. These results imply that differences in the biosynthetic pathways for biomass accumulation in sibling strains play a significant role in the intraspecific variation observed in pollutant sensitivity, pollutant degradation, and enzyme production. Categorical analysis of intraspecific differences was assessed according to four criterions. These included growth, extracellular peroxidase activities, tolerance to toxic pollutants and the biodegradation of model pollutants. Sibling strains showing the most variable responses in three or more of the selective criterion were recommended for further studies. These strains include P. chrysosporium ME446, BS 2.52, BS 13, BS 17, BS 18, and BS 24. Interestingly, BS 2.52 (a dikaryotic strain generating from the crossing of two haploid progeny) showed significantly lower degradation capabilities than the wildtype parent strain ME446. The inherited variability observed between sibling strains is to be further explored through proteome and transcriptome analysis and genetic linkage studies aimed at describing the mechanisms or pathways conferring tolerance to or degradation of environmental pollutants. In examining fewer organisms at this next level, the number of replicates examined can be increased and thus the power of detection of experimental procedures improved, enabling the detection of multigenic traits amongst genetically related organisms. Growth was shown to play a significant role in the intraspecific differences detected in pollutant sensitivity and degradation between sibling strains. Little is known about the mechanism of growth and differentiation, or the role of differentiation in regulating the lignolytic activity in this organism. The membrane gradostat bioreactor and a unique plug-flow membrane bioreactor were evaluated as novel tools with which to further explore the relationship between secondary metabolism, pollutant degradation and biofilm development in sibling strains. High yield MnP production at levels as high as 1478.8 U.l-1 was achieved using a laboratory scale membrane gradostat bioreactor. Furthermore, extensive mycelial differentiation and tissue formation are reported for P. chrysosporium in both the membrane gradostat bioreactor and plug-flow membrane bioreactor. Intraspecific differences in the extent of this differentiation were observed in strains ME446, BS 13, BS 17 and BS 26 cultured using the membrane gradostat bioreactor, highlighting the potential of these techniques as a platform for future strain improvement strategies.
- Full Text:
- Date Issued: 2005
Analysis of transcription factor binding specificity using ChIP-seq data.
- Authors: Kibet, Caleb Kipkurui
- Date: 2014
- Subjects: Transcription factors , Chronic myeloid leukemia , Antioncogenes , Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4115 , http://hdl.handle.net/10962/d1013131
- Description: Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.
- Full Text:
- Date Issued: 2014
- Authors: Kibet, Caleb Kipkurui
- Date: 2014
- Subjects: Transcription factors , Chronic myeloid leukemia , Antioncogenes , Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4115 , http://hdl.handle.net/10962/d1013131
- Description: Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.
- Full Text:
- Date Issued: 2014
Ericoid mycorrhizal fungi and potential for inoculation of commercial berry species (Vaccinium corymbosium L.)
- Authors: Bizabani, Christine
- Date: 2011
- Subjects: Ericaceae , Mycorrhizas , Fynbos
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4136 , http://hdl.handle.net/10962/d1016127
- Description: Ericaceous plants are the richest growth form of the fynbos vegetation of South Africa. The fynbos is characterized by highly leached acidic soils, low mineral nutrients and climatically it is a winter rainfall and dry summer region. Ericoid mycorrhizal fungi associate with Erica species enhancing their ability to access essential nutrients for survival under unfavourable growth conditions. The aim of this study was to select local Ericaceae plant species and to isolate, identify and characterize the ericoid endophytes and assess these isolates as potential inocula for commercial berry species. Two ericaceous plants Erica cerinthoides L. and Erica demmissa Klotzsch ex Benth. were identified from the Mountain Drive area of Grahamstown, Eastern Cape. Root staining was used to confirm the mycorrhizal status of both plants. Hyphal coils typical of ericoid association were observed within the epidermal cells of the hair roots under a light microscope. The endophytes were successfully isolated in pure culture on 2% malt extract agar (MEA) and modified Fontana medium. Cultural morphology and microscopy were used for initial identification. Two slow growing isolates were selected. These isolates were further subjected to molecular identification; extracted DNA was amplified using ITS1 and ITS4 fungal primers. The rDNA gene internal transcriber spacer (ITS) was then sequenced and analyzed by comparison to sequences in the GenBank. On the basis of percentage sequence identity Lachnum Retz. species and Cadophora Lagerb. & Melin species were identified as the ericoid endophytes of E. cerinthoides and E. demmissa respectively. The optimum growth parameters of the fungal isolates were determined in 2% MEA incubated at varying temperatures and pH. It was established that both species had optimum growth at 27⁰C and pH 5. The Ericaceae species are sometimes found in metal contaminated sites were ericoid fungi have been proved to alleviate toxicity of their host. The fungal isolates were grown in increasing concentration of Cu²⁺ and Zn²⁺ in 2% MEA. The growth of Lachnum species decreased with increasing Zn²⁺ ions above 2.7 mM while Cadophora species showed a change in morphology and also decreased in growth with increased ion concentration. However there were no significant differences recorded in the growth of Cadophora and Lachnum species on increasing Cu²⁺ concentration. Lachnum and Cadophora isolates were formulated into a semi solid inoculum and inoculated onto micropropagated Vaccinni corymbosum L. plantlets of 5 different varieties. Colonization was low for all varieties, Elliott and Brightwell varieties recorded the highest colonization of 35% and 31% respectively. Lachnum species infected roots showed potential ericoid structures while the Cadophora inoculated plantlets had hyphal coils within the cortical cells typical of ericoid mycorrhizas. Inoculation significantly enhanced the shoot growth of Brightwell and Elliott varieties. The Chandler variety inoculated with Lachnum species showed improved shoot dry weight. The Bluecrop and Elliott varieties inoculated with Cadophora and Lachnum accumulated more root biomass. All inoculated Bluecrop plantlets had an improved canopy growth index. Brightwell plantlets inoculated with Lachnum species also had an enhanced canopy growth index. The growth responses were variable within varieties and between varieties. Treatments with the Cadophora and Lachnum have shown potential in the promotion of growth of the Blueberry species. The findings indicate the need to conduct trials under conditions which simulate the commercial growth conditions so as explore the optimum potential of the isolates.
- Full Text:
- Date Issued: 2011
- Authors: Bizabani, Christine
- Date: 2011
- Subjects: Ericaceae , Mycorrhizas , Fynbos
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4136 , http://hdl.handle.net/10962/d1016127
- Description: Ericaceous plants are the richest growth form of the fynbos vegetation of South Africa. The fynbos is characterized by highly leached acidic soils, low mineral nutrients and climatically it is a winter rainfall and dry summer region. Ericoid mycorrhizal fungi associate with Erica species enhancing their ability to access essential nutrients for survival under unfavourable growth conditions. The aim of this study was to select local Ericaceae plant species and to isolate, identify and characterize the ericoid endophytes and assess these isolates as potential inocula for commercial berry species. Two ericaceous plants Erica cerinthoides L. and Erica demmissa Klotzsch ex Benth. were identified from the Mountain Drive area of Grahamstown, Eastern Cape. Root staining was used to confirm the mycorrhizal status of both plants. Hyphal coils typical of ericoid association were observed within the epidermal cells of the hair roots under a light microscope. The endophytes were successfully isolated in pure culture on 2% malt extract agar (MEA) and modified Fontana medium. Cultural morphology and microscopy were used for initial identification. Two slow growing isolates were selected. These isolates were further subjected to molecular identification; extracted DNA was amplified using ITS1 and ITS4 fungal primers. The rDNA gene internal transcriber spacer (ITS) was then sequenced and analyzed by comparison to sequences in the GenBank. On the basis of percentage sequence identity Lachnum Retz. species and Cadophora Lagerb. & Melin species were identified as the ericoid endophytes of E. cerinthoides and E. demmissa respectively. The optimum growth parameters of the fungal isolates were determined in 2% MEA incubated at varying temperatures and pH. It was established that both species had optimum growth at 27⁰C and pH 5. The Ericaceae species are sometimes found in metal contaminated sites were ericoid fungi have been proved to alleviate toxicity of their host. The fungal isolates were grown in increasing concentration of Cu²⁺ and Zn²⁺ in 2% MEA. The growth of Lachnum species decreased with increasing Zn²⁺ ions above 2.7 mM while Cadophora species showed a change in morphology and also decreased in growth with increased ion concentration. However there were no significant differences recorded in the growth of Cadophora and Lachnum species on increasing Cu²⁺ concentration. Lachnum and Cadophora isolates were formulated into a semi solid inoculum and inoculated onto micropropagated Vaccinni corymbosum L. plantlets of 5 different varieties. Colonization was low for all varieties, Elliott and Brightwell varieties recorded the highest colonization of 35% and 31% respectively. Lachnum species infected roots showed potential ericoid structures while the Cadophora inoculated plantlets had hyphal coils within the cortical cells typical of ericoid mycorrhizas. Inoculation significantly enhanced the shoot growth of Brightwell and Elliott varieties. The Chandler variety inoculated with Lachnum species showed improved shoot dry weight. The Bluecrop and Elliott varieties inoculated with Cadophora and Lachnum accumulated more root biomass. All inoculated Bluecrop plantlets had an improved canopy growth index. Brightwell plantlets inoculated with Lachnum species also had an enhanced canopy growth index. The growth responses were variable within varieties and between varieties. Treatments with the Cadophora and Lachnum have shown potential in the promotion of growth of the Blueberry species. The findings indicate the need to conduct trials under conditions which simulate the commercial growth conditions so as explore the optimum potential of the isolates.
- Full Text:
- Date Issued: 2011