Isolation, purification and characterization of a 'factor' from Fusarium oxysporum responsible for platinum nanoparticle formation
- Authors: Govender, Yageshni
- Date: 2008
- Subjects: Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3923 , http://hdl.handle.net/10962/d1003982 , Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Description: Nanoparticles are microscopic particles in the nanometre range of between 1-100 nm. A wide variety of metal nanoparticles have been found to be produced by prokaryotic and eukaryotic organisms including several fungal species, when exposed to solutions containing metal salts. Previous studies have suggested that this bioreduction of metal particles may occur via an active reductase/hydrogenase enzyme process where H2 is the electron donor and positively charged platinum species act as the electron acceptors becoming reduced to a neutral metal nanoparticle. In view of this on going research, the current study investigated the “factors” in the fungus Fusarium oxysporum which were responsible for platinum nanoparticle formation. The fungus F.oxysporum was used in this study as it has been previously shown to produce a variety of nanoparticles including gold and silver. During exposure of the biomass to H2PtCl6 the initial response to the platinum salts was metal internalisation and subsequent reduction of H2PtCI6 to produce platinum nanoparticles. The observed localization and distribution of platinum precipitates provided some evidence for a hydrogenase mediated bioreduction of platinum salts to produce nanoparticles. Factors secreted by the fungus into the extracellular fluids, were shown to be responsible for platinum nanoparticle formation. From the identification, purification and characterisation studies it was concluded that a hydrogenase and other “factors” were responsible for platinum nanoparticle formation in F.oxysporum. Purification of the hydrogenase by freeze-drying and Sephacryl S200 size exclusion- ion exchange chromatography revealed the enzyme to be a dimer with a 29.4 and 44.5 kDa when analysed by a 10 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 38 °C while it exhibited a poor thermal stability with a half life of 36 minutes. The kinetic parameters Vmax and Km were 3.16 U ml-1 and 3.64 mM respectively. The purified hydrogenase was used in subsequent experiments for the reduction of platinum salts, H2PtCl6 and PtCl2. the results indicated an over 90 % reduction of the platinum salts and TEM micrographs indicated the production of platinum nanoparticles under the various experimental conditions.
- Full Text:
- Date Issued: 2008
- Authors: Govender, Yageshni
- Date: 2008
- Subjects: Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3923 , http://hdl.handle.net/10962/d1003982 , Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Description: Nanoparticles are microscopic particles in the nanometre range of between 1-100 nm. A wide variety of metal nanoparticles have been found to be produced by prokaryotic and eukaryotic organisms including several fungal species, when exposed to solutions containing metal salts. Previous studies have suggested that this bioreduction of metal particles may occur via an active reductase/hydrogenase enzyme process where H2 is the electron donor and positively charged platinum species act as the electron acceptors becoming reduced to a neutral metal nanoparticle. In view of this on going research, the current study investigated the “factors” in the fungus Fusarium oxysporum which were responsible for platinum nanoparticle formation. The fungus F.oxysporum was used in this study as it has been previously shown to produce a variety of nanoparticles including gold and silver. During exposure of the biomass to H2PtCl6 the initial response to the platinum salts was metal internalisation and subsequent reduction of H2PtCI6 to produce platinum nanoparticles. The observed localization and distribution of platinum precipitates provided some evidence for a hydrogenase mediated bioreduction of platinum salts to produce nanoparticles. Factors secreted by the fungus into the extracellular fluids, were shown to be responsible for platinum nanoparticle formation. From the identification, purification and characterisation studies it was concluded that a hydrogenase and other “factors” were responsible for platinum nanoparticle formation in F.oxysporum. Purification of the hydrogenase by freeze-drying and Sephacryl S200 size exclusion- ion exchange chromatography revealed the enzyme to be a dimer with a 29.4 and 44.5 kDa when analysed by a 10 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 38 °C while it exhibited a poor thermal stability with a half life of 36 minutes. The kinetic parameters Vmax and Km were 3.16 U ml-1 and 3.64 mM respectively. The purified hydrogenase was used in subsequent experiments for the reduction of platinum salts, H2PtCl6 and PtCl2. the results indicated an over 90 % reduction of the platinum salts and TEM micrographs indicated the production of platinum nanoparticles under the various experimental conditions.
- Full Text:
- Date Issued: 2008
The development of a putative microbial product for use in crop production
- Authors: Gumede, Halalisani
- Date: 2008
- Subjects: Agricultural productivity , Agriculture -- Economic aspects , Microbial products , Bacterial diseases of plants , Biological pest control agents , Lettuce -- Diseases and pests , Crops -- Nutrition , Bacillus (Bacteria) , Phytopathogenic microorganisms -- Control
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3945 , http://hdl.handle.net/10962/d1004004 , Agricultural productivity , Agriculture -- Economic aspects , Microbial products , Bacterial diseases of plants , Biological pest control agents , Lettuce -- Diseases and pests , Crops -- Nutrition , Bacillus (Bacteria) , Phytopathogenic microorganisms -- Control
- Description: The challenges faced by the agricultural sector especially around improving production yields using environmentally friendly solutions have received market attention. Biological intervention can range from application of biological products to enhance the nutritional value of crops or to control plant pathogens. Biostart, a biological product that demonstrated growth enhancement when applied in lettuce crops is currently in the market. The product is comprised of a consortium of bacterial isolates (Bacillus licheniformis, Brevibacillus laterosporus and Bacillus laterosporus) but the contribution of the individual isolates to growth enhancement had not been elucidated. Green house experiments on lettuce seedlings with individual and mixed treatments were commissioned to determine such contribution. There was either no or marginal growth enhancement observed in the experiments. The results showed that the product was effective as a consortium and not as individual isolates. Further isolation and screening for potential Bacilli with antifungal properties was undertaken. An isolate identified as Bacillus subtilis that demonstrated inhibition against a wide spectrum of fungi, and especially the phytopathogenic Verticillium dahliae and Fusarium oxysporum, was successfully identified. The isolate was cryo-preserved and cultivated to significant levels at bench scale. A characterized comparison of different putative products with known systematic fungicide showed potential application even of heat treated products. The product showed control V. dahliae when tested in green houses with potatoes and tomatoes as test crops. This isolate has been targeted for further development as a biological control product.
- Full Text:
- Date Issued: 2008
- Authors: Gumede, Halalisani
- Date: 2008
- Subjects: Agricultural productivity , Agriculture -- Economic aspects , Microbial products , Bacterial diseases of plants , Biological pest control agents , Lettuce -- Diseases and pests , Crops -- Nutrition , Bacillus (Bacteria) , Phytopathogenic microorganisms -- Control
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3945 , http://hdl.handle.net/10962/d1004004 , Agricultural productivity , Agriculture -- Economic aspects , Microbial products , Bacterial diseases of plants , Biological pest control agents , Lettuce -- Diseases and pests , Crops -- Nutrition , Bacillus (Bacteria) , Phytopathogenic microorganisms -- Control
- Description: The challenges faced by the agricultural sector especially around improving production yields using environmentally friendly solutions have received market attention. Biological intervention can range from application of biological products to enhance the nutritional value of crops or to control plant pathogens. Biostart, a biological product that demonstrated growth enhancement when applied in lettuce crops is currently in the market. The product is comprised of a consortium of bacterial isolates (Bacillus licheniformis, Brevibacillus laterosporus and Bacillus laterosporus) but the contribution of the individual isolates to growth enhancement had not been elucidated. Green house experiments on lettuce seedlings with individual and mixed treatments were commissioned to determine such contribution. There was either no or marginal growth enhancement observed in the experiments. The results showed that the product was effective as a consortium and not as individual isolates. Further isolation and screening for potential Bacilli with antifungal properties was undertaken. An isolate identified as Bacillus subtilis that demonstrated inhibition against a wide spectrum of fungi, and especially the phytopathogenic Verticillium dahliae and Fusarium oxysporum, was successfully identified. The isolate was cryo-preserved and cultivated to significant levels at bench scale. A characterized comparison of different putative products with known systematic fungicide showed potential application even of heat treated products. The product showed control V. dahliae when tested in green houses with potatoes and tomatoes as test crops. This isolate has been targeted for further development as a biological control product.
- Full Text:
- Date Issued: 2008
The potential roles of interactions between STAT3, Hsp90, and Hop in the maintenance of self-renewal in mouse embryonic stem cells
- Authors: Setati, Mokgadi Michael
- Date: 2008
- Subjects: Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3981 , http://hdl.handle.net/10962/d1004040 , Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Description: Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.
- Full Text:
- Date Issued: 2008
- Authors: Setati, Mokgadi Michael
- Date: 2008
- Subjects: Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3981 , http://hdl.handle.net/10962/d1004040 , Embryonic stem cells , Leukemia inhibitory factor , Cellular signal transduction , Heat shock proteins , Molecular chaperones
- Description: Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.
- Full Text:
- Date Issued: 2008
Characterisation of Human Hsj1a : an HSP40 molecular chaperone similar to Malarial Pfj4
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
Determination of the botanical composition of black rhinoceros (Diceros bicornis) dung using the rbcL gene as a molecular marker, and analysis of antioxidant and phenolic content of its browse
- Authors: Bulani, Siyavuya Ishmael
- Date: 2007 , 2013-06-25
- Subjects: Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4070 , http://hdl.handle.net/10962/d1006468 , Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Description: The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l-picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe³⁺-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce ferric ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides.
- Full Text:
- Date Issued: 2007
- Authors: Bulani, Siyavuya Ishmael
- Date: 2007 , 2013-06-25
- Subjects: Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4070 , http://hdl.handle.net/10962/d1006468 , Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Description: The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l-picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe³⁺-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce ferric ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides.
- Full Text:
- Date Issued: 2007
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
Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Mutambanengwe, Cecil Clifford Zvandada
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
Isolation, propagation and rapid molecular detection of the Kalahari truffle, a mycorrhizal fungus occurring in South Africa
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2007
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2007
Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
- Full Text:
- Date Issued: 2007
The role of arbuscular mycorrhizal fungi in the biotransformation of coal and application in dump rehabilitation
- Mukasa-Mugerwa, Thomas Tendo
- Authors: Mukasa-Mugerwa, Thomas Tendo
- Date: 2007
- Subjects: Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3999 , http://hdl.handle.net/10962/d1004059 , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Description: Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
- Full Text:
- Date Issued: 2007
- Authors: Mukasa-Mugerwa, Thomas Tendo
- Date: 2007
- Subjects: Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3999 , http://hdl.handle.net/10962/d1004059 , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Fungi -- Biotechnology , Bermuda grass , Coal mines and mining -- Environmental aspects , Acid mine drainage
- Description: Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.
- Full Text:
- Date Issued: 2007
Wheat stress responses during Russian wheat aphid and Bird Cherry Oat aphid infestation: an analysis of differential protein regulation during plant biotic stress responses
- Louw, Cassandra Alexandrovna
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
- Full Text:
- Date Issued: 2007
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
- Full Text:
- Date Issued: 2007
Isolation, purification and characterization of a novel glucose oxidase from Penicillium canescens Tt42
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
- Full Text:
- Date Issued: 2006
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
- Full Text:
- Date Issued: 2006
Voltammetric analysis of pesticides and their degradation: A case study of Amitraz and its degradants
- Authors: Brimecombe, Rory Dennis
- Date: 2006
- Subjects: Hydrolysis , Biodegradation , Voltammetry , Pesticides -- Biodegradation , Pesticides -- Environmental aspects , Acaricides , Acaricides -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4131 , http://hdl.handle.net/10962/d1015724
- Description: Amitraz is a formamide acaricide used predominantly in the control of ectoparasites in livestock and honeybees. Amitraz hydrolysis is rapid and occurs under acidic conditions, exposure to sunlight and biodegradation by microorganisms. The main hydrolysis product of amitraz, 2,4-dimethylaniline, is recalcitrant in the environment and toxic to humans. An electrochemical method for the determination of total amitraz residues and its final breakdown product, 2,4-dimethylaniline, in spent cattle dip, is presented. Cyclic voltammetry at a glassy carbon electrode showed the irreversible oxidation of amitraz and 2,4-dimethylaniline. A limit of detection in the range of 8.5 x 10⁻⁸ M for amitraz and 2 x 10⁻⁸ M for 2,4-dimethylaniline was determined using differential pulse voltammetry. Feasibility studies in which the effect of supporting electrolyte type and pH had on electroanalysis of amitraz and its degradants, showed that pH affects current response as well as the potential at which amitraz and its degradants are oxidised. Britton-Robinson buffer was found to be the most suitable supporting electrolyte for detection of amitraz and its degradants in terms of sensitivity and reproducibility. Studies performed using environmental samples showed that the sensitivity and reproducibility of amitraz and 2,4-dimethylaniline analyses in spent cattle dip were comparable to analyses of amitraz and 2,4-dimethylaniline performed in Britton-Robinson buffer. In addition, the feasibility qf measuring amitraz and 2,4-dimethylaniline in environmental samples was assessed and compared to amitraz and 2,4-dimethylaniline analyses in Britton-Robinson buffer. Amitraz and 2,4-dimethylaniline were readily detectable in milk and honey. Furthermore, it was elucidated that 2,4-dimethylaniline can be metabolised to 3-methylcatechol by Pseudomonas species and the proposed breakdown pathway is presented. The biological degradation of amitraz and subsequent formation of 2,4-dimethylaniline was readily monitored in spent cattle dip. The breakdown of amitraz to 2,4-dimethylaniline and then to 3-MC was monitored using cyclic voltammetry.
- Full Text:
- Date Issued: 2006
- Authors: Brimecombe, Rory Dennis
- Date: 2006
- Subjects: Hydrolysis , Biodegradation , Voltammetry , Pesticides -- Biodegradation , Pesticides -- Environmental aspects , Acaricides , Acaricides -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4131 , http://hdl.handle.net/10962/d1015724
- Description: Amitraz is a formamide acaricide used predominantly in the control of ectoparasites in livestock and honeybees. Amitraz hydrolysis is rapid and occurs under acidic conditions, exposure to sunlight and biodegradation by microorganisms. The main hydrolysis product of amitraz, 2,4-dimethylaniline, is recalcitrant in the environment and toxic to humans. An electrochemical method for the determination of total amitraz residues and its final breakdown product, 2,4-dimethylaniline, in spent cattle dip, is presented. Cyclic voltammetry at a glassy carbon electrode showed the irreversible oxidation of amitraz and 2,4-dimethylaniline. A limit of detection in the range of 8.5 x 10⁻⁸ M for amitraz and 2 x 10⁻⁸ M for 2,4-dimethylaniline was determined using differential pulse voltammetry. Feasibility studies in which the effect of supporting electrolyte type and pH had on electroanalysis of amitraz and its degradants, showed that pH affects current response as well as the potential at which amitraz and its degradants are oxidised. Britton-Robinson buffer was found to be the most suitable supporting electrolyte for detection of amitraz and its degradants in terms of sensitivity and reproducibility. Studies performed using environmental samples showed that the sensitivity and reproducibility of amitraz and 2,4-dimethylaniline analyses in spent cattle dip were comparable to analyses of amitraz and 2,4-dimethylaniline performed in Britton-Robinson buffer. In addition, the feasibility qf measuring amitraz and 2,4-dimethylaniline in environmental samples was assessed and compared to amitraz and 2,4-dimethylaniline analyses in Britton-Robinson buffer. Amitraz and 2,4-dimethylaniline were readily detectable in milk and honey. Furthermore, it was elucidated that 2,4-dimethylaniline can be metabolised to 3-methylcatechol by Pseudomonas species and the proposed breakdown pathway is presented. The biological degradation of amitraz and subsequent formation of 2,4-dimethylaniline was readily monitored in spent cattle dip. The breakdown of amitraz to 2,4-dimethylaniline and then to 3-MC was monitored using cyclic voltammetry.
- Full Text:
- Date Issued: 2006
A comparative bioinformatic analysis of zinc binuclear cluster proteins
- Authors: Mthombeni, Jabulani S
- Date: 2005
- Subjects: Bioinformatics , Zinc proteins , GABA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4004 , http://hdl.handle.net/10962/d1004064 , Bioinformatics , Zinc proteins , GABA
- Description: Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.
- Full Text:
- Date Issued: 2005
- Authors: Mthombeni, Jabulani S
- Date: 2005
- Subjects: Bioinformatics , Zinc proteins , GABA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4004 , http://hdl.handle.net/10962/d1004064 , Bioinformatics , Zinc proteins , GABA
- Description: Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.
- Full Text:
- Date Issued: 2005
Development of an experimental system to investigate the interaction between the Helicoverpa armigera stunt virus capsid protein and viral RNA
- Authors: Nel, Andrew James Mascré
- Date: 2005
- Subjects: Helicoverpa armigera , RNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3946 , http://hdl.handle.net/10962/d1004005 , Helicoverpa armigera , RNA viruses
- Description: Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.
- Full Text:
- Date Issued: 2005
- Authors: Nel, Andrew James Mascré
- Date: 2005
- Subjects: Helicoverpa armigera , RNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3946 , http://hdl.handle.net/10962/d1004005 , Helicoverpa armigera , RNA viruses
- Description: Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.
- Full Text:
- Date Issued: 2005
Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
Enzymatic recovery of rhodium(III) from aqueous solution and industrial effluent using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
- Full Text:
- Date Issued: 2005
Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
Investigation into the biological removal of sulphate from ethanol distillery wastewater using sulphate-reducing prokaryotes
- Authors: Smuts, Lizl
- Date: 2005
- Subjects: Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3941 , http://hdl.handle.net/10962/d1004000 , Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Description: Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.
- Full Text:
- Date Issued: 2005
- Authors: Smuts, Lizl
- Date: 2005
- Subjects: Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3941 , http://hdl.handle.net/10962/d1004000 , Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Description: Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.
- Full Text:
- Date Issued: 2005