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
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
Sulphide-enhanced hydrolysis of primary sewage sludge : implications for the bioremediation of sulphate-enriched wastewaters
- Whittington-Jones, Kevin John
- Authors: Whittington-Jones, Kevin John
- Date: 2000
- Subjects: Bioremediation Sewage sludge Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3910 , http://hdl.handle.net/10962/d1003969
- Description: The potential application of sulphate reducing bacteria for the bioremediation of acid mine drainage has already been recognised, and offers significant financial advantages over conventional chemical treatment approaches. Although the technology has been demonstrated successfully on both small- and large-scale, it’s extensive implementation has been constrained by the provision of suitable and cost effective electron donor and carbon sources. Primary sewage sludge is readily available in large quantities, but the slow rate of solubilization and low yield of soluble products do not apparently favour its use for this application. A number of pre-treatment steps have been introduced in an attempt to improve the yield and rates under methanogenic conditions. However, although early work suggested that degradation of lignocellulose and proteins may be more rapid under sulphate reducing conditions, the fate of primary sewage sludge under these conditions has been ignored. It was proposed that by combining the hydrolysis of primary sewage sludge and biological sulphate reduction, in a settling sludge bed, both processes would be enhanced. The aim of this study was to test this hypothesis on laboratory- and pilot-scale, and attempt to elucidate the underlying mechanism involved. The solubilization of primary sewage sludge was enhanced in the presence of sulphate reduction in continuous laboratory-scale reactors. Particulate matter accumulated in the bed of non-sulphidogenic systems, but not in sulphidogenic ones. This was attributed to increased solubilization and the smaller average floc size in the latter. Solubilization occurred within the settling sludge bed of the reactors, and offered a possible explanation for the better performance of the multiple- over single-stage reactor. A pilot-scale Falling Sludge Bed Reactor was constructed at Grootvlei Gold Mine, Springs, South Africa, and resulted in the solubilization of more than 70% of the influent primary sewage sludge. The system was also found to be highly resilient to severe perturbations, and returned rapidly to steady-state. Flask studies revealed that the hydrolysis of both proteins and complex carbohydrates was accelerated in the presence of biological sulphate reduction or sulphide. A study of the enzymology of sludge digestion revealed that sulphate reduction had little direct effect on the activity of the hydrolytic enzymes, but that reactor design was critical in the prevention of washout of these enzymes. Finally, a descriptive model was developed to explain the enhanced hydrolysis of primary sewage sludge. The model incorporated the effect of sulphidogenesis on floc fracture and reflocculation, and likely implications for mass transfer limitations.
- Full Text:
- Date Issued: 2000
- Authors: Whittington-Jones, Kevin John
- Date: 2000
- Subjects: Bioremediation Sewage sludge Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3910 , http://hdl.handle.net/10962/d1003969
- Description: The potential application of sulphate reducing bacteria for the bioremediation of acid mine drainage has already been recognised, and offers significant financial advantages over conventional chemical treatment approaches. Although the technology has been demonstrated successfully on both small- and large-scale, it’s extensive implementation has been constrained by the provision of suitable and cost effective electron donor and carbon sources. Primary sewage sludge is readily available in large quantities, but the slow rate of solubilization and low yield of soluble products do not apparently favour its use for this application. A number of pre-treatment steps have been introduced in an attempt to improve the yield and rates under methanogenic conditions. However, although early work suggested that degradation of lignocellulose and proteins may be more rapid under sulphate reducing conditions, the fate of primary sewage sludge under these conditions has been ignored. It was proposed that by combining the hydrolysis of primary sewage sludge and biological sulphate reduction, in a settling sludge bed, both processes would be enhanced. The aim of this study was to test this hypothesis on laboratory- and pilot-scale, and attempt to elucidate the underlying mechanism involved. The solubilization of primary sewage sludge was enhanced in the presence of sulphate reduction in continuous laboratory-scale reactors. Particulate matter accumulated in the bed of non-sulphidogenic systems, but not in sulphidogenic ones. This was attributed to increased solubilization and the smaller average floc size in the latter. Solubilization occurred within the settling sludge bed of the reactors, and offered a possible explanation for the better performance of the multiple- over single-stage reactor. A pilot-scale Falling Sludge Bed Reactor was constructed at Grootvlei Gold Mine, Springs, South Africa, and resulted in the solubilization of more than 70% of the influent primary sewage sludge. The system was also found to be highly resilient to severe perturbations, and returned rapidly to steady-state. Flask studies revealed that the hydrolysis of both proteins and complex carbohydrates was accelerated in the presence of biological sulphate reduction or sulphide. A study of the enzymology of sludge digestion revealed that sulphate reduction had little direct effect on the activity of the hydrolytic enzymes, but that reactor design was critical in the prevention of washout of these enzymes. Finally, a descriptive model was developed to explain the enhanced hydrolysis of primary sewage sludge. The model incorporated the effect of sulphidogenesis on floc fracture and reflocculation, and likely implications for mass transfer limitations.
- Full Text:
- Date Issued: 2000
Genetic characterization of conspecific populations of Tilapia Sparrmanii (A.Smith 1840) in the dolomitic sinkholes and springs of the North-West Province (South Africa), and their comparison to Tilapia Guinasana (Trewavas 1936)
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
- Full Text:
- Date Issued: 2002
- Authors: Nxomani, Clifford David
- Date: 2002
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4079 , http://hdl.handle.net/10962/d1007452
- Description: This study was undertaken to investigate the genetic relationships of allopatric populations of the cichlid fish, Tilapia sparrmanii (A. Smith 1840) inhabiting the sinkholes and springs of the North West Province, South Africa. It also examined the genetic relationships of T sparrmanii to its polychromatic sister species, Tilapia guinasana (Trewavas 1936) which is endemic to the Guinas sinkhole in Namibia. Finally, the study investigated whether there is a genetic basis for T guinasana's colour polymorphism. The research was prompted by the concern of conservation authorities about the possible loss of unique fauna given the high demand for use of the subterranean waters for agricultural, domestic and industrial purposes. Such demands have the potential to drain these habitats. Further concerns related to habitat destruction and the introduction of alien species in the ecosystems inhabited by both fish species. Three approaches were adopted in attempting to answer the above questions. First was the investigation of Sodium dodecylsulphate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) of total cellular proteins as a fast and relatively inexpensive indicator of genetic relatedness between the fish populations. Secondly, genetic differentiation between the T sparrmanii populations and its relationship to T guinasana were assayed using restriction endonuclease analysis of Polymerase Chain Reaction (PCR)-amplified regions of the cytochrome b gene and the d-Ioop of mitochondrial DNA, coupled with Temperature Gradient Gel Electrophoresis (TGGE) analysis of the same regions. The third approach involved the use of Random Amplified Polymorphic DNA (RAPD) fingerprinting of the populations ofT sparrmanii as an indicator of genetic differentiation between them. RAPD fingerprinting was further used to investigate the genetic relationships between T sparrmanii and T guinasana and to probe the genetic basis of the polychromatism of the latter. SDS-PAGE did not reveal any genetic differentiation between the T sparrmanii populations, nor could the analysis detect variation within them. It however clearly distinguished at a species level between T sparrmanii and T guinasana as well as between these and other fish species, thus indicating its possible utility as an indicator of genetic relatedness at a species level. Mitochondrial studies employing the Restriction Fragment Length Polymorphism (RFLP) of Polymerase Chain Reaction (PCR)-amplified cytochrome b (1.1 kb) and d-Ioop regions (0.9 kb) with six and five restriction enzymes respectively, failed to reveal genetic differences within and between the allopatric populations. TGGE of500 bp of the d-Ioop and 400 bp of the 12sRNA PCR-amplified fragments did not reveal any differences between the populations of T. sparrmanii, nor did the analysis reveal any differences between T. sparrmanii and T. guinasana. The lack of differentiation between the T. sparrmanii populations by these mitochondrial Dna analysis techniques, despite habitat fragmentation, indicated a recent origin of the populations from a common ancestral population. Failure to distinguish between T. sparrmanii and T. guinasana may be related to the sensitivity of the techniques utilized. RAPD fingerprinting analysis indicated that the populations are genetically differentiated from each other. Using a measure of coefficient of variation, the population with the highest variation was the Wondergat population (13.99%), followed by the Klerkskraal popUlation (8.29%), the Malmani and Marico Oog populations (each with 5.88%) and the least variation (4.95 and 4.83%) was with the Amalinda and Molopo Oog populations respectively. This high degree of intra population similarity points to the fact that this differentiation is still confined within the limits of con specificity. The genetic distances between all of the T. sparrmanii populations across all primers ranged from 0.09 to 0.234 and averaged 0.146, a value that falls in the upper end of conspecific population differentiation. Such results indicate populational sub-division below the species level. RAPD fingerprinting therefore proved more sensitive than protein or mitochondrial studies. The differentiation it detected between the populations is a reflection of their adaptation to local conditions of the unique ecosystems they inhabit. A comparison with a subset of primers between T. guinasana and T. sparrmanii confirmed the separate species status of the former from the latter. The mean genetic distance between the T. sparrmanii populations was 0.136, compared to that between T. sparrmanii and T. guinasana which was found to be 0.374. Statistical analysis of the difference between the mean genetic distances indicated significance with 95% confidence. The polychromatism of T guinasana was investigated to determine whether there were significant differences between its five colour morphs. RAPD fingerprinting indicated with 95% confidence that there were significant differences between the colour forms based on the genetic distances computed between them. These genetic differences appeared to correlate with the observed assortative mating between the colour forms of the species. The manifestation of the polychromatism at sexual maturity in T guinasana probably indicates that colouration plays an important role in the breeding process. The genetic uniqueness shown here between the populations of T sparrmanii and the colour forms of T guinasana indicate for protective measures to be put in place if the genetic resources of the isolated fish populations are to be preserved. These must be coupled with a thorough assessment of the temporal and spatial distribution of genetic variability of the populations as a guide to a long-term management strategy for the fish populations and the ecosystems they inhabit. This study therefore has shown that the allopatric populations of T sparrmanii in the sinkholes and springs of the North-West Province are genetically unique, as well as show that the colour forms of T guinasana are genetically distinct.
- Full Text:
- Date Issued: 2002
Investigating the role of heat shock proteins (Hsps) 40, 70 and 90 in the life cycle of Theiler's murine encephalomyelitis virus (TMEV)
- Mutsvunguma, Lorraine Zvichapera
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
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
Immobilisation of electric eel acetylcholinesterase on nanofibres electrospun from a nylon and chitosan blend
- Authors: Mafuma, Tendai Simbarashe
- Date: 2013
- Subjects: Acetylcholinesterase Acetylcholinesterase -- Inhibitors Electric eel Biosensors Immobilized enzymes Pesticides -- Environmental aspects Pesticides -- Toxicology Nylon Chitosan Nanofibers Electrospinning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3886 , http://hdl.handle.net/10962/d1001620
- Description: Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity
- Full Text:
- Date Issued: 2013
- Authors: Mafuma, Tendai Simbarashe
- Date: 2013
- Subjects: Acetylcholinesterase Acetylcholinesterase -- Inhibitors Electric eel Biosensors Immobilized enzymes Pesticides -- Environmental aspects Pesticides -- Toxicology Nylon Chitosan Nanofibers Electrospinning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3886 , http://hdl.handle.net/10962/d1001620
- Description: Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity
- Full Text:
- Date Issued: 2013
Characterisation of the plasmodium falciparum Hsp40 chaperones and their partnerships with Hsp70
- Authors: Botha, Melissa
- Date: 2009
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Molecular chaperones Malaria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3938 , http://hdl.handle.net/10962/d1003997
- Description: Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.
- Full Text:
- Date Issued: 2009
- Authors: Botha, Melissa
- Date: 2009
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Molecular chaperones Malaria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3938 , http://hdl.handle.net/10962/d1003997
- Description: Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.
- Full Text:
- Date Issued: 2009
Serotonin-melatonin interactions in acetaminophen and N,N-dimethylformamide toxicity
- Anoopkumar-Dukie, Shailendra
- Authors: Anoopkumar-Dukie, Shailendra
- Date: 2000
- Subjects: Serotonin , Acetaminophen , Melatonin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3898 , http://hdl.handle.net/10962/d1003957 , Serotonin , Acetaminophen , Melatonin
- Description: Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain serotonin levels. Using organ culture where exogenous (3H) tryptophan is metabolised to (3H) melatonin, acetaminophen (100mg/kg for three hours) was shown to significantly increase (p<0.05) pineal melatonin concentrations. However, the ELISA technique did not reveal any changes in endogenous pineal melatonin levels. The final experiment was the determination of urinary 5-hydroxyindole acetic acid (5- HIAA), the major metabolite of serotonin, following acetaminophen administration (100mg/kg for three hours). Acetaminophen was shown to significantly reduce 5-HIAA levels (p<0.05) suggesting reduced catabolism of serotonin. The findings of this study indicate that acetaminophen mimics the actions of an antidepressant. This compelling finding has important clinical implications, and needs to be examined further.
- Full Text:
- Date Issued: 2000
- Authors: Anoopkumar-Dukie, Shailendra
- Date: 2000
- Subjects: Serotonin , Acetaminophen , Melatonin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3898 , http://hdl.handle.net/10962/d1003957 , Serotonin , Acetaminophen , Melatonin
- Description: Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain serotonin levels. Using organ culture where exogenous (3H) tryptophan is metabolised to (3H) melatonin, acetaminophen (100mg/kg for three hours) was shown to significantly increase (p<0.05) pineal melatonin concentrations. However, the ELISA technique did not reveal any changes in endogenous pineal melatonin levels. The final experiment was the determination of urinary 5-hydroxyindole acetic acid (5- HIAA), the major metabolite of serotonin, following acetaminophen administration (100mg/kg for three hours). Acetaminophen was shown to significantly reduce 5-HIAA levels (p<0.05) suggesting reduced catabolism of serotonin. The findings of this study indicate that acetaminophen mimics the actions of an antidepressant. This compelling finding has important clinical implications, and needs to be examined further.
- Full Text:
- Date Issued: 2000
The toxic effect of heavy metals on algal biomass (Spirulina sp.) and carbonic anhydrase activity, an enzyme which is central to algal application in metal precipitation
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
The hydrolysis of primary sewage sludge under biosulphidogenic conditions
- Molwantwa, Jennifer Balatedi
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
- Full Text:
- Date Issued: 2003
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
- Full Text:
- Date Issued: 2003
A process for the detanning of chrome leather wastes utilising tannery effluents
- Authors: Glaum, Deanne Melanie
- Date: 1994
- Subjects: Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4029 , http://hdl.handle.net/10962/d1004089 , Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Description: The considerable volume of chromium-bearing wastes generated during the process of leather tanning, exacerbated by the potential for trivalent chromium in the wastes to be oxidised to the toxic hexavalent state, has created a major waste disposal dilemma for the tanning industry. While methods are available for the safe and effective treatment of residual chrome-tanning liquors, little has been done to address the issue of the chrome-bearing solid wastes. Given the increasingly stringent environmental compliance standards facing tanneries, unless an appropriate treatment process is developed in the immediate future, the continued use of chromium as a tanning agent could be compromised. Recent investigations have demonstrated the potential of heated alkaline conditions for dechroming these solid wastes. This study expanded upon these considerations and examined the feasibility of utilising the highly alkaline tannery waste effluents as cost-effective, substitute alkaline media. The three effluents considered in this study, classed as lime sulphide liquors, were shown to be capable of dechroming wet blue shavings, with resultant separation of the solid wastes into a protein and a concentrated chromium product. The solubilised protein product contained low chromium concentrations which comply with legal discharge limits. The precipitated chromium product offers opportunity for reutilisation in the tannery. A novel industrial-scale treatment process, based on these investigations, indicated the process to be capable of treating the quantity of shavings produced on a daily basis by a medium to large scale tannery. Application of this method for the dechroming of other chrome-tanned solid wastes was also shown to be feasible.
- Full Text:
- Date Issued: 1994
- Authors: Glaum, Deanne Melanie
- Date: 1994
- Subjects: Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4029 , http://hdl.handle.net/10962/d1004089 , Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Description: The considerable volume of chromium-bearing wastes generated during the process of leather tanning, exacerbated by the potential for trivalent chromium in the wastes to be oxidised to the toxic hexavalent state, has created a major waste disposal dilemma for the tanning industry. While methods are available for the safe and effective treatment of residual chrome-tanning liquors, little has been done to address the issue of the chrome-bearing solid wastes. Given the increasingly stringent environmental compliance standards facing tanneries, unless an appropriate treatment process is developed in the immediate future, the continued use of chromium as a tanning agent could be compromised. Recent investigations have demonstrated the potential of heated alkaline conditions for dechroming these solid wastes. This study expanded upon these considerations and examined the feasibility of utilising the highly alkaline tannery waste effluents as cost-effective, substitute alkaline media. The three effluents considered in this study, classed as lime sulphide liquors, were shown to be capable of dechroming wet blue shavings, with resultant separation of the solid wastes into a protein and a concentrated chromium product. The solubilised protein product contained low chromium concentrations which comply with legal discharge limits. The precipitated chromium product offers opportunity for reutilisation in the tannery. A novel industrial-scale treatment process, based on these investigations, indicated the process to be capable of treating the quantity of shavings produced on a daily basis by a medium to large scale tannery. Application of this method for the dechroming of other chrome-tanned solid wastes was also shown to be feasible.
- Full Text:
- Date Issued: 1994
Development and characterisation of a membrane gradostat bioreactor for the bioremediation of aromatic pollutants using white rot fungi
- Authors: Leukes, W
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
- Full Text:
- Date Issued: 1999
- Authors: Leukes, W
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
- Full Text:
- Date Issued: 1999
Fundamental investigations into the factors affecting the response of laccase-based electrochemical biosensors
- Authors: Fogel, Ronen
- Date: 2011
- Subjects: Laccase Phenols Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4073 , http://hdl.handle.net/10962/d1007166
- Description: Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes.
- Full Text:
- Date Issued: 2011
- Authors: Fogel, Ronen
- Date: 2011
- Subjects: Laccase Phenols Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4073 , http://hdl.handle.net/10962/d1007166
- Description: Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes.
- Full Text:
- Date Issued: 2011
Voltammetric investigation of microbiological growth media and carbon nanotube modified electrodes : a case study of oxytetracycline
- Authors: Kruid, Jan
- Date: 2013
- Subjects: Voltammetry , Electrodes , Oxytetracycline , Carbon nanotubes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4156 , http://hdl.handle.net/10962/d1018238
- Description: Oxytetracycline (OTC) is a broad spectrum antibiotic used extensively in the agricultural and human-health sector, and is effective against various gram positive and –negative bacteria as well as large viruses and certain pathogenic Rickettsiae. This study addresses the lack of voltammetric knowledge regarding the electroanalytical characterisation of OTC and its analysis in complex matrices. Cyclic voltammetry (CV) revealed several irreversible anodic peaks for OTC at a bare glassy carbon electrode (GCE). These current responses were improved through the selection of a diluent for OTC stock preparation, electrolyte solution and electrolyte pH, stir time and applied preconditioning potential. Under enhanced adsorptive conditions and using square wave voltammetry (SWV), a detection limit of 24.3 nM was achieved. The electrode surface could be renewed in vitro for 10 successive scans. OTC oxidation was characterised as a one electron:one proton ECiE mechanisms. Next, investigating the viability of voltammetry in various complex microbiological growth media revealed that selected growth media contained interfering redox active components, which, while simultaneously coating the electrode surface, effectively reduced GCE performance and lowered the active electrode surface area, as ascertained through CV and electrochemical impedance spectroscopy (EIS) studies. This interference lowered OTC current response in the presence of growth media which was partially recovered by appropriate growth media selection and sample dilution. In testing the use of acid functionalised multi-walled carbon nanotubes (MWCNTs) to improve anodic OTC response, charge-based attraction was observed between the MWCNT dispersal agent Nafion® and OTC, while increased surface area associated with prolonged acid functionalisation time aided in improving OTC current response.
- Full Text:
- Date Issued: 2013
- Authors: Kruid, Jan
- Date: 2013
- Subjects: Voltammetry , Electrodes , Oxytetracycline , Carbon nanotubes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4156 , http://hdl.handle.net/10962/d1018238
- Description: Oxytetracycline (OTC) is a broad spectrum antibiotic used extensively in the agricultural and human-health sector, and is effective against various gram positive and –negative bacteria as well as large viruses and certain pathogenic Rickettsiae. This study addresses the lack of voltammetric knowledge regarding the electroanalytical characterisation of OTC and its analysis in complex matrices. Cyclic voltammetry (CV) revealed several irreversible anodic peaks for OTC at a bare glassy carbon electrode (GCE). These current responses were improved through the selection of a diluent for OTC stock preparation, electrolyte solution and electrolyte pH, stir time and applied preconditioning potential. Under enhanced adsorptive conditions and using square wave voltammetry (SWV), a detection limit of 24.3 nM was achieved. The electrode surface could be renewed in vitro for 10 successive scans. OTC oxidation was characterised as a one electron:one proton ECiE mechanisms. Next, investigating the viability of voltammetry in various complex microbiological growth media revealed that selected growth media contained interfering redox active components, which, while simultaneously coating the electrode surface, effectively reduced GCE performance and lowered the active electrode surface area, as ascertained through CV and electrochemical impedance spectroscopy (EIS) studies. This interference lowered OTC current response in the presence of growth media which was partially recovered by appropriate growth media selection and sample dilution. In testing the use of acid functionalised multi-walled carbon nanotubes (MWCNTs) to improve anodic OTC response, charge-based attraction was observed between the MWCNT dispersal agent Nafion® and OTC, while increased surface area associated with prolonged acid functionalisation time aided in improving OTC current response.
- Full Text:
- Date Issued: 2013
Characterization of amide bond hydrolysis in novel hydantoinase-producing bacteria
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
- Full Text:
- Date Issued: 2000
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
- Full Text:
- Date Issued: 2000
High pressure liquid chromatographic quantification of nitrile biocatalysis
- Authors: Mathiba, Kgama
- Date: 2012
- Subjects: High performance liquid chromatography , Rhodococcus , Biocatalysis , Organic compounds -- Industrial applications
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4129 , http://hdl.handle.net/10962/d1015710
- Description: Nitrile biocatalysts are of use in the chemical and pharmaceutical industries for the synthesis of carboxyamides and carboxylic acids. In particular, the application of biocatalysts in the synthesis of single enantiomer compounds is of increasing interest, but requires novel substrate specific highly stereoselective biocatalysts. Addition to the limited toolbox of known nitrile biocatalysts requires definitive characterisation of the biocatalysts through accurate determination of the substrate profiles and quantification of activity. The accurate quantification of stereoisomers chiral mixtures to determine biocatalyst stereoselectivity remains a significant challenge due to the difficulty in separating stereoisomers by physical methods. The known nitrile metabolising organism, Rhodococcus rhodochrous ATCC BAA-870, was grown in a defined medium and harvested, providing whole cell biocatalyst. Additional biomass was disrupted to provide a cell free enzyme extract, which was put through an enzyme purification protocol to provide a solution with specific activity of 351 U.mg⁻¹. A portion of the enzyme was self immobilised using the SphereZyme™ technique. The nitrile hydratase SphereZymes™ (1.2 U.mg⁻¹ initial activity) that were prepared had pH and temperature optima of 6 and 30°C respectively, and could be recovered by repeated washing. The particles retained activity in the presence of the organic solvents isooctane and n-hexadecane saturated with 50 mM phosphate buffer (pH 7.5). An initial analytical system was devised for quantification of the nitrile hydratase activity using the non-chiral substrate benzonitrile. An improved reversed phase high performance liquid chromatography method was developed to separate and quantify benzamide, benzoic acid and benzonitrile. The mobile phase consisting of 0.1% trifluoroacetic acid in H₂O and acetonitrile (70:30, %v/v), at a flow rate of 0.5 ml.ml⁻¹, 25°C, resolved all three analytes in 3.5 minutes on a Waters X-Terra MS C18 3.5μm column. UV detection was carried out at 210 nm. Analytical methods to determine activity and enantioselectivity of the whole cell biocatalyst were subsequently developed for both β-amino nitriles and β-hydroxy nitrile substrates and hydrolysis products.
- Full Text:
- Date Issued: 2012
- Authors: Mathiba, Kgama
- Date: 2012
- Subjects: High performance liquid chromatography , Rhodococcus , Biocatalysis , Organic compounds -- Industrial applications
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4129 , http://hdl.handle.net/10962/d1015710
- Description: Nitrile biocatalysts are of use in the chemical and pharmaceutical industries for the synthesis of carboxyamides and carboxylic acids. In particular, the application of biocatalysts in the synthesis of single enantiomer compounds is of increasing interest, but requires novel substrate specific highly stereoselective biocatalysts. Addition to the limited toolbox of known nitrile biocatalysts requires definitive characterisation of the biocatalysts through accurate determination of the substrate profiles and quantification of activity. The accurate quantification of stereoisomers chiral mixtures to determine biocatalyst stereoselectivity remains a significant challenge due to the difficulty in separating stereoisomers by physical methods. The known nitrile metabolising organism, Rhodococcus rhodochrous ATCC BAA-870, was grown in a defined medium and harvested, providing whole cell biocatalyst. Additional biomass was disrupted to provide a cell free enzyme extract, which was put through an enzyme purification protocol to provide a solution with specific activity of 351 U.mg⁻¹. A portion of the enzyme was self immobilised using the SphereZyme™ technique. The nitrile hydratase SphereZymes™ (1.2 U.mg⁻¹ initial activity) that were prepared had pH and temperature optima of 6 and 30°C respectively, and could be recovered by repeated washing. The particles retained activity in the presence of the organic solvents isooctane and n-hexadecane saturated with 50 mM phosphate buffer (pH 7.5). An initial analytical system was devised for quantification of the nitrile hydratase activity using the non-chiral substrate benzonitrile. An improved reversed phase high performance liquid chromatography method was developed to separate and quantify benzamide, benzoic acid and benzonitrile. The mobile phase consisting of 0.1% trifluoroacetic acid in H₂O and acetonitrile (70:30, %v/v), at a flow rate of 0.5 ml.ml⁻¹, 25°C, resolved all three analytes in 3.5 minutes on a Waters X-Terra MS C18 3.5μm column. UV detection was carried out at 210 nm. Analytical methods to determine activity and enantioselectivity of the whole cell biocatalyst were subsequently developed for both β-amino nitriles and β-hydroxy nitrile substrates and hydrolysis products.
- Full Text:
- Date Issued: 2012
Bacterial degradation of waste coal
- Authors: Madikiza, Lwazikazi
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54576 , vital:26590
- Description: As an energy source coal has one of the largest agglomerations in the world. Consequently mining of coal creates large volumes of waste in the form of low ranks coals. The complex structure of coal makes it difficult for the microorganisms to degrade and relatively few bacteria and fungi have been shown to break down coal. This study aimed to investigate bacteria not previously known to degrade coal. In this study bacteria were isolated from hydrocarbon contaminated sites and inoculated in coal medium where coal served as the only carbon source. Three strains produced a yellow – brown supernatant after 14 d of incubation at 30 °C. Bacteria generating a yellow – brown coloured supernatant were presumed to possess coal degrading capabilities and the best performing of these bacterial species was identified using 16s rDNA as Bacillus flexus. Scanning electron microscopy showed that there was a close association between the bacterium and substrate coal. The close association of bacteria to substrate suggested that these organisms were able to maximize solubilisation. FT-IR spectroscopic analysis demonstrated the addition of single bonded compounds COOH, OH, CN and CH that were absent prior to bacterial interaction. The increase in oxygen rich regions indicated degradation of the coal substrate. Elemental analysis showed that there was a decrease in carbon content from 47 % to 24 % during the 14 day incubation period. Reduction in coal carbon content was assumed to be due to bacterial utilization for metabolism and growth particularly as untreated coal substrate showed minimal loss of carbon. Analysis of the residual culture medium revealed that there was a linear increase in humic-like substance concentration for 8 d, coincident with increased coal biosolubilisation and colour change. Laccase activity was insignificant, and at 13 d enzyme activity was only 5×10-3 U/L suggesting that B. flexus may use a different mechanism to degrade coal. Residual culture medium remaining after bacterial action on the coal substrate appeared to possess plant growth promoting activity. This soluble biodegradation product with characteristics similar to humic acid-like substances was shown to impact growth of radish cotyledons. Expansion of isolated radish cotyledons was enhanced by 140% when incubated in coal biodegradation product. In conclusion, this study has yielded B. flexus and two other unidentified bacteria, isolated from polyaromatic hydrocarbon contaminated soils, and demonstrated the ability of these microorganisms to degrade waste coal. Further studies to elucidate the mechanism of coal breakdown by B. flexus, synergies with other coal degrading microorganisms, and incorporation of bacterium into Fungcoal bioprocess technology is imminent.
- Full Text:
- Date Issued: 2014
- Authors: Madikiza, Lwazikazi
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54576 , vital:26590
- Description: As an energy source coal has one of the largest agglomerations in the world. Consequently mining of coal creates large volumes of waste in the form of low ranks coals. The complex structure of coal makes it difficult for the microorganisms to degrade and relatively few bacteria and fungi have been shown to break down coal. This study aimed to investigate bacteria not previously known to degrade coal. In this study bacteria were isolated from hydrocarbon contaminated sites and inoculated in coal medium where coal served as the only carbon source. Three strains produced a yellow – brown supernatant after 14 d of incubation at 30 °C. Bacteria generating a yellow – brown coloured supernatant were presumed to possess coal degrading capabilities and the best performing of these bacterial species was identified using 16s rDNA as Bacillus flexus. Scanning electron microscopy showed that there was a close association between the bacterium and substrate coal. The close association of bacteria to substrate suggested that these organisms were able to maximize solubilisation. FT-IR spectroscopic analysis demonstrated the addition of single bonded compounds COOH, OH, CN and CH that were absent prior to bacterial interaction. The increase in oxygen rich regions indicated degradation of the coal substrate. Elemental analysis showed that there was a decrease in carbon content from 47 % to 24 % during the 14 day incubation period. Reduction in coal carbon content was assumed to be due to bacterial utilization for metabolism and growth particularly as untreated coal substrate showed minimal loss of carbon. Analysis of the residual culture medium revealed that there was a linear increase in humic-like substance concentration for 8 d, coincident with increased coal biosolubilisation and colour change. Laccase activity was insignificant, and at 13 d enzyme activity was only 5×10-3 U/L suggesting that B. flexus may use a different mechanism to degrade coal. Residual culture medium remaining after bacterial action on the coal substrate appeared to possess plant growth promoting activity. This soluble biodegradation product with characteristics similar to humic acid-like substances was shown to impact growth of radish cotyledons. Expansion of isolated radish cotyledons was enhanced by 140% when incubated in coal biodegradation product. In conclusion, this study has yielded B. flexus and two other unidentified bacteria, isolated from polyaromatic hydrocarbon contaminated soils, and demonstrated the ability of these microorganisms to degrade waste coal. Further studies to elucidate the mechanism of coal breakdown by B. flexus, synergies with other coal degrading microorganisms, and incorporation of bacterium into Fungcoal bioprocess technology is imminent.
- Full Text:
- Date Issued: 2014
Structural bioinformatics analysis of the Hsp40 and Hsp70 molecular chaperones from humans
- Authors: Adeyemi, Samson Adebowale
- Date: 2014
- Subjects: Structural bioinformatics , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Biomolecules
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4171 , http://hdl.handle.net/10962/d1020962
- Description: HSP70 is one of the most important families of molecular chaperone that regulate the folding and transport of client proteins in an ATP dependent manner. The ATPase activity of HSP70 is stimulated through an interaction with its family of HSP40 co-chaperones. There is evidence to suggest that specific partnerships occur between the different HSP40 and HSP70 isoforms. While some of the residues involved in the interaction are known, many of the residues governing the specificity of HSP40-HSP70 partnerships are not precisely defined. It is not currently possible to predict which HSP40 and HSP70 isoforms will interact. We attempted to use bioinformatics to identify residues involved in the specificity of the interaction between the J domain from HSP40 and the ATPase domain from the HSP70 isoforms from humans. A total of 49 HSP40 and 13 HSP70 sequences from humans were retrieved and used for subsequent analyses. The HSP40 J domains and HSP70 ATPase domains were extracted using python scripts and classified according to the subcellular localization of the proteins using localization prediction programs. Motif analysis was carried out using the full length HSP40 proteins and Multiple Sequence Alignment (MSA) was performed to identify conserved residues that may contribute to the J domain – ATPase domain interactions. Phylogenetic inference of the proteins was also performed in order to study their evolutionary relationship. Homology models of the J domains and ATPase domains were generated. The corresponding models were docked using HADDOCK server in order to analyze possible putative interactions between the partner proteins using the Protein Interactions Calculator (PIC). The level of residue conservation was found to be higher in Type I and II HSP40 than in Type III J proteins. While highly conserved residues on helixes II and III could play critical roles in J domain interactions with corresponding HSP70s, conserved residues on helixes I and IV seemed to be significant in keeping the J domain in its right orientation for functional interactions with HSP70s. Our results also showed that helixes II and III formed the interaction interface for binding to HSP70 ATPase domain as well as the linker residues. Finally, data based docking procedures, such as applied in this study, could be an effective method to investigate protein-protein interactions complex of biomolecules.
- Full Text:
- Date Issued: 2014
- Authors: Adeyemi, Samson Adebowale
- Date: 2014
- Subjects: Structural bioinformatics , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Biomolecules
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4171 , http://hdl.handle.net/10962/d1020962
- Description: HSP70 is one of the most important families of molecular chaperone that regulate the folding and transport of client proteins in an ATP dependent manner. The ATPase activity of HSP70 is stimulated through an interaction with its family of HSP40 co-chaperones. There is evidence to suggest that specific partnerships occur between the different HSP40 and HSP70 isoforms. While some of the residues involved in the interaction are known, many of the residues governing the specificity of HSP40-HSP70 partnerships are not precisely defined. It is not currently possible to predict which HSP40 and HSP70 isoforms will interact. We attempted to use bioinformatics to identify residues involved in the specificity of the interaction between the J domain from HSP40 and the ATPase domain from the HSP70 isoforms from humans. A total of 49 HSP40 and 13 HSP70 sequences from humans were retrieved and used for subsequent analyses. The HSP40 J domains and HSP70 ATPase domains were extracted using python scripts and classified according to the subcellular localization of the proteins using localization prediction programs. Motif analysis was carried out using the full length HSP40 proteins and Multiple Sequence Alignment (MSA) was performed to identify conserved residues that may contribute to the J domain – ATPase domain interactions. Phylogenetic inference of the proteins was also performed in order to study their evolutionary relationship. Homology models of the J domains and ATPase domains were generated. The corresponding models were docked using HADDOCK server in order to analyze possible putative interactions between the partner proteins using the Protein Interactions Calculator (PIC). The level of residue conservation was found to be higher in Type I and II HSP40 than in Type III J proteins. While highly conserved residues on helixes II and III could play critical roles in J domain interactions with corresponding HSP70s, conserved residues on helixes I and IV seemed to be significant in keeping the J domain in its right orientation for functional interactions with HSP70s. Our results also showed that helixes II and III formed the interaction interface for binding to HSP70 ATPase domain as well as the linker residues. Finally, data based docking procedures, such as applied in this study, could be an effective method to investigate protein-protein interactions complex of biomolecules.
- Full Text:
- Date Issued: 2014
Bacterial interaction in hide biodeterioration with special reference to selected Clostridium species
- Authors: Thompson, Gillian Ann
- Date: 1995
- Subjects: Hides and skins -- Preservation Aerobic bacteria Pseudomonas aeruginosa Clostridium Halobacterium
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4041 , http://hdl.handle.net/10962/d1004102
- Description: Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.
- Full Text:
- Date Issued: 1995
- Authors: Thompson, Gillian Ann
- Date: 1995
- Subjects: Hides and skins -- Preservation Aerobic bacteria Pseudomonas aeruginosa Clostridium Halobacterium
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4041 , http://hdl.handle.net/10962/d1004102
- Description: Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.
- Full Text:
- Date Issued: 1995