Characterisation of the cellulolytic and hemicellulolytic system of Bacillus Licheniformis SVD1 and the isolation and characterisation of a multi-enzyme complex
- Authors: Van Dyk, Jacoba Susanna
- Date: 2009
- Subjects: Lignocellulose Lignocellulose -- Biotechnology Lignocellulose -- Biodegradation Plant biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3936 , http://hdl.handle.net/10962/d1003995
- Description: The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.
- Full Text:
- Date Issued: 2009
- Authors: Van Dyk, Jacoba Susanna
- Date: 2009
- Subjects: Lignocellulose Lignocellulose -- Biotechnology Lignocellulose -- Biodegradation Plant biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3936 , http://hdl.handle.net/10962/d1003995
- Description: The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.
- Full Text:
- Date Issued: 2009
The development and evaluation of gaze selection techniques
- Authors: Van Tonder, Martin Stephen
- Date: 2009
- Subjects: Human-computer interaction , User interfaces (Computer systems) , Gaze
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10469 , http://hdl.handle.net/10948/882 , Human-computer interaction , User interfaces (Computer systems) , Gaze
- Description: Eye gaze interaction enables users to interact with computers using their eyes. A wide variety of eye gaze interaction techniques have been developed to support this type of interaction. Gaze selection techniques, a class of eye gaze interaction techniques which support target selection, are the subject of this research. Researchers developing these techniques face a number of challenges. The most significant challenge is the limited accuracy of eye tracking equipment (due to the properties of the human eye). The design of gaze selection techniques is dominated by this constraint. Despite decades of research, existing techniques are still significantly less accurate than the mouse. A recently developed technique, EyePoint, represents the state of the art in gaze selection techniques. EyePoint combines gaze input with keyboard input. Evaluation results for this technique are encouraging, but accuracy is still a concern. Early trigger errors, resulting from users triggering a selection before looking at the intended target, were found to be the most commonly occurring errors for this technique. The primary goal of this research was to improve the usability of gaze selection techniques. In order to achieve this goal, novel gaze selection techniques were developed. New techniques were developed by combining elements of existing techniques in novel ways. Seven novel gaze selection techniques were developed. Three of these techniques were selected for evaluation. A software framework was developed for implementing and evaluating gaze selection techniques. This framework was used to implement the gaze selection techniques developed during this research. Implementing and evaluating all of the techniques using a common framework ensured consistency when comparing the techniques. The novel techniques which were developed were evaluated against EyePoint and the mouse using the framework. The three novel techniques evaluated were named TargetPoint, StaggerPoint and ScanPoint. TargetPoint combines motor space expansion with a visual feedback highlight whereas the StaggerPoint and TargetPoint designs explore novel approaches to target selection disambiguation. A usability evaluation of the three novel techniques alongside EyePoint and the mouse revealed some interesting trends. TargetPoint was found to be more usable and accurate than EyePoint. This novel technique also proved more popular with test participants. One aspect of TargetPoint which proved particularly popular was the visual feedback highlight, a feature which was found to be a more effective method of combating early trigger errors than existing approaches. StaggerPoint was more efficient than EyePoint, but was less effective and satisfying. ScanPoint was the least popular technique. The benefits of providing a visual feedback highlight and test participants' positive views thereof contradict views expressed in existing research regarding the usability of visual feedback. These results have implications for the design of future gaze selection techniques. A set of design principles was developed for designing new gaze selection techniques. The designers of gaze selection techniques can benefit from these design principles by applying them to their techniques
- Full Text:
- Date Issued: 2009
- Authors: Van Tonder, Martin Stephen
- Date: 2009
- Subjects: Human-computer interaction , User interfaces (Computer systems) , Gaze
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10469 , http://hdl.handle.net/10948/882 , Human-computer interaction , User interfaces (Computer systems) , Gaze
- Description: Eye gaze interaction enables users to interact with computers using their eyes. A wide variety of eye gaze interaction techniques have been developed to support this type of interaction. Gaze selection techniques, a class of eye gaze interaction techniques which support target selection, are the subject of this research. Researchers developing these techniques face a number of challenges. The most significant challenge is the limited accuracy of eye tracking equipment (due to the properties of the human eye). The design of gaze selection techniques is dominated by this constraint. Despite decades of research, existing techniques are still significantly less accurate than the mouse. A recently developed technique, EyePoint, represents the state of the art in gaze selection techniques. EyePoint combines gaze input with keyboard input. Evaluation results for this technique are encouraging, but accuracy is still a concern. Early trigger errors, resulting from users triggering a selection before looking at the intended target, were found to be the most commonly occurring errors for this technique. The primary goal of this research was to improve the usability of gaze selection techniques. In order to achieve this goal, novel gaze selection techniques were developed. New techniques were developed by combining elements of existing techniques in novel ways. Seven novel gaze selection techniques were developed. Three of these techniques were selected for evaluation. A software framework was developed for implementing and evaluating gaze selection techniques. This framework was used to implement the gaze selection techniques developed during this research. Implementing and evaluating all of the techniques using a common framework ensured consistency when comparing the techniques. The novel techniques which were developed were evaluated against EyePoint and the mouse using the framework. The three novel techniques evaluated were named TargetPoint, StaggerPoint and ScanPoint. TargetPoint combines motor space expansion with a visual feedback highlight whereas the StaggerPoint and TargetPoint designs explore novel approaches to target selection disambiguation. A usability evaluation of the three novel techniques alongside EyePoint and the mouse revealed some interesting trends. TargetPoint was found to be more usable and accurate than EyePoint. This novel technique also proved more popular with test participants. One aspect of TargetPoint which proved particularly popular was the visual feedback highlight, a feature which was found to be a more effective method of combating early trigger errors than existing approaches. StaggerPoint was more efficient than EyePoint, but was less effective and satisfying. ScanPoint was the least popular technique. The benefits of providing a visual feedback highlight and test participants' positive views thereof contradict views expressed in existing research regarding the usability of visual feedback. These results have implications for the design of future gaze selection techniques. A set of design principles was developed for designing new gaze selection techniques. The designers of gaze selection techniques can benefit from these design principles by applying them to their techniques
- Full Text:
- Date Issued: 2009
Ecological role of estuarine brachyuran crabs in mangrove and salt marsh estuaries, Eastern Cape, South Africa
- Authors: Vorsatz, Jeanne Pauline
- Date: 2009
- Subjects: Crabs -- South Africa -- Mangrove Estuary , Crabs -- South Africa -- Salt Marsh Estuary , Salt marsh ecology , Mangrove ecology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10698 , http://hdl.handle.net/10948/1108 , Crabs -- South Africa -- Mangrove Estuary , Crabs -- South Africa -- Salt Marsh Estuary , Salt marsh ecology , Mangrove ecology
- Description: Crabs are conspicuous inhabitants of temperate salt marshes and tropical mangroves and interact with their environment through several processes. However, detailed information on crab community processes is absent for most South African estuaries and nearshore coastal regions. This study evaluated the primary producers supporting crab species in the salt marsh dominated Swartkops estuary and the mangrove Mngazana estuary. Various methods estimating crab abundances were also assessed in different microhabitats and the larval distribution of crabs in the coastal zone was also investigated. Various methods for estimating crab abundance have been employed in the past, each with its inherent biases. The microhabitat of a mangrove forest in Australia was structurally altered by the manipulation of the litter, pneumatophores and the associated algae. These alterations did not affect the behavioural activity or the numbers of crabs recorded in any of the experimental treatments by either visual counts or pitfall traps. However, the number of crabs caught in the pitfall traps differed between the sites. Species-specific behaviour which was not investigated in this study may bias crab abundance estimates when using pitfall traps and therefore requires further investigation. Benthic consumers inhabiting shallow coastal environments may ultimately have the origin of their nutrition in a number of possible sources. Isotopic and gut content analysis of Thalamita crenata and juvenile Scylla serrata in the Mngazana estuary in South Africa revealed that these two portunids are able to share a habitat by resource partitioning. Differences were noted for species-specific utilization of primary producers not only between seasons within a site, but also between sites. This highlighted the use of locally produced primary producers sustaining food webs in estuaries. Mangrove production in the Mngazana estuary is very important and contributes to most of the carbon in the underlying sediments in the mangrove forest. However, the relatively large number of species and biomass encountered in this estuary may also be attributed to the fact that the different species are able to exploit of a number of different resources. The variation in stable isotope analysis of the different crab species throughout the estuary indicated that these crabs able to occupy the same habitat by feeding on a number of different resources and may preferentially select for a specific primary producer. A stable isotope of crabs in the salt marsh Swartkops estuary indicated that the dominant primary producer sustaining crab communities may even take place on a relatively smallscale. Sesarma catenata found at the inner marsh site recorded more depleted carbon signatures than those encountered in the other sites approximately 100 m away, and reflected signatures similar to the locally-encountered inner marsh plants. The relatively enriched nitrogen signatures of the anthropogenically-impacted Swartkops estuary is an indication of extensive inputs due to urbanization and industrialization, in contrast to the relatively pristine Mngazana estuary which exhibited low nitrogen signatures. Emphasis has been placed on the abiotic component of the exchange of nutrients and energy, although living organisms may also be transported, both actively and passively, between ecosystems. Little variation in either species composition or abundance was found between seasons for the larval distribution of brachyuran crabs on the east coast of South Africa. Due to the lack of published larval descriptions, larvae could not be identified to species level and it was therefore not possible to identify whether the larvae were hatched or spawned in an estuary or in a marine environment, or whether the larvae originated in the northern tropical regions. Frequent wind-reversals which are common in this region may retain larvae close inshore and supply the southern temperate locations with larvae from the northern locations. In conclusion, this study has shown that in highly productive systems with a number of potential primary producers, the crabs that inhabit the estuary show a marked diversity in resource utilization which could potentially allow a number of closely related species to occupy different trophic levels. This study also highlights the importance of locally produced sources in an estuary, which may occur on very small scales and this needs to be factored in with the design of any future stable isotope studies of this nature.
- Full Text:
- Date Issued: 2009
- Authors: Vorsatz, Jeanne Pauline
- Date: 2009
- Subjects: Crabs -- South Africa -- Mangrove Estuary , Crabs -- South Africa -- Salt Marsh Estuary , Salt marsh ecology , Mangrove ecology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10698 , http://hdl.handle.net/10948/1108 , Crabs -- South Africa -- Mangrove Estuary , Crabs -- South Africa -- Salt Marsh Estuary , Salt marsh ecology , Mangrove ecology
- Description: Crabs are conspicuous inhabitants of temperate salt marshes and tropical mangroves and interact with their environment through several processes. However, detailed information on crab community processes is absent for most South African estuaries and nearshore coastal regions. This study evaluated the primary producers supporting crab species in the salt marsh dominated Swartkops estuary and the mangrove Mngazana estuary. Various methods estimating crab abundances were also assessed in different microhabitats and the larval distribution of crabs in the coastal zone was also investigated. Various methods for estimating crab abundance have been employed in the past, each with its inherent biases. The microhabitat of a mangrove forest in Australia was structurally altered by the manipulation of the litter, pneumatophores and the associated algae. These alterations did not affect the behavioural activity or the numbers of crabs recorded in any of the experimental treatments by either visual counts or pitfall traps. However, the number of crabs caught in the pitfall traps differed between the sites. Species-specific behaviour which was not investigated in this study may bias crab abundance estimates when using pitfall traps and therefore requires further investigation. Benthic consumers inhabiting shallow coastal environments may ultimately have the origin of their nutrition in a number of possible sources. Isotopic and gut content analysis of Thalamita crenata and juvenile Scylla serrata in the Mngazana estuary in South Africa revealed that these two portunids are able to share a habitat by resource partitioning. Differences were noted for species-specific utilization of primary producers not only between seasons within a site, but also between sites. This highlighted the use of locally produced primary producers sustaining food webs in estuaries. Mangrove production in the Mngazana estuary is very important and contributes to most of the carbon in the underlying sediments in the mangrove forest. However, the relatively large number of species and biomass encountered in this estuary may also be attributed to the fact that the different species are able to exploit of a number of different resources. The variation in stable isotope analysis of the different crab species throughout the estuary indicated that these crabs able to occupy the same habitat by feeding on a number of different resources and may preferentially select for a specific primary producer. A stable isotope of crabs in the salt marsh Swartkops estuary indicated that the dominant primary producer sustaining crab communities may even take place on a relatively smallscale. Sesarma catenata found at the inner marsh site recorded more depleted carbon signatures than those encountered in the other sites approximately 100 m away, and reflected signatures similar to the locally-encountered inner marsh plants. The relatively enriched nitrogen signatures of the anthropogenically-impacted Swartkops estuary is an indication of extensive inputs due to urbanization and industrialization, in contrast to the relatively pristine Mngazana estuary which exhibited low nitrogen signatures. Emphasis has been placed on the abiotic component of the exchange of nutrients and energy, although living organisms may also be transported, both actively and passively, between ecosystems. Little variation in either species composition or abundance was found between seasons for the larval distribution of brachyuran crabs on the east coast of South Africa. Due to the lack of published larval descriptions, larvae could not be identified to species level and it was therefore not possible to identify whether the larvae were hatched or spawned in an estuary or in a marine environment, or whether the larvae originated in the northern tropical regions. Frequent wind-reversals which are common in this region may retain larvae close inshore and supply the southern temperate locations with larvae from the northern locations. In conclusion, this study has shown that in highly productive systems with a number of potential primary producers, the crabs that inhabit the estuary show a marked diversity in resource utilization which could potentially allow a number of closely related species to occupy different trophic levels. This study also highlights the importance of locally produced sources in an estuary, which may occur on very small scales and this needs to be factored in with the design of any future stable isotope studies of this nature.
- Full Text:
- Date Issued: 2009
Establishing experimental systems for studying the replication biology of Providence virus
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Date Issued: 2009
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Date Issued: 2009
Mathematical requirements for first-year BCOM students at NMMU
- Authors: Walton, Marguerite
- Date: 2009
- Subjects: Business mathematics -- South Africa , Mathematics -- Study and teaching (Higher) -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10502 , http://hdl.handle.net/10948/886 , Business mathematics -- South Africa , Mathematics -- Study and teaching (Higher) -- South Africa
- Description: These studies have focused on identifying the mathematical requirements of first-year BCom students at Nelson Mandela Metropolitan University. The research methodology used in this quantitative study was to make use of interviewing, questionnaire investigation, and document analysis in the form of textbook, test and examination analysis. These methods provided data that fitted into a grounded theory approach. The study concluded by identifying the list of mathematical topics required for the first year of the core subjects in the BCom degree programme. In addition, the study found that learners who study Mathematics in the National Senior Certificate should be able to cope with the mathematical content included in their BCom degree programme, while learners studying Mathematical Literacy would probably need support in some of the areas of mathematics, especially algebra, in order to cope with the mathematical content included in their BCom degree programme. It makes a valuable contribution towards elucidating the mathematical requirements needed to improve the chances of successful BCom degree programme studies at South African universities. It also draws the contours for starting to design an efficient support course for future “at-risk” students who enter higher education studies.
- Full Text:
- Date Issued: 2009
- Authors: Walton, Marguerite
- Date: 2009
- Subjects: Business mathematics -- South Africa , Mathematics -- Study and teaching (Higher) -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10502 , http://hdl.handle.net/10948/886 , Business mathematics -- South Africa , Mathematics -- Study and teaching (Higher) -- South Africa
- Description: These studies have focused on identifying the mathematical requirements of first-year BCom students at Nelson Mandela Metropolitan University. The research methodology used in this quantitative study was to make use of interviewing, questionnaire investigation, and document analysis in the form of textbook, test and examination analysis. These methods provided data that fitted into a grounded theory approach. The study concluded by identifying the list of mathematical topics required for the first year of the core subjects in the BCom degree programme. In addition, the study found that learners who study Mathematics in the National Senior Certificate should be able to cope with the mathematical content included in their BCom degree programme, while learners studying Mathematical Literacy would probably need support in some of the areas of mathematics, especially algebra, in order to cope with the mathematical content included in their BCom degree programme. It makes a valuable contribution towards elucidating the mathematical requirements needed to improve the chances of successful BCom degree programme studies at South African universities. It also draws the contours for starting to design an efficient support course for future “at-risk” students who enter higher education studies.
- Full Text:
- Date Issued: 2009
Compensation for polarization mode dispersion and nonlinear birefringence in a multichannel optical fibre system
- Authors: Waswa, David Wafula
- Date: 2009
- Subjects: Fiber optics , Nonlinear optics , Polarization (Light)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10375 , http://hdl.handle.net/10948/885 , Fiber optics , Nonlinear optics , Polarization (Light)
- Description: Polarization mode dispersion (PMD) is stochastic in nature and continues evolving in an unpredictable manner according to the changing environment. Nonlinear birefringence in multichannel systems alters the polarization states of the bits, so that they vary from one bit to the next in a way that is difficult to predict. These are the two major signal-impairment effects that are inherent in optical fibre transmission links which can seriously degrade network performance. It is therefore extremely challenging to compensate for both linear and nonlinear birefringence in multichannel systems. The purpose of this thesis is to investigate the interaction between PMD and nonlinear induced birefringence in a fibre with consideration of mode coupling. A sound knowledge of this interaction is necessary in designing a linear and nonlinear polarization mode dispersion compensator for WDM systems, as was successfully carried out in this study. The investigation shows that the effect of nonlinear birefringence alone depolarizes the signal, while in high PMD links where polarization mode coupling is high, the nonlinear birefringence effect couples with second-order PMD such that it may reduce the penalty and improve the signal DOP. Further investigation shows that when nonlinear birefringence becomes significant, asymmetry arises between the two principal axes of the fibre, such that it is only one axis which experiences the effect of nonlinear birefringence. It is found out that along this vii axis, there exists a critical point in pump power where the nonlinear birefringence cancels PMD in the link and improves the signal. An adaptive compensator to cancel PMD and nonlinear birefringence was designed based on feedforward DOP-monitoring signal. The compensator was tested both at laboratory level and on the Telkom buried fibre link and found to be functioning as intended. It was able to adaptively track and compensate PMD in the link in less than a second. The compensator was able to cancel PMD in the link up to a maximum of 30 ps. The compensator improved the DOP of the worst signal by more than 100 percent.
- Full Text:
- Date Issued: 2009
- Authors: Waswa, David Wafula
- Date: 2009
- Subjects: Fiber optics , Nonlinear optics , Polarization (Light)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10375 , http://hdl.handle.net/10948/885 , Fiber optics , Nonlinear optics , Polarization (Light)
- Description: Polarization mode dispersion (PMD) is stochastic in nature and continues evolving in an unpredictable manner according to the changing environment. Nonlinear birefringence in multichannel systems alters the polarization states of the bits, so that they vary from one bit to the next in a way that is difficult to predict. These are the two major signal-impairment effects that are inherent in optical fibre transmission links which can seriously degrade network performance. It is therefore extremely challenging to compensate for both linear and nonlinear birefringence in multichannel systems. The purpose of this thesis is to investigate the interaction between PMD and nonlinear induced birefringence in a fibre with consideration of mode coupling. A sound knowledge of this interaction is necessary in designing a linear and nonlinear polarization mode dispersion compensator for WDM systems, as was successfully carried out in this study. The investigation shows that the effect of nonlinear birefringence alone depolarizes the signal, while in high PMD links where polarization mode coupling is high, the nonlinear birefringence effect couples with second-order PMD such that it may reduce the penalty and improve the signal DOP. Further investigation shows that when nonlinear birefringence becomes significant, asymmetry arises between the two principal axes of the fibre, such that it is only one axis which experiences the effect of nonlinear birefringence. It is found out that along this vii axis, there exists a critical point in pump power where the nonlinear birefringence cancels PMD in the link and improves the signal. An adaptive compensator to cancel PMD and nonlinear birefringence was designed based on feedforward DOP-monitoring signal. The compensator was tested both at laboratory level and on the Telkom buried fibre link and found to be functioning as intended. It was able to adaptively track and compensate PMD in the link in less than a second. The compensator was able to cancel PMD in the link up to a maximum of 30 ps. The compensator improved the DOP of the worst signal by more than 100 percent.
- Full Text:
- Date Issued: 2009
Chang liver cell line as a model for Type II Diabetes in the liver and possible reversal of this condition by an indigenous medicinal plant
- Authors: Williams, Saralene Iona
- Date: 2009
- Subjects: Diabetes -- Alternative treatment , Medicinal plants , Traditional medicine , Liver -- Diseases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10339 , http://hdl.handle.net/10948/d1016179
- Description: The incidence of Type 2 Diabetes Mellittus (T2DM) is increasing world wide. In Africa the limited access to health care and the insidious course of the disease lead to more severe illness and diabetic complications. There is a need to find alternative approaches to treatment and prevention that address the problems and needs of Africa. Sutherlandia frutescens (S.frutescens) is a traditional herbal plant with known anti-diabetic properties, the precise mechanism of action of S.frutescens is not known. In order to develop new approaches for treatment and prevention of T2DM the pathophysiology of T2DM must be understood. T2DM is the final outcome of a multi-organ disease characterized by early defects in muscle, adipocytes, hepatocytes and pancreatic β-cells. In this study the role of the liver was investigated because of its central role in glucose and lipid metabolism. It is hard to differentiate between all the influences in an in vivo model, so the aim of this study was to develop an in vitro model of T2DM in Chang liver cells and to determine if S.frutescens can reverse the state of insulin resistance in this model. Different culture media conditions were screened to identify a method that can be used as the T2DM model in Chang liver cells. Serum free medium (MCBD-201) supplemented with human diabetic serum, (2.5%-10%), high insulin concentrations (0.1μM-1μM), high fructose concentrations (1-10mM). and a combination of high insulin and high fructose was used for this screening. Chang liver cells cultured in MCBD-201 medium supplemented with 1mM fructose and 0.1μM insulin showed reduced glucose uptake and increased lipid accumulation. The effect of two S.frutescens extracts, two anti-diabetic drugs, metformin and ciglitazone, and a hypolipidemic drug ciprofibrate were determined and shown to increase glucose uptake and reduce lipid accumulation. It was postulated that exposing the cells to excess nutrients in the form of high fructose would stimulate the cells to become adipogenic and accumulate lipids, which would interfere with the glucose uptake and induce insulin resistance. Gene expression of PPARγ, PPARα, and SREBP-1 transcription factors regulating lipid metabolism was determined in Chang liver cells cultured in insulin resistance inducing medium over a 48 hour time course. The expression of PPARγ, known to stimulate adipogenesis was increased after 6, 24 and 48 hours of exposure (P(H1)<0.0001). The expression of PPARα, known to stimulate β-oxidation expression, was significantly decreased after 24 hours of exposure (P(H1)<0.0001). The presence of the plant extracts in the insulin resistance inducing media protect against this increase in adipogenesis and decrease in β-oxidation after 48 hours of exposure by increasing PPARα expression and decreasing PPARγ expression. A PCR Array was performed which identified 32 more potential molecular targets of S.frutescens. Five of the 32 targets identified with the PCR Array were validated using qRT-PCR. These genes play a role in lipid and glucose metabolism and protection against oxidative stress and inflammation. In summary a cellular model of insulin resistace in hepatocytes has been established and the capacity of S.frutescens to reverse this process has been demonstrated by acting as a dual PPARγ/α agonist. New genes have been identified in the development of insulin resistance and as targets of S.frutescens.
- Full Text:
- Date Issued: 2009
- Authors: Williams, Saralene Iona
- Date: 2009
- Subjects: Diabetes -- Alternative treatment , Medicinal plants , Traditional medicine , Liver -- Diseases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10339 , http://hdl.handle.net/10948/d1016179
- Description: The incidence of Type 2 Diabetes Mellittus (T2DM) is increasing world wide. In Africa the limited access to health care and the insidious course of the disease lead to more severe illness and diabetic complications. There is a need to find alternative approaches to treatment and prevention that address the problems and needs of Africa. Sutherlandia frutescens (S.frutescens) is a traditional herbal plant with known anti-diabetic properties, the precise mechanism of action of S.frutescens is not known. In order to develop new approaches for treatment and prevention of T2DM the pathophysiology of T2DM must be understood. T2DM is the final outcome of a multi-organ disease characterized by early defects in muscle, adipocytes, hepatocytes and pancreatic β-cells. In this study the role of the liver was investigated because of its central role in glucose and lipid metabolism. It is hard to differentiate between all the influences in an in vivo model, so the aim of this study was to develop an in vitro model of T2DM in Chang liver cells and to determine if S.frutescens can reverse the state of insulin resistance in this model. Different culture media conditions were screened to identify a method that can be used as the T2DM model in Chang liver cells. Serum free medium (MCBD-201) supplemented with human diabetic serum, (2.5%-10%), high insulin concentrations (0.1μM-1μM), high fructose concentrations (1-10mM). and a combination of high insulin and high fructose was used for this screening. Chang liver cells cultured in MCBD-201 medium supplemented with 1mM fructose and 0.1μM insulin showed reduced glucose uptake and increased lipid accumulation. The effect of two S.frutescens extracts, two anti-diabetic drugs, metformin and ciglitazone, and a hypolipidemic drug ciprofibrate were determined and shown to increase glucose uptake and reduce lipid accumulation. It was postulated that exposing the cells to excess nutrients in the form of high fructose would stimulate the cells to become adipogenic and accumulate lipids, which would interfere with the glucose uptake and induce insulin resistance. Gene expression of PPARγ, PPARα, and SREBP-1 transcription factors regulating lipid metabolism was determined in Chang liver cells cultured in insulin resistance inducing medium over a 48 hour time course. The expression of PPARγ, known to stimulate adipogenesis was increased after 6, 24 and 48 hours of exposure (P(H1)<0.0001). The expression of PPARα, known to stimulate β-oxidation expression, was significantly decreased after 24 hours of exposure (P(H1)<0.0001). The presence of the plant extracts in the insulin resistance inducing media protect against this increase in adipogenesis and decrease in β-oxidation after 48 hours of exposure by increasing PPARα expression and decreasing PPARγ expression. A PCR Array was performed which identified 32 more potential molecular targets of S.frutescens. Five of the 32 targets identified with the PCR Array were validated using qRT-PCR. These genes play a role in lipid and glucose metabolism and protection against oxidative stress and inflammation. In summary a cellular model of insulin resistace in hepatocytes has been established and the capacity of S.frutescens to reverse this process has been demonstrated by acting as a dual PPARγ/α agonist. New genes have been identified in the development of insulin resistance and as targets of S.frutescens.
- Full Text:
- Date Issued: 2009
Regulating franchise operations in South Africa : a study of the existing legal framework with suggestions for reform.
- Authors: Woker, Tanya Ann
- Date: 2009
- Subjects: Franchise Association of Southern Africa , Franchises (Retail trade) -- Law and legislation -- South Africa , South Africa. Dept. of Trade and Industry , Common law -- South Africa , Commercial law -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3721 , http://hdl.handle.net/10962/d1015719
- Description: This thesis analyses the existing legal framework that applies to franchising in South Africa today. The study begins with an examination of the history and nature of the franchise contract, focusing particularly on the nature of the franchise relationship. This study is undertaken in order to substantiate the argument that franchising is a unique method of doing business. There is a need therefore to recognise that the franchise contract is a special contract in its own right, just like contracts of sale, lease, insurance and suretyship. The study then goes on to examine the problems which are experienced in the sector, as well as the law which must provide solutions to these problems. The research will show that in a modern commercial world the existing legal framework, especially the common law, cannot adequately deal with many of these problems. The complex relationship between franchising and competition law is also explored. A common thread that emerges from franchise disputes is the lack of protection afforded to the interests of franchisees. Franchisees tend to be at the mercy of economically stronger franchisors, hence the belief that there is a need for a stronger regulatory framework. The study then shifts to proposals for reform. In 2000 the Department of Trade and Industry (DTI) established the Franchise Steering Committee in conjunction with the Franchise Association of South Africa (FASA) to review the regulatory environment. This Committee drafted franchise legislation which aimed to bring the regulation of the sector under the control of the DTI. This legislation has not been implemented and the DTI has changed its strategy. Instead of dealing with franchising independently, franchising will fall within the scope of consumer protection legislation. Both the consumer protection legislation and the legislation proposed by the Franchise Steering Committee are thoroughly examined and explained. Shortcomings in the proposals are highlighted and an alternative approach is recommended. It is proposed that franchise-specific legislation should be introduced but that this legislation should establish a system of co-regulation between the government and the franchise sector.
- Full Text:
- Date Issued: 2009
- Authors: Woker, Tanya Ann
- Date: 2009
- Subjects: Franchise Association of Southern Africa , Franchises (Retail trade) -- Law and legislation -- South Africa , South Africa. Dept. of Trade and Industry , Common law -- South Africa , Commercial law -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3721 , http://hdl.handle.net/10962/d1015719
- Description: This thesis analyses the existing legal framework that applies to franchising in South Africa today. The study begins with an examination of the history and nature of the franchise contract, focusing particularly on the nature of the franchise relationship. This study is undertaken in order to substantiate the argument that franchising is a unique method of doing business. There is a need therefore to recognise that the franchise contract is a special contract in its own right, just like contracts of sale, lease, insurance and suretyship. The study then goes on to examine the problems which are experienced in the sector, as well as the law which must provide solutions to these problems. The research will show that in a modern commercial world the existing legal framework, especially the common law, cannot adequately deal with many of these problems. The complex relationship between franchising and competition law is also explored. A common thread that emerges from franchise disputes is the lack of protection afforded to the interests of franchisees. Franchisees tend to be at the mercy of economically stronger franchisors, hence the belief that there is a need for a stronger regulatory framework. The study then shifts to proposals for reform. In 2000 the Department of Trade and Industry (DTI) established the Franchise Steering Committee in conjunction with the Franchise Association of South Africa (FASA) to review the regulatory environment. This Committee drafted franchise legislation which aimed to bring the regulation of the sector under the control of the DTI. This legislation has not been implemented and the DTI has changed its strategy. Instead of dealing with franchising independently, franchising will fall within the scope of consumer protection legislation. Both the consumer protection legislation and the legislation proposed by the Franchise Steering Committee are thoroughly examined and explained. Shortcomings in the proposals are highlighted and an alternative approach is recommended. It is proposed that franchise-specific legislation should be introduced but that this legislation should establish a system of co-regulation between the government and the franchise sector.
- Full Text:
- Date Issued: 2009