Exploring pre-service teachers’ reflective practice in the context of video-based lesson analysis
- Authors: Chikiwa, Samukeliso
- Date: 2020-04-30
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/355357 , vital:64492
- Description: This study explored the development of reflective practice in foundation phase pre-service teachers in the context of video-based lesson analysis at a university in South Africa. The study was conducted in the field of mathematics education, responding to the urgent need to equip pre-service South African teachers with the knowledge and skills for effective mathematics teaching. The research is foregrounded by the continuing poor performance of South African learners in mathematics at all levels of education in the country, which has been linked to the inadequate knowledge and skills of mathematics teachers. Pre-service teacher education is putting considerable effort into improving the preparation of mathematics teachers and developing their ability to reflect on their teaching practice is one of the strategies being employed for this purpose. Research has demonstrated the importance of reflective practice (RP) in both developing and extending teachers’ mathematical knowledge for teaching. This study therefore contributes to current research that supports the development of RP as a professional skill for promoting the acquisition of knowledge for teaching in pre-service teacher education. The study adopted a qualitative case study approach with two phases of data collection. In Phase 1 I collected and analysed three sets of 19 pre-service teachers’ written reflections to establish the nature of the reflections that they developed when analysing video-recorded mathematics lessons of experienced teachers’ practice. Phase 2 was conducted with four PSTs who reflected on video-recorded mathematics lessons of their own practice, and similarly sought to investigate the nature of the reflections they developed when reflecting on practice. The four PSTs wrote one set of reflections on their own lessons, went through three sessions of facilitator-guided reflections, then wrote another set of reflections to establish if the support provided in small group facilitator-guided sessions improved their reflections. Iterative content analysis was employed to analyse the PSTs’ written reflections, using an analytic tool that I developed for this purpose through merging Lee’s (2007) and Muir and Beswick’s (2007) levels of reflection frameworks. My model had four levels of reflection: description, explanation, suggestion and reflectivity. The names of each of the levels connect to the key indicator for that level. PSTs’ written reflections were coded and analysed according to these levels. The study found that PSTs’ initial reflections were mostly description of general classroom events with little reflection at the levels of explanation and suggestion, and an absence of reflectivity. Most reflections focused on general events in the lesson rather than mathematical events, even though the six lens framework they were given to guide their reflections prompted them to steer their attention towards mathematical events. The second and third sets of reflections, although mostly still at level 1, showed some shifts towards explanation and suggestion, although an increased focus on mathematical events though reflectivity was still largely absent. No PST reached the fourth level of reflectivity in Phase 1. However, in Phase 2, the PSTs’ reflections after the three small group facilitator-guided sessions included some evidence of reflectivity. The findings suggest the need for pre-service teacher educators to make a concerted effort to teach PSTs what reflection is and how to reflect on their practice. The findings also showed the need for small group facilitator-guided support in the development of PSTs’ reflective practice. , Thesis (PhD) -- Faculty of Education, Education, 2020
- Full Text:
- Date Issued: 2020-04-30
- Authors: Chikiwa, Samukeliso
- Date: 2020-04-30
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/355357 , vital:64492
- Description: This study explored the development of reflective practice in foundation phase pre-service teachers in the context of video-based lesson analysis at a university in South Africa. The study was conducted in the field of mathematics education, responding to the urgent need to equip pre-service South African teachers with the knowledge and skills for effective mathematics teaching. The research is foregrounded by the continuing poor performance of South African learners in mathematics at all levels of education in the country, which has been linked to the inadequate knowledge and skills of mathematics teachers. Pre-service teacher education is putting considerable effort into improving the preparation of mathematics teachers and developing their ability to reflect on their teaching practice is one of the strategies being employed for this purpose. Research has demonstrated the importance of reflective practice (RP) in both developing and extending teachers’ mathematical knowledge for teaching. This study therefore contributes to current research that supports the development of RP as a professional skill for promoting the acquisition of knowledge for teaching in pre-service teacher education. The study adopted a qualitative case study approach with two phases of data collection. In Phase 1 I collected and analysed three sets of 19 pre-service teachers’ written reflections to establish the nature of the reflections that they developed when analysing video-recorded mathematics lessons of experienced teachers’ practice. Phase 2 was conducted with four PSTs who reflected on video-recorded mathematics lessons of their own practice, and similarly sought to investigate the nature of the reflections they developed when reflecting on practice. The four PSTs wrote one set of reflections on their own lessons, went through three sessions of facilitator-guided reflections, then wrote another set of reflections to establish if the support provided in small group facilitator-guided sessions improved their reflections. Iterative content analysis was employed to analyse the PSTs’ written reflections, using an analytic tool that I developed for this purpose through merging Lee’s (2007) and Muir and Beswick’s (2007) levels of reflection frameworks. My model had four levels of reflection: description, explanation, suggestion and reflectivity. The names of each of the levels connect to the key indicator for that level. PSTs’ written reflections were coded and analysed according to these levels. The study found that PSTs’ initial reflections were mostly description of general classroom events with little reflection at the levels of explanation and suggestion, and an absence of reflectivity. Most reflections focused on general events in the lesson rather than mathematical events, even though the six lens framework they were given to guide their reflections prompted them to steer their attention towards mathematical events. The second and third sets of reflections, although mostly still at level 1, showed some shifts towards explanation and suggestion, although an increased focus on mathematical events though reflectivity was still largely absent. No PST reached the fourth level of reflectivity in Phase 1. However, in Phase 2, the PSTs’ reflections after the three small group facilitator-guided sessions included some evidence of reflectivity. The findings suggest the need for pre-service teacher educators to make a concerted effort to teach PSTs what reflection is and how to reflect on their practice. The findings also showed the need for small group facilitator-guided support in the development of PSTs’ reflective practice. , Thesis (PhD) -- Faculty of Education, Education, 2020
- Full Text:
- Date Issued: 2020-04-30
Exploring the structural integrity of a picornavirus capsid
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
Orchid mycorrhizal and endophytic fungal diversity of three co-occurring terrestrial orchids in the large African genus Disa (Orchidaceae)
- Khambule, Nondumiso Venessia
- Authors: Khambule, Nondumiso Venessia
- Date: 2020
- Subjects: Orchids South Africa , Mycorrhizal fungi South Africa , Endomycorrhizas South Africa , Endophytes , Orchids Roots
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163341 , vital:41030
- Description: Orchids (in the family Orchidaceous) are one of the richest plant families and approximately 500 species are found in South Africa. A number of orchid species are found on disturbed areas and many of the terrestrial species grow in poor soils with low mineral nutrient availability. Most orchid species are thought to be associated with mycorrhizal fungi for germination and mycorrhiza provides nutrients for the survival of adult plants. The aim of this study was to select Orchidaceous plant species and to isolate, identify and characterize the orchid endophytes and assess these isolates for potential antimicrobial and enzymatic activities Isa is the largest genus in South Africa and three Disa species co-occurring in a small geographical area were selected. These included Disa bracteata, D. cornuta and D. polygonoides which span three sections of the genus. Roots were stained to confirm the mycorrhizal status of the Disa species. Mycorrhizal pelotons structures were microscopically observed inside root cells. The presence of pelotons is indictive of mycorrhizal fungal interactions within the orchid roots and areas associated with the site of nutrient exchange between plant and fungus. The presence of pelotons, however, does not give n indication of the fungal species involved. The endophytes were successfully isolated in pure cultures on potato dextrose agar (PDA). All slow growing isolates were selected, and further molecular identification undertaken; DNA was extracted, and PCR amplified using internal transcribed spacer (ITS1F and ITS4) fungal primers. The amplified products were then sequenced and analysed by comparison to sequences in the GenBank database. Trichoderma, Penicillium, Metapochonia, Talaromyces, Oidiodendron Neopestalotiopsis, and Chaetomium were identified from these sequences. The presence of other fungal root endophytes was suspected despite the rigorous surface sterilization procedure used. The primers used to amplify the ITS region are the universal barcoding primers which are specific to fungi. ITS1F is one of the primers designed to amplify a broad range of fungi. DNA was extracted from orchid roots and amplicons were cloned into a pGEMT plasmid vector. Individual clones were sequenced and aligned with Mega software and compared to sequences in the GenBank and UNITE database. Based on percentage sequence identity, unidentified Tulasnella species, Tullasnela colaspora, and various Ascomycota endophytes were identified as contributing to the endophytic root fungal diversity of the selected Disa species. The Disa species investigated in this study were associated with several soil endophytes. D. bracteata, D. polygonoides were collected from the same site along the road verge which is regarded as being disturbed. Based on both culture – dependent and independent techniques employed Oidiodendron was found associated with both species. Antimicrobial activity was determined using a well diffusion method using extracts from the isolated fungi against the bacterial isolates Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas puptida. Most of the isolated fungi showed at least one potential inhibition effect against one of the bacterial isolates. The extracts that showed potential antimicrobial activity could be further screened to determine the compounds produced as secondary metabolites using techniques such as LC-MS Enzymatic activities of protease, cellulose and amylase were determined using solid media amended with milk protein, carboxymethylcellulose (CMC) and starch. The majority of fungal isolates tested positive with amylase and cellulose with only a few fungal isolates testing positive for protease activity. Broth cultures containing CMC and starch were shown to enhance biomass production in approximately 40 % of the fungal isolates. Degradation of the substrates is required in order to provide carbon to the fungus under test in order to optimize fungal growth as well as to gain insight into their ecological role. Enzyme activity was evident particularly when cellulose and starch were provided as substrates. All the fungal isolates tested grew on the amended medium, with 40% of the isolates preferring to utilize CMC and/or starch, indicating the ability of these fungi to utilize various resources for carbon acquisitions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Khambule, Nondumiso Venessia
- Date: 2020
- Subjects: Orchids South Africa , Mycorrhizal fungi South Africa , Endomycorrhizas South Africa , Endophytes , Orchids Roots
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163341 , vital:41030
- Description: Orchids (in the family Orchidaceous) are one of the richest plant families and approximately 500 species are found in South Africa. A number of orchid species are found on disturbed areas and many of the terrestrial species grow in poor soils with low mineral nutrient availability. Most orchid species are thought to be associated with mycorrhizal fungi for germination and mycorrhiza provides nutrients for the survival of adult plants. The aim of this study was to select Orchidaceous plant species and to isolate, identify and characterize the orchid endophytes and assess these isolates for potential antimicrobial and enzymatic activities Isa is the largest genus in South Africa and three Disa species co-occurring in a small geographical area were selected. These included Disa bracteata, D. cornuta and D. polygonoides which span three sections of the genus. Roots were stained to confirm the mycorrhizal status of the Disa species. Mycorrhizal pelotons structures were microscopically observed inside root cells. The presence of pelotons is indictive of mycorrhizal fungal interactions within the orchid roots and areas associated with the site of nutrient exchange between plant and fungus. The presence of pelotons, however, does not give n indication of the fungal species involved. The endophytes were successfully isolated in pure cultures on potato dextrose agar (PDA). All slow growing isolates were selected, and further molecular identification undertaken; DNA was extracted, and PCR amplified using internal transcribed spacer (ITS1F and ITS4) fungal primers. The amplified products were then sequenced and analysed by comparison to sequences in the GenBank database. Trichoderma, Penicillium, Metapochonia, Talaromyces, Oidiodendron Neopestalotiopsis, and Chaetomium were identified from these sequences. The presence of other fungal root endophytes was suspected despite the rigorous surface sterilization procedure used. The primers used to amplify the ITS region are the universal barcoding primers which are specific to fungi. ITS1F is one of the primers designed to amplify a broad range of fungi. DNA was extracted from orchid roots and amplicons were cloned into a pGEMT plasmid vector. Individual clones were sequenced and aligned with Mega software and compared to sequences in the GenBank and UNITE database. Based on percentage sequence identity, unidentified Tulasnella species, Tullasnela colaspora, and various Ascomycota endophytes were identified as contributing to the endophytic root fungal diversity of the selected Disa species. The Disa species investigated in this study were associated with several soil endophytes. D. bracteata, D. polygonoides were collected from the same site along the road verge which is regarded as being disturbed. Based on both culture – dependent and independent techniques employed Oidiodendron was found associated with both species. Antimicrobial activity was determined using a well diffusion method using extracts from the isolated fungi against the bacterial isolates Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas puptida. Most of the isolated fungi showed at least one potential inhibition effect against one of the bacterial isolates. The extracts that showed potential antimicrobial activity could be further screened to determine the compounds produced as secondary metabolites using techniques such as LC-MS Enzymatic activities of protease, cellulose and amylase were determined using solid media amended with milk protein, carboxymethylcellulose (CMC) and starch. The majority of fungal isolates tested positive with amylase and cellulose with only a few fungal isolates testing positive for protease activity. Broth cultures containing CMC and starch were shown to enhance biomass production in approximately 40 % of the fungal isolates. Degradation of the substrates is required in order to provide carbon to the fungus under test in order to optimize fungal growth as well as to gain insight into their ecological role. Enzyme activity was evident particularly when cellulose and starch were provided as substrates. All the fungal isolates tested grew on the amended medium, with 40% of the isolates preferring to utilize CMC and/or starch, indicating the ability of these fungi to utilize various resources for carbon acquisitions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Repurposing a polymer precursor scaffold for medicinal application: Synthesis, characterization and biological evaluation of ferrocenyl 1,3-benzoxazine derivatives as potential antiprotozoal and anticancer agents
- Authors: Mbaba, Mziyanda
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/164502 , vital:41124 , DOI 10.21504/10962/164502
- Description: The benzoxazines are a prominent class of heterocyclic compounds that possess a multitude of properties. To this end, benzoxazine derivatives have been used as versatile compounds for various utilities ranging from biological applications to the fabrication of polymers. Particularly, the 1,3-benzoxazine scaffold has featured in several bioactive compounds showing antimalarial, anticancer and antibacterial activities. Traditionally, it has been employed as a substrate in the synthesis of polymers with appealing physical and chemical properties. Due to the increasing interest in the polymer application of 1,3-benzoxazines, research of the 1,3-benzoxazine motif for polymer synthesis has been prioritized over other applications including its medicinal potential. The continuous development of resistance to clinical anticancer and antimalarial drugs has necessitated the need for the search of innovative bioactive compounds as potential alternative medicinal agents. To address this, the field of medicinal chemistry is adapting new approaches to counter resistance by incorporating nonconventional chemical moieties such as organometallic complexes, like ferrocene, into bioactive chemical motifs to serve as novel compounds with medicinal benefits. Incorporation of ferrocene into known bioactive chemical moieties has been shown to impart beneficial biological effects into the resultant compounds, which include the introduction of novel, and sometimes varied, mechanistic modalities and enhanced potency. Presented with the benefits of this strategy, the current work aims to design and evaluate the pharmaceutical capacity of novel derivatives containing 1,3-benzoxazine scaffold (traditionally applied in polymer synthesis) hybridized with the organometallic ferrocene unit as bioactive agents. Using a combination of expedient synthetic procedures such as the Burke three-component Mannich-type condensation, Vilsmeier-Haack formylation and reductive amination, four series of ferrocenyl 1,3-benzoxazine derivatives were synthesized and their structures confirmed by common spectroscopic techniques: nuclear magnetic resonance (NMR), infrared spectroscopy (IR) and high-resolution mass spectrometry (HRMS). The target compounds were evaluated in vitro for potential antimalarial and anticancer activities against strains of the malaria parasite (Plasmodium falciparum 3D7 and Dd2) and the triple-negative breast cancer cell line HCC70. Compounds exhibited higher potency towards the Plasmodium falciparum strains with IC50 values in the low and sub-micromolar range in comparison to the breast cancer cell line against for which mid-molar activities were observed. To gain insight into the possible mode of action of ferrocenyl 1,3-benzoxazines, representative compounds showing most efficacy from each series were assessed for DNA binding affinity by employing UV-Vis and fluorescence DNA titration experiments. The selected compounds were found to interact with the DNA by binding to the minor groove, and these findings were confirmed by in silico ligand docking studies using a B-DNA structure as the receptor. Compound 3.16c (IC50: 0.261 μM [3D7], 0.599 μM [Dd2], 11.0 μM [HCC70]), which emerged as the most promising compound, was found to induce DNA damage in HCC70 cancer cells when investigated for effects of DNA interaction. Additionally, compound 3.16c displayed a higher binding constant (Kb) against DNA isolated from 3D7 Plasmodium falciparum trophozoites (Kb = 1.88×106 M-1) than the mammalian DNA (Kb = 6.33×104 M-1) from calf thymus, thus explaining the preferred selectivity of the compounds for the malaria parasite. Moreover, the investigated compounds demonstrated binding affinity for synthetic hemozoin, β-hematin. Collectively, these data suggest that the compounds possess a dual mode of action for antimalarial activity involving DNA interaction and hemozoin inhibition. , Thesis (PhD) -- Faculty of Science, Chemistry, 2020
- Full Text:
- Date Issued: 2020
- Authors: Mbaba, Mziyanda
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/164502 , vital:41124 , DOI 10.21504/10962/164502
- Description: The benzoxazines are a prominent class of heterocyclic compounds that possess a multitude of properties. To this end, benzoxazine derivatives have been used as versatile compounds for various utilities ranging from biological applications to the fabrication of polymers. Particularly, the 1,3-benzoxazine scaffold has featured in several bioactive compounds showing antimalarial, anticancer and antibacterial activities. Traditionally, it has been employed as a substrate in the synthesis of polymers with appealing physical and chemical properties. Due to the increasing interest in the polymer application of 1,3-benzoxazines, research of the 1,3-benzoxazine motif for polymer synthesis has been prioritized over other applications including its medicinal potential. The continuous development of resistance to clinical anticancer and antimalarial drugs has necessitated the need for the search of innovative bioactive compounds as potential alternative medicinal agents. To address this, the field of medicinal chemistry is adapting new approaches to counter resistance by incorporating nonconventional chemical moieties such as organometallic complexes, like ferrocene, into bioactive chemical motifs to serve as novel compounds with medicinal benefits. Incorporation of ferrocene into known bioactive chemical moieties has been shown to impart beneficial biological effects into the resultant compounds, which include the introduction of novel, and sometimes varied, mechanistic modalities and enhanced potency. Presented with the benefits of this strategy, the current work aims to design and evaluate the pharmaceutical capacity of novel derivatives containing 1,3-benzoxazine scaffold (traditionally applied in polymer synthesis) hybridized with the organometallic ferrocene unit as bioactive agents. Using a combination of expedient synthetic procedures such as the Burke three-component Mannich-type condensation, Vilsmeier-Haack formylation and reductive amination, four series of ferrocenyl 1,3-benzoxazine derivatives were synthesized and their structures confirmed by common spectroscopic techniques: nuclear magnetic resonance (NMR), infrared spectroscopy (IR) and high-resolution mass spectrometry (HRMS). The target compounds were evaluated in vitro for potential antimalarial and anticancer activities against strains of the malaria parasite (Plasmodium falciparum 3D7 and Dd2) and the triple-negative breast cancer cell line HCC70. Compounds exhibited higher potency towards the Plasmodium falciparum strains with IC50 values in the low and sub-micromolar range in comparison to the breast cancer cell line against for which mid-molar activities were observed. To gain insight into the possible mode of action of ferrocenyl 1,3-benzoxazines, representative compounds showing most efficacy from each series were assessed for DNA binding affinity by employing UV-Vis and fluorescence DNA titration experiments. The selected compounds were found to interact with the DNA by binding to the minor groove, and these findings were confirmed by in silico ligand docking studies using a B-DNA structure as the receptor. Compound 3.16c (IC50: 0.261 μM [3D7], 0.599 μM [Dd2], 11.0 μM [HCC70]), which emerged as the most promising compound, was found to induce DNA damage in HCC70 cancer cells when investigated for effects of DNA interaction. Additionally, compound 3.16c displayed a higher binding constant (Kb) against DNA isolated from 3D7 Plasmodium falciparum trophozoites (Kb = 1.88×106 M-1) than the mammalian DNA (Kb = 6.33×104 M-1) from calf thymus, thus explaining the preferred selectivity of the compounds for the malaria parasite. Moreover, the investigated compounds demonstrated binding affinity for synthetic hemozoin, β-hematin. Collectively, these data suggest that the compounds possess a dual mode of action for antimalarial activity involving DNA interaction and hemozoin inhibition. , Thesis (PhD) -- Faculty of Science, Chemistry, 2020
- Full Text:
- Date Issued: 2020
Synthesis of peptidomimetic compounds as HIV-1 protease inhibitors
- Authors: Kayembe, Jean-Pierre
- Date: 2020
- Subjects: Protease inhibitors , HIV (Viruses) , HIV infections Treatment , Peptidomimetics
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/124397 , vital:35604 , DOI https://dx.doi.org/10.21504/10962/124397
- Description: This research project has involved the design, synthesis and evaluation of novel peptidomimetics compounds as HIV-1 protease inhibitors. Here is presented one-step, two-step and three-step syntheses and the in vitro bio-assay studies of a series of fully characterized peptidomimetics as HIV-1 protease inhibitors candidate using the shortest and most cost effective synthetic routes. The first series of compounds were accessed via a synthetic elaboration of Morita-Baylis-Hillman adducts by a Michael addition with benzylamine, proline or glycine esters to afford a series of β-amino-β’-hydroxycarboxylate esters in moderate to good yields. Base-catalyzed cyclization of non-benzylated aza-Michael adducts afforded a series of coumarin-3-hydroxy-2-methylenepropanoate esters in moderate yields. The uncatalyzed direct amidation of diethyl tartrate/tartaric acid and tartaric acid osazone with selected amines/amino acids afforded a series of C2-symmetrical and unsymmetrical 1,2-dihydroxycarboxylates in moderate to very high yields. All the synthesized compounds were fully characterized using spectroscopic techniques. These conjugates, designed as potential HIV-1 inhibitors, were tested against the HIV-1 protease enzyme. A number of these ligands have exhibited inhibition levels and IC50 values comparable to ritonavir, permitting, therefore, their identification as lead compounds for the development of novel inhibitors. , Thesis (PhD) -- Faculty of Science, Chemistry, 2020
- Full Text: false
- Date Issued: 2020
- Authors: Kayembe, Jean-Pierre
- Date: 2020
- Subjects: Protease inhibitors , HIV (Viruses) , HIV infections Treatment , Peptidomimetics
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/124397 , vital:35604 , DOI https://dx.doi.org/10.21504/10962/124397
- Description: This research project has involved the design, synthesis and evaluation of novel peptidomimetics compounds as HIV-1 protease inhibitors. Here is presented one-step, two-step and three-step syntheses and the in vitro bio-assay studies of a series of fully characterized peptidomimetics as HIV-1 protease inhibitors candidate using the shortest and most cost effective synthetic routes. The first series of compounds were accessed via a synthetic elaboration of Morita-Baylis-Hillman adducts by a Michael addition with benzylamine, proline or glycine esters to afford a series of β-amino-β’-hydroxycarboxylate esters in moderate to good yields. Base-catalyzed cyclization of non-benzylated aza-Michael adducts afforded a series of coumarin-3-hydroxy-2-methylenepropanoate esters in moderate yields. The uncatalyzed direct amidation of diethyl tartrate/tartaric acid and tartaric acid osazone with selected amines/amino acids afforded a series of C2-symmetrical and unsymmetrical 1,2-dihydroxycarboxylates in moderate to very high yields. All the synthesized compounds were fully characterized using spectroscopic techniques. These conjugates, designed as potential HIV-1 inhibitors, were tested against the HIV-1 protease enzyme. A number of these ligands have exhibited inhibition levels and IC50 values comparable to ritonavir, permitting, therefore, their identification as lead compounds for the development of novel inhibitors. , Thesis (PhD) -- Faculty of Science, Chemistry, 2020
- Full Text: false
- Date Issued: 2020
The Role of HSP70/HSP90 Organizing Protein (Hop) in the Heat Shock Factor 1 (HSF1)-mediated Stress Response
- Authors: Chakraborty, Abantika
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163204 , vital:41018 , doi:10.21504/10962/163204
- Description: Molecular chaperones regulate cellular proteostasis. They control protein conformation and prevent misfolding and aggregation under both normal and stressful environments, ultimately resulting in cell survival. The project aimed to understand the role of the HSP70 – HSP90 Organizing Protein (Hop/STIP1) in the survival of stressed cells and the function of the stress-responsive transcription factor, Heat Shock Factor 1 (HSF1). HSF1 protein levels were significantly reduced in Hop-depleted HEK293T cells compared to controls by ELISA, western blot, and mass spectrometry. HSF1 transcriptional activity at the HSP70 promoter, and binding of a biotinylated HSE oligonucleotide under basal conditions were significantly reduced, consistent with the reduced levels of HSF1. In response to heat shock, HSF1 levels in Hop-depleted cells increased to that of controls, but there was still significantly lowerHSF1 transcriptional activity and HSE binding. Hop-depleted HEK293T cells were more sensitive than controls to the HSF1 inhibitor KRIBB11 and showed reduced short-term and long-term proliferation. Unlike the HSP90 inhibitor 17-DMAG, which had no effect, the HSP70 inhibitor JG98, further decreased the levels of HSF1 in Hop-depleted cells, suggesting a role for HSP70 in the Hop-mediated effects. There was punctate nuclear staining for HSF1 in Hop-depleted cells under both basal and heat shock conditions, as well as reduced nuclear localization and increased cytoplasmic accumulation of HSF1 in response to heat shock. Hop and HSF1 colocalized in cells, and HSF1 could be isolated in complex with Hop and HSP70. Loss of Hop reduced HSF1 in HSP70complexes but did not affect HSF1 abundance in HSP90 complexes. Hop-depleted cells showed reduced short-term and long-term survival compared to controls, an effect that was potentiated by the JG98 HSP70 inhibitor. Taken together, these data suggest that Hop regulation of HSF1activity is via a mechanism involving reductions in HSP70 interaction, as well as reduced nuclear localization, and DNA binding, and is consistent with reduced cellular fitness under basal and stress conditions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Chakraborty, Abantika
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163204 , vital:41018 , doi:10.21504/10962/163204
- Description: Molecular chaperones regulate cellular proteostasis. They control protein conformation and prevent misfolding and aggregation under both normal and stressful environments, ultimately resulting in cell survival. The project aimed to understand the role of the HSP70 – HSP90 Organizing Protein (Hop/STIP1) in the survival of stressed cells and the function of the stress-responsive transcription factor, Heat Shock Factor 1 (HSF1). HSF1 protein levels were significantly reduced in Hop-depleted HEK293T cells compared to controls by ELISA, western blot, and mass spectrometry. HSF1 transcriptional activity at the HSP70 promoter, and binding of a biotinylated HSE oligonucleotide under basal conditions were significantly reduced, consistent with the reduced levels of HSF1. In response to heat shock, HSF1 levels in Hop-depleted cells increased to that of controls, but there was still significantly lowerHSF1 transcriptional activity and HSE binding. Hop-depleted HEK293T cells were more sensitive than controls to the HSF1 inhibitor KRIBB11 and showed reduced short-term and long-term proliferation. Unlike the HSP90 inhibitor 17-DMAG, which had no effect, the HSP70 inhibitor JG98, further decreased the levels of HSF1 in Hop-depleted cells, suggesting a role for HSP70 in the Hop-mediated effects. There was punctate nuclear staining for HSF1 in Hop-depleted cells under both basal and heat shock conditions, as well as reduced nuclear localization and increased cytoplasmic accumulation of HSF1 in response to heat shock. Hop and HSF1 colocalized in cells, and HSF1 could be isolated in complex with Hop and HSP70. Loss of Hop reduced HSF1 in HSP70complexes but did not affect HSF1 abundance in HSP90 complexes. Hop-depleted cells showed reduced short-term and long-term survival compared to controls, an effect that was potentiated by the JG98 HSP70 inhibitor. Taken together, these data suggest that Hop regulation of HSF1activity is via a mechanism involving reductions in HSP70 interaction, as well as reduced nuclear localization, and DNA binding, and is consistent with reduced cellular fitness under basal and stress conditions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Characterization of termite Trinervitermes trinervoides metagenome-derived glycoside hydrolases, the formulation of synergistic core enzyme sets for effective sweet sorghum and corncob saccharification, and their potential industrial applications
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
Ectomycorrhizal fungal assessment of South African Pinus patula seedlings and their biological control potential to enhance seedling growth
- FitzGerald, Véronique Chartier
- Authors: FitzGerald, Véronique Chartier
- Date: 2019
- Subjects: Ectomycorrhizal fungi , Pinus patula Diseases and pests Biological control South Africa , Fusarium , Forest nurseries , Seedlings Growth , Seedlings Diseases and pests
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/67902 , vital:29167
- Description: The South Africa forestry industry, covering 1.3 million hectares, is dependent on exotic pine and eucalyptus species. Nursery seedlings are not inoculated with ectomycorrhizal (ECM) fungi or other beneficial microbes. Fusarium circinatum is an economically important pathogen affecting seedling survival. The purpose of this investigation was to assess levels of naturally occurring ECM colonisation in South African nurseries and to determine the effects of seedling inoculation with selected ECM and bacterial isolates on plant growth and resistance to the fungal pathogen F. circiantum. Pinus patula seedlings from 10 different South African nurseries were assessed for ECM colonisation using a grid line intersect method and molecularly identified using morphological and next-generation Illumina sequencing. Explants from ECM basidiocarps, collected from Pinus stands, were plated onto MMN medium to obtain isolates which were verified using molecular techniques. Mycorrhizal helper bacteria (MHB) were also isolated from these basiocarps, tested for MHB properties, siderophore production, phospahte solubilising and IAA production. ECM and associated bacterial isolates were used to inoculate seedlings and growth was assessed over a 5 month period. Colonisation of seedlings in production nurseries was low (2-21%). Morphologically the ECM fungi T. terrestris, Suillus sibiricus, and the genera Russula, Pseudotomentella were identified. Molecularly the ECM fungi T. terrestris, Inocybe jacobi and the genera Sphaerosporella and several other ECM containing families were identified along with many saprotrophic/endophytic fungi belonging to genera such as, Penicillium, Ramasonia and Talaromyces. Inoculated seedlings showed a significant increase in growth in comparison to the un-inoculated control seedlings. ECM fungal colonisation levels of these seedlings were significantly increased and colonisation was promoted by the Suillus isolate, Salmon Suillus. Seedling growth in the presence of the pathogen F. circinatum was significantly increased and promoted by the Lactarius isolate Lactarius quieticolor. Inoculation of seedlings in the nursery would ensure the production of stronger healthy plants which may be more tolerant to fusarial infection increasing survival in the plantation. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: FitzGerald, Véronique Chartier
- Date: 2019
- Subjects: Ectomycorrhizal fungi , Pinus patula Diseases and pests Biological control South Africa , Fusarium , Forest nurseries , Seedlings Growth , Seedlings Diseases and pests
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/67902 , vital:29167
- Description: The South Africa forestry industry, covering 1.3 million hectares, is dependent on exotic pine and eucalyptus species. Nursery seedlings are not inoculated with ectomycorrhizal (ECM) fungi or other beneficial microbes. Fusarium circinatum is an economically important pathogen affecting seedling survival. The purpose of this investigation was to assess levels of naturally occurring ECM colonisation in South African nurseries and to determine the effects of seedling inoculation with selected ECM and bacterial isolates on plant growth and resistance to the fungal pathogen F. circiantum. Pinus patula seedlings from 10 different South African nurseries were assessed for ECM colonisation using a grid line intersect method and molecularly identified using morphological and next-generation Illumina sequencing. Explants from ECM basidiocarps, collected from Pinus stands, were plated onto MMN medium to obtain isolates which were verified using molecular techniques. Mycorrhizal helper bacteria (MHB) were also isolated from these basiocarps, tested for MHB properties, siderophore production, phospahte solubilising and IAA production. ECM and associated bacterial isolates were used to inoculate seedlings and growth was assessed over a 5 month period. Colonisation of seedlings in production nurseries was low (2-21%). Morphologically the ECM fungi T. terrestris, Suillus sibiricus, and the genera Russula, Pseudotomentella were identified. Molecularly the ECM fungi T. terrestris, Inocybe jacobi and the genera Sphaerosporella and several other ECM containing families were identified along with many saprotrophic/endophytic fungi belonging to genera such as, Penicillium, Ramasonia and Talaromyces. Inoculated seedlings showed a significant increase in growth in comparison to the un-inoculated control seedlings. ECM fungal colonisation levels of these seedlings were significantly increased and colonisation was promoted by the Suillus isolate, Salmon Suillus. Seedling growth in the presence of the pathogen F. circinatum was significantly increased and promoted by the Lactarius isolate Lactarius quieticolor. Inoculation of seedlings in the nursery would ensure the production of stronger healthy plants which may be more tolerant to fusarial infection increasing survival in the plantation. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
Gem-bearing granitic pegmatites in Malawi: their mineralogy, geochemistry, age, and fluid compositional variations
- Kankuzi, Charles Frienderson
- Authors: Kankuzi, Charles Frienderson
- Date: 2019
- Subjects: Granite , Pegmatites , Geochemistry , Fluid inclusions , Nonferrous metals
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/97905 , vital:31505 , DOI https://doi.org/10.21504/10962/97905
- Description: The gem bearing granitic pegmatites from different pegmatite fields across Malawi intrude all important geological entities from the Palaeoproterozoic in the north, the Mesoproterozoic in central Malawi and the Pan-African basement in the south. U/Pb zircon and Rb/Sr mineral isochron ages indicate pegmatite emplacement from the Palaeoproterozoic to Pan-African and Mesozoic time. Most pegmatites are related to the Pan-African cycle; no Mesoproterozoic pegmatites were observed in this study. Within the Pan-African pegmatite groups there are two important subgroups. Some pegmatites show Sr isotopic compositions that indicate mantle components contributing to the parental granites from which the pegmatites evolved. Others show higher Sr initials, indicating crustal granites as primary pegmatite sources or significant crustal contamination. Only for few pegmatites, such as the Palaeoproterozoic and Ordovician gem tourmaline pegmatites in the Chitipa and Dowa Districts, the granitic source is evident from their field context. For all others the granitic origin is interpreted by mineralogical and geochemical evidence. All analysed pegmatites belong to either the Rare Element Class or the Miarolitic Class, but they vary in their degree of fractionation. The more evolved pegmatites are more enriched in incompatible elements such as Be, Li, B, and Ta, which resulted in the formation of gem minerals such as beryl, aquamarine, tourmaline and topaz, which may or may not be associated with tantalite. The Rare Element pegmatites can be further subdivided into the REL-Li subclass, beryl type, beryl-columbite subtype, and in the complex type and elbaite subtype. The Miarolitic pegmatites include Mi-Li subclass and beryl-topaz type. Fluid inclusion studies (heating-cooling stage, Raman spectroscopy) identified a variety of fluid compositions that were present at different times and different places, indicating a variety of fluid sources. They range from aqueous-saline to CO2–rich carbonic fluids (CO2 +C3H8+ N2), or aqueous-carbonic fluids (H2O-CO2-CH4 and H2O-CO2-H2-H2S-CH4). The dominant solutes and species for the pegmatites show genetic variations over time and orogen (Paleo-/Meso-/Neoproterozoic). Uniform homogenisation temperatures and salinities in individual samples indicate that the gem-bearing pegmatites contained homogeneous fluids at the time of their capturing in quartz. Based on fluid inclusion data, the estimated trapping conditions of inclusions in quartz for all studied pegmatites except for one pegmatite suggest low pressures between 0.9 to 2.6 kb at temperatures of 400-600 C. The other pegmatite formed at slightly higher pressures of 2.2 to 3.6 kb. However, the pressure range for all the pegmatites is in agreement with the known liquidus conditions of Rare-Element pegmatite crystallisation. The shallow crustal emplacement level (3.4-9.8 km) and the greater depth (8.3 to 13.6 km) favoured the formation of gemstones. , Thesis (PhD) -- Faculty of Science, Geology, 2019
- Full Text: false
- Date Issued: 2019
- Authors: Kankuzi, Charles Frienderson
- Date: 2019
- Subjects: Granite , Pegmatites , Geochemistry , Fluid inclusions , Nonferrous metals
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/97905 , vital:31505 , DOI https://doi.org/10.21504/10962/97905
- Description: The gem bearing granitic pegmatites from different pegmatite fields across Malawi intrude all important geological entities from the Palaeoproterozoic in the north, the Mesoproterozoic in central Malawi and the Pan-African basement in the south. U/Pb zircon and Rb/Sr mineral isochron ages indicate pegmatite emplacement from the Palaeoproterozoic to Pan-African and Mesozoic time. Most pegmatites are related to the Pan-African cycle; no Mesoproterozoic pegmatites were observed in this study. Within the Pan-African pegmatite groups there are two important subgroups. Some pegmatites show Sr isotopic compositions that indicate mantle components contributing to the parental granites from which the pegmatites evolved. Others show higher Sr initials, indicating crustal granites as primary pegmatite sources or significant crustal contamination. Only for few pegmatites, such as the Palaeoproterozoic and Ordovician gem tourmaline pegmatites in the Chitipa and Dowa Districts, the granitic source is evident from their field context. For all others the granitic origin is interpreted by mineralogical and geochemical evidence. All analysed pegmatites belong to either the Rare Element Class or the Miarolitic Class, but they vary in their degree of fractionation. The more evolved pegmatites are more enriched in incompatible elements such as Be, Li, B, and Ta, which resulted in the formation of gem minerals such as beryl, aquamarine, tourmaline and topaz, which may or may not be associated with tantalite. The Rare Element pegmatites can be further subdivided into the REL-Li subclass, beryl type, beryl-columbite subtype, and in the complex type and elbaite subtype. The Miarolitic pegmatites include Mi-Li subclass and beryl-topaz type. Fluid inclusion studies (heating-cooling stage, Raman spectroscopy) identified a variety of fluid compositions that were present at different times and different places, indicating a variety of fluid sources. They range from aqueous-saline to CO2–rich carbonic fluids (CO2 +C3H8+ N2), or aqueous-carbonic fluids (H2O-CO2-CH4 and H2O-CO2-H2-H2S-CH4). The dominant solutes and species for the pegmatites show genetic variations over time and orogen (Paleo-/Meso-/Neoproterozoic). Uniform homogenisation temperatures and salinities in individual samples indicate that the gem-bearing pegmatites contained homogeneous fluids at the time of their capturing in quartz. Based on fluid inclusion data, the estimated trapping conditions of inclusions in quartz for all studied pegmatites except for one pegmatite suggest low pressures between 0.9 to 2.6 kb at temperatures of 400-600 C. The other pegmatite formed at slightly higher pressures of 2.2 to 3.6 kb. However, the pressure range for all the pegmatites is in agreement with the known liquidus conditions of Rare-Element pegmatite crystallisation. The shallow crustal emplacement level (3.4-9.8 km) and the greater depth (8.3 to 13.6 km) favoured the formation of gemstones. , Thesis (PhD) -- Faculty of Science, Geology, 2019
- Full Text: false
- Date Issued: 2019
Political constellations: an analysis of the use of language in positioning South African political parties in the Daily Sun
- Authors: Siebörger, Ian
- Date: 2019
- Subjects: Knowledge, Theory of , Political culture South Africa , Systemic grammar , Political parties South Africa , Functionalism (Linguistics) , Mass media and language South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/113934 , vital:33846 , http://doi.org/10.21504/10962/113934
- Description: As a young democracy, post-apartheid South Africa has both a complex and rapidly changing political landscape, and a developing system of interconnected public spheres in which ordinary citizens engage in political discourses. In this context, this thesis examines the ways in which language is used to position South Africa's political parties in the Daily Sun, the country's most widely-read tabloid newspaper. It uses a complex, multi-stage method of analysis to develop tools for describing how different policy positions and moral evaluations are associated with political parties. This is accomplished through complementary analyses using Systemic Functional Linguistics (SFL) and Legitimation Code Theory (LCT). The study describes current political discourses in South Africa as the products of a long process of historical development extending through the segregationist and apartheid eras to the post-apartheid present. It also situates the Daily Sun within South Africa's current media landscape and argues that the newspaper is to some extent successful in facilitating a vibrant alternative public sphere in which readers engage with the news. The study's multi-stage method of analysis begins with a corpus analysis of all political news articles from the Daily Sun dating from January to June 2015. This analysis reveals various tendencies in the positioning of South Africa's three largest political parties that are investigated further in later stages of analysis. Three selected individual articles are subjected to _ne-grained analysis in an exploratory analysis stage, and three others in a targeted analysis stage. The exploratory analyses are designed to investigate the contribution of various linguistic and discursive resources to political positioning in the data, while the targeted analyses demonstrate the contribution of these resources to positioning in articles that were carefully selected to reflect as many of the trends found in the corpus analysis as possible. These analyses enact the concept of axiological-semantic density from LCT to show the ways in which political knowledge is condensed in the articles. This condensation produces constellations of people, policy positions and moral evaluations in each article. These constellations, in turn, give indications of the cosmologies, or organizing principles, behind the Daily Sun's political news coverage. On the basis of the exploratory analyses, a multi-level translation device for axiological-semantic density is developed to describe the ways in which this concept is enacted in the analysis. The use of this translation device is then demonstrated using the targeted analyses. This study adapts the concepts of iconization, aggregation and technicality from SFL, collectively referred to as 'mass', to show how language works to position political parties. It finds that the use of intertextual references and lexical strings makes a considerable contribution toward political positioning in the data. Repeated couplings of the abovementioned resources in complexes (also known as syndromes) drive the process of political positioning. The Daily Sun is found to report primarily on concrete actions by individual politicians, and contains little to no information on policy decisions. No one political party is favoured, but coverage tends to emphasize accusations against politicians. This may reinforce distrust in politics and a withdrawal from engagement in political processes among readers. By contrast, this study shows that engagement in South Africa's public discourses can be encouraged through stronger promotion of critical language awareness in education, the growth of civil society organizations as vehicles for dialogue and social action on matters of public interest, and a variety of initiatives by the media designed to show the relevance of policy decisions to individuals' lives and increase participation in the news-making process. , Thesis (PhD) -- Faculty of Humanities, English Language and Linguistics, 2019
- Full Text: false
- Date Issued: 2019
- Authors: Siebörger, Ian
- Date: 2019
- Subjects: Knowledge, Theory of , Political culture South Africa , Systemic grammar , Political parties South Africa , Functionalism (Linguistics) , Mass media and language South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/113934 , vital:33846 , http://doi.org/10.21504/10962/113934
- Description: As a young democracy, post-apartheid South Africa has both a complex and rapidly changing political landscape, and a developing system of interconnected public spheres in which ordinary citizens engage in political discourses. In this context, this thesis examines the ways in which language is used to position South Africa's political parties in the Daily Sun, the country's most widely-read tabloid newspaper. It uses a complex, multi-stage method of analysis to develop tools for describing how different policy positions and moral evaluations are associated with political parties. This is accomplished through complementary analyses using Systemic Functional Linguistics (SFL) and Legitimation Code Theory (LCT). The study describes current political discourses in South Africa as the products of a long process of historical development extending through the segregationist and apartheid eras to the post-apartheid present. It also situates the Daily Sun within South Africa's current media landscape and argues that the newspaper is to some extent successful in facilitating a vibrant alternative public sphere in which readers engage with the news. The study's multi-stage method of analysis begins with a corpus analysis of all political news articles from the Daily Sun dating from January to June 2015. This analysis reveals various tendencies in the positioning of South Africa's three largest political parties that are investigated further in later stages of analysis. Three selected individual articles are subjected to _ne-grained analysis in an exploratory analysis stage, and three others in a targeted analysis stage. The exploratory analyses are designed to investigate the contribution of various linguistic and discursive resources to political positioning in the data, while the targeted analyses demonstrate the contribution of these resources to positioning in articles that were carefully selected to reflect as many of the trends found in the corpus analysis as possible. These analyses enact the concept of axiological-semantic density from LCT to show the ways in which political knowledge is condensed in the articles. This condensation produces constellations of people, policy positions and moral evaluations in each article. These constellations, in turn, give indications of the cosmologies, or organizing principles, behind the Daily Sun's political news coverage. On the basis of the exploratory analyses, a multi-level translation device for axiological-semantic density is developed to describe the ways in which this concept is enacted in the analysis. The use of this translation device is then demonstrated using the targeted analyses. This study adapts the concepts of iconization, aggregation and technicality from SFL, collectively referred to as 'mass', to show how language works to position political parties. It finds that the use of intertextual references and lexical strings makes a considerable contribution toward political positioning in the data. Repeated couplings of the abovementioned resources in complexes (also known as syndromes) drive the process of political positioning. The Daily Sun is found to report primarily on concrete actions by individual politicians, and contains little to no information on policy decisions. No one political party is favoured, but coverage tends to emphasize accusations against politicians. This may reinforce distrust in politics and a withdrawal from engagement in political processes among readers. By contrast, this study shows that engagement in South Africa's public discourses can be encouraged through stronger promotion of critical language awareness in education, the growth of civil society organizations as vehicles for dialogue and social action on matters of public interest, and a variety of initiatives by the media designed to show the relevance of policy decisions to individuals' lives and increase participation in the news-making process. , Thesis (PhD) -- Faculty of Humanities, English Language and Linguistics, 2019
- Full Text: false
- Date Issued: 2019
Towards a biological profile for South African perinatal remains: osteological and genetic perspectives
- Authors: Thornton, Roxanne
- Date: 2019
- Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
- Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Thornton, Roxanne
- Date: 2019
- Subjects: Identification , Forensic osteology , Methylation , RNA , Autopsy
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198 , DOI 10.21504/10962/68102
- Description: Forensic identification of abandoned and suspected infanticide cases admitted to the South African Forensic Pathology Services is often impossible due to decomposition of the remains. In these cases, investigation of suspected criminal activity is almost never pursued. Ancillary tests in the form of anthropological and molecular analyses can assist with the forensic identification of perinatal remains. To provide fundamental information about bone development of perinatal skeleton, osteological and genetic techniques focusing on the pars basilaris, pars lateralis, sternal rib and left femur were used. Samples were obtained from unidentified and unclaimed remains originating from the Johannesburg Forensic Pathology Service (JFPS). To provide a biological age to individuals in the collection, dental aging was used to categorize remains for comparisons with anthropological and molecular data. A molecular protocol was designed to sex individuals using the X-linked G6PD and Y-linked SRY genes. Bone development was studied using osteometric and morphological data of dry bone remains coupled with bone mineral density analysis (Micro-CT). The methylation levels of CpG rich sites within the promoter region of selected bone-associated genes were incorporated to examine silencing of genes during development. Osteological results support the use of the pars basilaris, pars lateralis and femur for age-at-death estimations as well as provide the foundation for dry bone aging criteria for South African individuals. Data compared with established skeletal aging standards indicated developmental differences between populations. Through the use of animal models and the perinatal sternal rib tissue, insights and precautions into the use of post mortem bone derived RNA for forensic applications is communicated. The methylation status of CpG rich sites within the promoter regions support the hypothesis for interdependent machinery involving selected genes during early bone development. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
Bioprospecting for amylases, cellulases and xylanases from ericoid associated fungi, their production and characterisation for the bio-economy
- Authors: Adeoyo, Olusegun Richard
- Date: 2018
- Subjects: Mycorrhizal fungi , Hydrolases , Ericaceae South Africa , Ericaceae Molecular aspects
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64327 , vital:28533
- Description: South Africa is one of the most productive areas for ericaceous plants with about 850 identified species in the Cape Floral Region. The Albany Centre of Endemism where all fungi used in this study were isolated from, falls within this region. Ericaceous plants interact with some fungi via an association called the ericoid mycorrhizal (ERM) association. All fungi used in this study were isolated from roots of six ericaceous plants; Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Fungal enzymes are known to play a significant role in the food, brewing, detergent, pharmaceutical and biofuel industries. The enzyme industry is among the major sectors of the world, and additional novel sources are being explored from time to time. This study focussed on amylases (amyloglucosidase, AMG), cellulases (endoglucanase) and xylanases (endo-1,4-P-xylanase) production from ERM fungal isolates. Out of the fifty-one (51), fungal isolates screened, ChemRU330 (Leohumicola sp.), EdRU083 and EdRU002 were among the fungi that had the highest activities of all the enzymes. They were tested for the ability to produce amylases and cellulases under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28°C in a modified Melin-Norkrans (MMN) liquid medium. Cellulase specific activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and ChemRU330, respectively, was produced at an optimal pH of 5.0. For amylase, ChemRU330 had the highest specific activity of 1.11 U/mg protein while EdRU083 and EdRU02 had a specific activity of 0.80 and 0.92 U/mg protein, respectively, at the same pH with corresponding biomass yield of 113, 125 and 97 mg/50 ml, respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone, while NaFe-EDTA and cobalt inhibited enzyme activity. ChemRU330 was selected to determine the consistency and amount of amylase, cellulase and xylanase formed after several in vitro subculturing events. AMG and endo-1,4-P-xylanase were found to have the most consistent production throughout the study period. The AMG was stable at 45oC (pH 5.0), retaining approximately 65% activity over a period of 24 h. The molecular mass of AMG and endo-1,4-P-xylanase were estimated to be 101 kDa and 72 kDa, respectively. The Km and kcat were 0.38 mg/ml and 70 s-1, respectively, using soluble starch (AMG). For endo-1,4-P-xylanase, the Km and Vmax were 0.93 mg/ml and 8.54 U/ml, respectively, using beechwood xylan (endo-1,4-P-xylanase) as substrate. Additionally, crude extracts of five root endophytes with unique morphological characteristics were screened for antibacterial properties and was followed by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). L. incrustata (ChemRU330) and Chaetomium sp. extracts exhibited varying degrees of inhibition against two Gram-positive and Gram-negative bacteria. The crude extract of L. incrustata was the most effective which was found to inhibit Staphylococcus aureus (MIC: 1 mg/ml), Bacillus subtilis (MIC: 2 mg/ml) and Proteus vulgaris (MIC: 16 mg/ml). The L. incrustata displayed potential for antibacterial production and could be considered as an additional source of new antimicrobial agents in drug and food preservation. Also, the three isolates used for enzyme production were identified to genus and species levels, i.e., Leohumicola incrustata (ChemRU330), Leohumicola sp. (EdRU083) and Oidiodendron sp. (EdRU002) using both ITS and Cox1 DNA regions. The molecular analysis results indicated that these ERM mycorrhizal fungi were similar to those successfully described by some researchers in South Africa and Australia. Therefore, this study opens new opportunities for exploring ERM fungal biomolecules for the bio-economy. The promising physicochemical properties, starch and xylan hydrolysis end- products, and being non-pathogenic make AMG and endo-1,4-P-xylanase potential candidates for future applications as additives in the food industry for the production of glucose, glucose syrups, high-fructose corn syrups, and as well as the production of bioethanol. Finally, the findings of this study revealed that it is possible to produce hydrolytic enzymes from ERM fungi in vitro using chemically defined media. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Adeoyo, Olusegun Richard
- Date: 2018
- Subjects: Mycorrhizal fungi , Hydrolases , Ericaceae South Africa , Ericaceae Molecular aspects
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64327 , vital:28533
- Description: South Africa is one of the most productive areas for ericaceous plants with about 850 identified species in the Cape Floral Region. The Albany Centre of Endemism where all fungi used in this study were isolated from, falls within this region. Ericaceous plants interact with some fungi via an association called the ericoid mycorrhizal (ERM) association. All fungi used in this study were isolated from roots of six ericaceous plants; Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Fungal enzymes are known to play a significant role in the food, brewing, detergent, pharmaceutical and biofuel industries. The enzyme industry is among the major sectors of the world, and additional novel sources are being explored from time to time. This study focussed on amylases (amyloglucosidase, AMG), cellulases (endoglucanase) and xylanases (endo-1,4-P-xylanase) production from ERM fungal isolates. Out of the fifty-one (51), fungal isolates screened, ChemRU330 (Leohumicola sp.), EdRU083 and EdRU002 were among the fungi that had the highest activities of all the enzymes. They were tested for the ability to produce amylases and cellulases under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28°C in a modified Melin-Norkrans (MMN) liquid medium. Cellulase specific activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and ChemRU330, respectively, was produced at an optimal pH of 5.0. For amylase, ChemRU330 had the highest specific activity of 1.11 U/mg protein while EdRU083 and EdRU02 had a specific activity of 0.80 and 0.92 U/mg protein, respectively, at the same pH with corresponding biomass yield of 113, 125 and 97 mg/50 ml, respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone, while NaFe-EDTA and cobalt inhibited enzyme activity. ChemRU330 was selected to determine the consistency and amount of amylase, cellulase and xylanase formed after several in vitro subculturing events. AMG and endo-1,4-P-xylanase were found to have the most consistent production throughout the study period. The AMG was stable at 45oC (pH 5.0), retaining approximately 65% activity over a period of 24 h. The molecular mass of AMG and endo-1,4-P-xylanase were estimated to be 101 kDa and 72 kDa, respectively. The Km and kcat were 0.38 mg/ml and 70 s-1, respectively, using soluble starch (AMG). For endo-1,4-P-xylanase, the Km and Vmax were 0.93 mg/ml and 8.54 U/ml, respectively, using beechwood xylan (endo-1,4-P-xylanase) as substrate. Additionally, crude extracts of five root endophytes with unique morphological characteristics were screened for antibacterial properties and was followed by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). L. incrustata (ChemRU330) and Chaetomium sp. extracts exhibited varying degrees of inhibition against two Gram-positive and Gram-negative bacteria. The crude extract of L. incrustata was the most effective which was found to inhibit Staphylococcus aureus (MIC: 1 mg/ml), Bacillus subtilis (MIC: 2 mg/ml) and Proteus vulgaris (MIC: 16 mg/ml). The L. incrustata displayed potential for antibacterial production and could be considered as an additional source of new antimicrobial agents in drug and food preservation. Also, the three isolates used for enzyme production were identified to genus and species levels, i.e., Leohumicola incrustata (ChemRU330), Leohumicola sp. (EdRU083) and Oidiodendron sp. (EdRU002) using both ITS and Cox1 DNA regions. The molecular analysis results indicated that these ERM mycorrhizal fungi were similar to those successfully described by some researchers in South Africa and Australia. Therefore, this study opens new opportunities for exploring ERM fungal biomolecules for the bio-economy. The promising physicochemical properties, starch and xylan hydrolysis end- products, and being non-pathogenic make AMG and endo-1,4-P-xylanase potential candidates for future applications as additives in the food industry for the production of glucose, glucose syrups, high-fructose corn syrups, and as well as the production of bioethanol. Finally, the findings of this study revealed that it is possible to produce hydrolytic enzymes from ERM fungi in vitro using chemically defined media. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Exploring the potential of imines as antiprotozoan agents with focus on t. Brucei and p. Falciparum
- Authors: Oluwafemi, Kola Augustus
- Date: 2018
- Subjects: Protozoa , Parasites , Imines , Nuclear magnetic resonance , HeLa cells , Plasmodium falciparum , Trypanosoma brucei , Isomerism
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62235 , vital:28145 , DOI 10.21504/10962/62235
- Description: This work focuses on the design, synthesis and evaluation of imine-containing heterocyclic and acyclic compounds with special focus on their bioactivity against parasitic protozoans (P. falciparum and T. brucei) - given the context of drug resistance in the treatment of malaria and Human African sleeping sickness and the fact that several bioactive organic compounds have been reported to possess the imino group. Starting from 2-aminopyridine, novel #-alkylated-5-bromo-7-azabenzimidazoles and substituted 5-bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives were prepared, and their bioactivity against parasitic protozoans was assessed. NMR spectra of the substituted 5- bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives exhibited rotational isomerism, and a dynamic NMR study was used in the estimation of the rate constants and the free- energies of activation for rotation. The free-energy differences between the two rotamers were determined and the more stable conformations were predicted. Novel 2-phenyl-7-azabenzimidazoles were also synthesised from 2-aminopyridine. A convenient method for the regioselective formylation of 2,3-diaminopyridines into 2-amino- 7-(benzylimino)pyridine analogues of 2-phenyl-7-azabenzimidazole was developed, and some of the resulting imino derivatives were hydrogenated to verify the importance of the imino moiety for bioactivity. The 2-phenyl-7-azabenzimidazoles and the 2-amino-7- (benzylimino)pyridine analogues were screened for their anti-protozoal activity and their cytotoxicity level was determined against the HeLa cell line. In order to validate the importance of the pyridine moiety, novel #-(phenyl)-2- hydroxybenzylimines, #-(benzyl)-2-hydroxybenzylimines and (±)-trans-1,2-bis[2- hydroxybenzylimino]cyclohexanes were also synthesized and screened for activity against the parasitic protozoans and for cytotoxicity against the HeLa cell line. The biological assay results indicated that these compounds are not significantly cytotoxic and a good number of them show potential as lead compounds for the development of new malaria and trypanosomiasis drugs. , Thesis (PhD) -- Faculty of Science, Chemistry, 2018
- Full Text:
- Date Issued: 2018
- Authors: Oluwafemi, Kola Augustus
- Date: 2018
- Subjects: Protozoa , Parasites , Imines , Nuclear magnetic resonance , HeLa cells , Plasmodium falciparum , Trypanosoma brucei , Isomerism
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62235 , vital:28145 , DOI 10.21504/10962/62235
- Description: This work focuses on the design, synthesis and evaluation of imine-containing heterocyclic and acyclic compounds with special focus on their bioactivity against parasitic protozoans (P. falciparum and T. brucei) - given the context of drug resistance in the treatment of malaria and Human African sleeping sickness and the fact that several bioactive organic compounds have been reported to possess the imino group. Starting from 2-aminopyridine, novel #-alkylated-5-bromo-7-azabenzimidazoles and substituted 5-bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives were prepared, and their bioactivity against parasitic protozoans was assessed. NMR spectra of the substituted 5- bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives exhibited rotational isomerism, and a dynamic NMR study was used in the estimation of the rate constants and the free- energies of activation for rotation. The free-energy differences between the two rotamers were determined and the more stable conformations were predicted. Novel 2-phenyl-7-azabenzimidazoles were also synthesised from 2-aminopyridine. A convenient method for the regioselective formylation of 2,3-diaminopyridines into 2-amino- 7-(benzylimino)pyridine analogues of 2-phenyl-7-azabenzimidazole was developed, and some of the resulting imino derivatives were hydrogenated to verify the importance of the imino moiety for bioactivity. The 2-phenyl-7-azabenzimidazoles and the 2-amino-7- (benzylimino)pyridine analogues were screened for their anti-protozoal activity and their cytotoxicity level was determined against the HeLa cell line. In order to validate the importance of the pyridine moiety, novel #-(phenyl)-2- hydroxybenzylimines, #-(benzyl)-2-hydroxybenzylimines and (±)-trans-1,2-bis[2- hydroxybenzylimino]cyclohexanes were also synthesized and screened for activity against the parasitic protozoans and for cytotoxicity against the HeLa cell line. The biological assay results indicated that these compounds are not significantly cytotoxic and a good number of them show potential as lead compounds for the development of new malaria and trypanosomiasis drugs. , Thesis (PhD) -- Faculty of Science, Chemistry, 2018
- Full Text:
- Date Issued: 2018
Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core-sets for effective degradation of various pre-treated hardwoods
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Interaction of catechol O-methyltransferase with gold and silver nanoparticles
- Authors: Usman, Aminu
- Date: 2018
- Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
- Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Usman, Aminu
- Date: 2018
- Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
- Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
The development, manufacture and evaluation of sustained release gastric-resistant isoniazid and gastroretentive microporous rifampicin microspheres
- Authors: Mwila, Chiluba
- Date: 2018
- Subjects: Biodegradation , Microspheres (Pharmacy) , Drug delivery systems , Rifampin , Isoniazid
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/63497 , vital:28421 , DOI 10.21504/10962/63497
- Description: According to the World Health Organization Global Tuberculosis (TB) 2017 Report, there were an estimated 10.4 million new TB cases worldwide of which, in 2016, 65 % occurred in men, 28.1 % in women and 6.9 % in children. TB is the ninth leading cause of death globally and is the leading cause due to an infectious organism surpassing HIV/AIDS. Treatment is long-term and the use of a combination of medicines is required for success. The concern related to the use of fixed dose combination products for the treatment of TB is the issue of low bioavailability of rifampicin observed from a number of fixed dose combination (FDC) formulations. The hydrolysis of rifampicin, in acidic media, to form insoluble 3-formyl rifamycin SV contributes to poor bioavailability of rifampicin. The degradation of rifampicin to form this poorly absorbed compound is accelerated in the presence of isoniazid via the reversible formation of isonicotinyl hydrazone is a further factor contributing to the poor bioavailability of rifampicin. Therefore, the development of a novel drug delivery technology that prevents interactions between rifampicin and isoniazid in an acidic medium is required. A Box Behnken design was successfully used for the optimisation of a rapid and accurate stability-indicating gradient elution RP-HPLC method for the simultaneous analysis of isoniazid, pyrazinamide and rifampicin. The method was validated using ICH guidelines and the results indicate it can be used for the rapid analysis of commercially available TB FDC formulations containing the active pharmaceutical ingredients, API. The method is precise, sensitive and has the necessary selectivity for use during formulation development and optimisation studies for a combination of rifampicin, isoniazid and pyrazinamide. Initially formulation activities were undertaken with rifampicin and isoniazid for the development of an approach to enhance the effective delivery of these compounds. The characterisation of rifampicin and isoniazid was undertaken using spectroscopic, thermal and microscopic analysis. The studies revealed that the compounds are crystalline and exhibit distinct characteristic sharp peaks in X-ray diffractograms and Differential Scanning Calorimetry thermograms. The thermograms, 13C Nuclear Magnetic Resonance and Fourier Transform Infrared spectroscopy results identified that rifampicin occurs as the form II polymorph however, as there are no significant biopharmaceutic differences between the polymorphic forms of rifampicin this information was used for identification purposes only. The results were used as baseline data for comparative purposes to monitor changes that may occur when rifampicin and isoniazid are used in formulation development, dosage form manufacture and characterisation activities for a FDC technology designed to deliver both compounds simultaneously. Hydroxypropylmethylcellulose acetate succinate (HPMC-AS) and Eudragit® L100 polymers were successfully used for manufacture of isoniazid loaded gastric-resistant sustained release microspheres using an o/o solvent emulsification and evaporation approach. A Hybrid experimental design was used to investigate the influence of input variables viz., homogenisation speed and amount of HPMC-AS and Eudragit® L100 on gastric-resistance, INH release and encapsulation efficiency. The approach of using coating polymers viz., HPMC-AS and Eudragit® L100, to manufacture gastric resistant sustained release microspheres of isoniazid is unique and was efficient for preventing the release of isoniazid in an acidic environment. Only 0.523 % isoniazid was released from the optimised formulation after 2 h exposure to pH 1.2 0.1 M HCl suggesting there is also the possibility of minimising the accelerated degradation of rifampicin that occurs in the presence of isoniazid in acidic media. The microspheres also exhibited sustained release properties without burst release in pH 6.8 0.1 M phosphate buffer as < 5 % isoniazid was released at 0.5 h and only 11 % isoniazid was released at 2 h. The release of isoniazid was sustained over the entire period of dissolution testing with > 85 % isoniazid released at 24 h, implying that the majority of encapsulated isoniazid would be available for absorption. The manufacturing process resulted in the production of hard spherical particles and particle size analysis revealed that the microspheres ranged between 415.76 ± 76.93 μm and 903.35 ± 197.10 μm in diameter. The microspheres exhibited excellent flow properties attributed to the spherical nature of particles. Carr‟s index (CI) was 4.934 ± 0.775 % and the Hausner ratio (HR) was 1.148 ± 0.033 indicating good packability of the microspheres that would help in achieving weight and content uniformity of capsule dosage units. The manufacturing process however produced a low % yield suggesting that scale up difficulties may be encountered. However the high encapsulation efficiency observed may counter the challenges associated with the low yield. The DSC thermograms and FT Raman spectra of 1:1 mixtures of isoniazid, excipients and the microspheres did not reveal any potential detrimental interactions. Microporous floating sustained release microspheres for the delivery of rifampicin in the stomach have been successfully manufactured using emulsification and a diffusion/evaporation process. A novel approach using solvent mixture of acetone and dichloromethane that has not been reported for the manufacture of rifampicin microspheres was successfully used and resulted in the formation of a stable emulsion and the manufacture of rifampicin-loaded microspheres with uniform characteristics. In addition the manufacturing process was shorter than most other reported methods. A Box-Behnken experimental design was successfully used to study the influence of ethylcellulose, Eudragit® RLPO and d-glucose content on the floating properties, encapsulation efficiency and % yield of microspheres. The optimised formulation did not yield desired floating characteristics as the % buoyancy was low and floating lag times were high. The optimised formulation was modified by addition of NaHCO3 to increase the % buoyancy and reduce the floating lag time. Rifampicin release from the microspheres of the modified batch was 87.10 % at 12 h and the microspheres exhibited a % buoyancy of 87.66 ± 1.28 % (n = 6) and floating lag time of 15 ± 3.2 (n = 6) seconds. The microspheres remained buoyant for up to 12 h and an encapsulation efficiency of 88.26 ± 1.25 % was achieved. SEM images of microspheres following exposure to dissolution fluid revealed that the microspheres had numerous pores on their surface. The mean particle size distribution ranged between 423.19 ± 121.86 μm to 620.07 ± 102.67 μm. The microspheres exhibited similar flow characteristics to isoniazid microspheres with a CI of 1.422 ± 0.074 %, and HR of 1.034 ± 0.002. The excellent flow characteristics indicate that filling of the microspheres into hard gelatin capsules was unlikely to pose a challenge in respect of producing a product with uniform content. Rifampicin-excipient compatibility studies did not reveal any potential or significant interactions suggesting that the excipients used for the manufacture of the microspheres were compatible, although long term stability studies would be required to ascertain this is, indeed the case. The microporous floating sustained release microspheres manufactured in these studies has the potential to increase the bioavailability of rifampicin as they may be retained in the stomach where the solubility of rifampicin is high and from which absorption is best achieved. The degradation of rifampicin after 12 h dissolution testing in pH 1.2 0.1 M HCl in the presence of isoniazid gastric-resistant sustained release microspheres was only 4.44%. These results indicate that the degradation of rifampicin in the presence of isoniazid in acidic media can be overcome by encapsulation of both active pharmaceutical ingredients in a manner that ensure release in different segments of the gastrointestinal tract. The use of sustained release microporous gastroretentive rifampicin microspheres in combination with sustained release isoniazid gastric-resistant microspheres revealed that accelerated degradation of rifampicin in the presence of isoniazid is reduced significantly when using this approach and a FDC of rifampicin and isoniazid microspheres has the potential to improve the bioavailability of rifampicin thereby enhancing therapeutic outcomes. In vivo studies would be required to confirm the potential benefits of using this approach to deliver rifampicin in combination with isoniazid. , Thesis (PhD) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
- Authors: Mwila, Chiluba
- Date: 2018
- Subjects: Biodegradation , Microspheres (Pharmacy) , Drug delivery systems , Rifampin , Isoniazid
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/63497 , vital:28421 , DOI 10.21504/10962/63497
- Description: According to the World Health Organization Global Tuberculosis (TB) 2017 Report, there were an estimated 10.4 million new TB cases worldwide of which, in 2016, 65 % occurred in men, 28.1 % in women and 6.9 % in children. TB is the ninth leading cause of death globally and is the leading cause due to an infectious organism surpassing HIV/AIDS. Treatment is long-term and the use of a combination of medicines is required for success. The concern related to the use of fixed dose combination products for the treatment of TB is the issue of low bioavailability of rifampicin observed from a number of fixed dose combination (FDC) formulations. The hydrolysis of rifampicin, in acidic media, to form insoluble 3-formyl rifamycin SV contributes to poor bioavailability of rifampicin. The degradation of rifampicin to form this poorly absorbed compound is accelerated in the presence of isoniazid via the reversible formation of isonicotinyl hydrazone is a further factor contributing to the poor bioavailability of rifampicin. Therefore, the development of a novel drug delivery technology that prevents interactions between rifampicin and isoniazid in an acidic medium is required. A Box Behnken design was successfully used for the optimisation of a rapid and accurate stability-indicating gradient elution RP-HPLC method for the simultaneous analysis of isoniazid, pyrazinamide and rifampicin. The method was validated using ICH guidelines and the results indicate it can be used for the rapid analysis of commercially available TB FDC formulations containing the active pharmaceutical ingredients, API. The method is precise, sensitive and has the necessary selectivity for use during formulation development and optimisation studies for a combination of rifampicin, isoniazid and pyrazinamide. Initially formulation activities were undertaken with rifampicin and isoniazid for the development of an approach to enhance the effective delivery of these compounds. The characterisation of rifampicin and isoniazid was undertaken using spectroscopic, thermal and microscopic analysis. The studies revealed that the compounds are crystalline and exhibit distinct characteristic sharp peaks in X-ray diffractograms and Differential Scanning Calorimetry thermograms. The thermograms, 13C Nuclear Magnetic Resonance and Fourier Transform Infrared spectroscopy results identified that rifampicin occurs as the form II polymorph however, as there are no significant biopharmaceutic differences between the polymorphic forms of rifampicin this information was used for identification purposes only. The results were used as baseline data for comparative purposes to monitor changes that may occur when rifampicin and isoniazid are used in formulation development, dosage form manufacture and characterisation activities for a FDC technology designed to deliver both compounds simultaneously. Hydroxypropylmethylcellulose acetate succinate (HPMC-AS) and Eudragit® L100 polymers were successfully used for manufacture of isoniazid loaded gastric-resistant sustained release microspheres using an o/o solvent emulsification and evaporation approach. A Hybrid experimental design was used to investigate the influence of input variables viz., homogenisation speed and amount of HPMC-AS and Eudragit® L100 on gastric-resistance, INH release and encapsulation efficiency. The approach of using coating polymers viz., HPMC-AS and Eudragit® L100, to manufacture gastric resistant sustained release microspheres of isoniazid is unique and was efficient for preventing the release of isoniazid in an acidic environment. Only 0.523 % isoniazid was released from the optimised formulation after 2 h exposure to pH 1.2 0.1 M HCl suggesting there is also the possibility of minimising the accelerated degradation of rifampicin that occurs in the presence of isoniazid in acidic media. The microspheres also exhibited sustained release properties without burst release in pH 6.8 0.1 M phosphate buffer as < 5 % isoniazid was released at 0.5 h and only 11 % isoniazid was released at 2 h. The release of isoniazid was sustained over the entire period of dissolution testing with > 85 % isoniazid released at 24 h, implying that the majority of encapsulated isoniazid would be available for absorption. The manufacturing process resulted in the production of hard spherical particles and particle size analysis revealed that the microspheres ranged between 415.76 ± 76.93 μm and 903.35 ± 197.10 μm in diameter. The microspheres exhibited excellent flow properties attributed to the spherical nature of particles. Carr‟s index (CI) was 4.934 ± 0.775 % and the Hausner ratio (HR) was 1.148 ± 0.033 indicating good packability of the microspheres that would help in achieving weight and content uniformity of capsule dosage units. The manufacturing process however produced a low % yield suggesting that scale up difficulties may be encountered. However the high encapsulation efficiency observed may counter the challenges associated with the low yield. The DSC thermograms and FT Raman spectra of 1:1 mixtures of isoniazid, excipients and the microspheres did not reveal any potential detrimental interactions. Microporous floating sustained release microspheres for the delivery of rifampicin in the stomach have been successfully manufactured using emulsification and a diffusion/evaporation process. A novel approach using solvent mixture of acetone and dichloromethane that has not been reported for the manufacture of rifampicin microspheres was successfully used and resulted in the formation of a stable emulsion and the manufacture of rifampicin-loaded microspheres with uniform characteristics. In addition the manufacturing process was shorter than most other reported methods. A Box-Behnken experimental design was successfully used to study the influence of ethylcellulose, Eudragit® RLPO and d-glucose content on the floating properties, encapsulation efficiency and % yield of microspheres. The optimised formulation did not yield desired floating characteristics as the % buoyancy was low and floating lag times were high. The optimised formulation was modified by addition of NaHCO3 to increase the % buoyancy and reduce the floating lag time. Rifampicin release from the microspheres of the modified batch was 87.10 % at 12 h and the microspheres exhibited a % buoyancy of 87.66 ± 1.28 % (n = 6) and floating lag time of 15 ± 3.2 (n = 6) seconds. The microspheres remained buoyant for up to 12 h and an encapsulation efficiency of 88.26 ± 1.25 % was achieved. SEM images of microspheres following exposure to dissolution fluid revealed that the microspheres had numerous pores on their surface. The mean particle size distribution ranged between 423.19 ± 121.86 μm to 620.07 ± 102.67 μm. The microspheres exhibited similar flow characteristics to isoniazid microspheres with a CI of 1.422 ± 0.074 %, and HR of 1.034 ± 0.002. The excellent flow characteristics indicate that filling of the microspheres into hard gelatin capsules was unlikely to pose a challenge in respect of producing a product with uniform content. Rifampicin-excipient compatibility studies did not reveal any potential or significant interactions suggesting that the excipients used for the manufacture of the microspheres were compatible, although long term stability studies would be required to ascertain this is, indeed the case. The microporous floating sustained release microspheres manufactured in these studies has the potential to increase the bioavailability of rifampicin as they may be retained in the stomach where the solubility of rifampicin is high and from which absorption is best achieved. The degradation of rifampicin after 12 h dissolution testing in pH 1.2 0.1 M HCl in the presence of isoniazid gastric-resistant sustained release microspheres was only 4.44%. These results indicate that the degradation of rifampicin in the presence of isoniazid in acidic media can be overcome by encapsulation of both active pharmaceutical ingredients in a manner that ensure release in different segments of the gastrointestinal tract. The use of sustained release microporous gastroretentive rifampicin microspheres in combination with sustained release isoniazid gastric-resistant microspheres revealed that accelerated degradation of rifampicin in the presence of isoniazid is reduced significantly when using this approach and a FDC of rifampicin and isoniazid microspheres has the potential to improve the bioavailability of rifampicin thereby enhancing therapeutic outcomes. In vivo studies would be required to confirm the potential benefits of using this approach to deliver rifampicin in combination with isoniazid. , Thesis (PhD) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
Synthesis and biolgical screening of potential plasmodium falciparum DXR inhibitors
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017-04
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017-04
A systems thinking approach to e-government strategy formulation for water service delivery in South African local municipalities
- Authors: Osah, Umeoniso Joshua
- Date: 2017
- Subjects: Internet in public administration South Africa , Electronic government information South Africa , Communication in public administration South Africa , Municipal services South Africa , Municipal services Information technology , System theory
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64471 , vital:28547 , DOI 10.21504/10962/64471
- Description: E-Government deployments by stakeholders within the South African water service sector, provide certain benefits for the sector. While deemed beneficial and of considerable value, e-Government deployments and implementations in the water sector of local governments of South Africa have not always been successful. One important reason for e-Government failures, among several others, is the lack of coherent strategies, informed by key representing stakeholder views, to guide implementation and deployment of e-Government tools. Without strategies, it is highly likely that ICT integration will be conducted haphazardly. As a point of note, more than the deliverable (strategy document) that represents the output of the strategy development process, it serves more purpose to understand the process that results in the strategy. Importantly, understanding the process helps to account for the formed relationships between the various stakeholders that need to buy into the strategy. The research study develops an e-Government strategy formulation framework based on a systems thinking approach, intended to support the strategy formulation process of e-Government strategies – to underpin the effective integration, deployment and sustained use of ICT solutions for water service delivery at the local government level. A systems thinking approach is considered due to its emphasis on the strategy being informed by a holistic assessment. Where there is some knowledge about the processes by which a strategy is formulated – over time ideas may be derived on the types of processes that may produce efficient e-Government strategies. The research is conducted using the Design Science research paradigm. The Design Science paradigm is comprised of two processes – build and evaluate (Hevner et al., 2004). The build process, as related to this research concentrates on the progression through which the theoretical e-Government strategy formulation framework is derived. Weick’s (1989) theorizing approach is ascribed, supporting the design of the theoretical framework. In applying Weick’s theorizing approach, firstly, the lack of knowledge on how e-Government strategies should be formulated in South African local municipalities – is explicated in an intelligible manner. Once the problem is properly articulated, a trial and error selection process is undertaken of existing approaches on strategy formulation – thought to possess the potential to contribute to the development of an e-Government strategy formulation framework, suited to local governments in South Africa. With good reason, 10 (ten) strategy approaches are selected from, e-Government programmes in developed countries, Non-Governmental organizational strategy approaches, and business related strategy formulation approaches. Lastly, as all possible approaches that may contribute to the framework development process cannot be selected, criteria is specified to limit the number of possible selections. Furthermore, in deriving the framework, foundations for systematically dealing with unstructured problems, such as, strategy formulation are consulted. This foundation along with the research goals, informs the development of a template used to comparatively analyse the 10 selected approaches on strategy formulation. This analysis aids in revealing the components of an e-Government strategy formulation process. With the developed framework, the evaluation process of the design science research commences, seeking to determine the utility of the framework (suitability and shortcomings). The framework is applied to the procedural formulation of a strategy for a tentative e-Government project called MobiSAM, which aims to enhance citizen engagement with local government through the use of mobile phones. The strategy formulation application process in the project environment and local government reveals lessons that inform revisions to the framework. The e-Government strategy formulation framework, therefore represents a fundamental tool for e-Government strategy development in local municipalities, and may be customized to fit the requirements of varying local municipalities. , Thesis (PhD) -- Faculty of Commerce, Information Systems, 2017
- Full Text:
- Date Issued: 2017
- Authors: Osah, Umeoniso Joshua
- Date: 2017
- Subjects: Internet in public administration South Africa , Electronic government information South Africa , Communication in public administration South Africa , Municipal services South Africa , Municipal services Information technology , System theory
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64471 , vital:28547 , DOI 10.21504/10962/64471
- Description: E-Government deployments by stakeholders within the South African water service sector, provide certain benefits for the sector. While deemed beneficial and of considerable value, e-Government deployments and implementations in the water sector of local governments of South Africa have not always been successful. One important reason for e-Government failures, among several others, is the lack of coherent strategies, informed by key representing stakeholder views, to guide implementation and deployment of e-Government tools. Without strategies, it is highly likely that ICT integration will be conducted haphazardly. As a point of note, more than the deliverable (strategy document) that represents the output of the strategy development process, it serves more purpose to understand the process that results in the strategy. Importantly, understanding the process helps to account for the formed relationships between the various stakeholders that need to buy into the strategy. The research study develops an e-Government strategy formulation framework based on a systems thinking approach, intended to support the strategy formulation process of e-Government strategies – to underpin the effective integration, deployment and sustained use of ICT solutions for water service delivery at the local government level. A systems thinking approach is considered due to its emphasis on the strategy being informed by a holistic assessment. Where there is some knowledge about the processes by which a strategy is formulated – over time ideas may be derived on the types of processes that may produce efficient e-Government strategies. The research is conducted using the Design Science research paradigm. The Design Science paradigm is comprised of two processes – build and evaluate (Hevner et al., 2004). The build process, as related to this research concentrates on the progression through which the theoretical e-Government strategy formulation framework is derived. Weick’s (1989) theorizing approach is ascribed, supporting the design of the theoretical framework. In applying Weick’s theorizing approach, firstly, the lack of knowledge on how e-Government strategies should be formulated in South African local municipalities – is explicated in an intelligible manner. Once the problem is properly articulated, a trial and error selection process is undertaken of existing approaches on strategy formulation – thought to possess the potential to contribute to the development of an e-Government strategy formulation framework, suited to local governments in South Africa. With good reason, 10 (ten) strategy approaches are selected from, e-Government programmes in developed countries, Non-Governmental organizational strategy approaches, and business related strategy formulation approaches. Lastly, as all possible approaches that may contribute to the framework development process cannot be selected, criteria is specified to limit the number of possible selections. Furthermore, in deriving the framework, foundations for systematically dealing with unstructured problems, such as, strategy formulation are consulted. This foundation along with the research goals, informs the development of a template used to comparatively analyse the 10 selected approaches on strategy formulation. This analysis aids in revealing the components of an e-Government strategy formulation process. With the developed framework, the evaluation process of the design science research commences, seeking to determine the utility of the framework (suitability and shortcomings). The framework is applied to the procedural formulation of a strategy for a tentative e-Government project called MobiSAM, which aims to enhance citizen engagement with local government through the use of mobile phones. The strategy formulation application process in the project environment and local government reveals lessons that inform revisions to the framework. The e-Government strategy formulation framework, therefore represents a fundamental tool for e-Government strategy development in local municipalities, and may be customized to fit the requirements of varying local municipalities. , Thesis (PhD) -- Faculty of Commerce, Information Systems, 2017
- Full Text:
- Date Issued: 2017
Assessing the potential role of microorganisms in the production of seedlings for the restoration of Albany Thicket
- Authors: Mpama, Nelisa
- Date: 2017
- Subjects: Vesicular-arbuscular mycorrhizas , Rhizobacteria , Restoration ecology South Africa Albany , Microorganisms
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64555 , vital:28558
- Description: The role of microorganisms in restoration of the Albany Thicket has not been well documented, although the benefits to plants of these various interactions has been well documented. Microorganisms are chief ecological engineers and assist in resolving environmental problems and act to restore degraded ecosystem function by forming mutual relationships with the roots of the plants. The aim of this study was to assess the potential of microorganisms for the improved biomass production of selected woody and succulent seedlings used in mesic thicket restoration. Three tree species were selected for propagation in this study namely; Mystroxylon aethiopicum Scutia myrtina and Aloe ferox. Soil samples were collected from a degraded and intact thicket site from Bathurst, South Africa. Soils were evaluated for number of arbuscular mycorrhizal (AM) spores, mycorrhizal infectivity potential and nutrient availability both before and after seedling propagation. Pasteurized soil from the degraded site was used in a pot trial. Ten replicates seedling for plant species were planted and subjected to four treatments which included inoculation with AM fungi and the rhizobacterium, Enterobacter sp., alone and in combination; the fourth treatment was an un-inouclated control. Plant growth parameters were recorded at regular intervals where appropriate and seedlings were harvested after 24 weeks for biomass measurements and AM colonisation assessments. Although generally low (< 1 spore per gram) the density of AM fungal spores was significantly higher in soils from the intact site when compared with soils from the degraded site. The mycorrhizal potential of the soils was however not significantly different. Mystroxylon aethiopicum seedling shoot height, canopy diameter and shoot biomass showed a significant increase when inoculated with AM fungi while S. myrtina seedlings showed increased shoot height when inoculated with both AM fungi and Enterobacter sp. Aloe ferox seedlings did not respond to microbial inoculation. The concentration of soil P and Na increased in treatments with Enterobacter sp. alone and in combination with AM fungi. Mystroxylon aethiopicum and S. myrtina seedlings showed a dependency on microbial inoculants indicating the importance of inoculation in the nursery before planting out into the field. Overall AM fungal inoculants applied to seedlings can be used to compensate for nutrient deficiency in soils. Although the Enterobacter isolate used was known to have various plant growth promoting capabilities. It is recommended that other rhizobacterial isolates be investigated. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2017
- Full Text:
- Date Issued: 2017
- Authors: Mpama, Nelisa
- Date: 2017
- Subjects: Vesicular-arbuscular mycorrhizas , Rhizobacteria , Restoration ecology South Africa Albany , Microorganisms
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64555 , vital:28558
- Description: The role of microorganisms in restoration of the Albany Thicket has not been well documented, although the benefits to plants of these various interactions has been well documented. Microorganisms are chief ecological engineers and assist in resolving environmental problems and act to restore degraded ecosystem function by forming mutual relationships with the roots of the plants. The aim of this study was to assess the potential of microorganisms for the improved biomass production of selected woody and succulent seedlings used in mesic thicket restoration. Three tree species were selected for propagation in this study namely; Mystroxylon aethiopicum Scutia myrtina and Aloe ferox. Soil samples were collected from a degraded and intact thicket site from Bathurst, South Africa. Soils were evaluated for number of arbuscular mycorrhizal (AM) spores, mycorrhizal infectivity potential and nutrient availability both before and after seedling propagation. Pasteurized soil from the degraded site was used in a pot trial. Ten replicates seedling for plant species were planted and subjected to four treatments which included inoculation with AM fungi and the rhizobacterium, Enterobacter sp., alone and in combination; the fourth treatment was an un-inouclated control. Plant growth parameters were recorded at regular intervals where appropriate and seedlings were harvested after 24 weeks for biomass measurements and AM colonisation assessments. Although generally low (< 1 spore per gram) the density of AM fungal spores was significantly higher in soils from the intact site when compared with soils from the degraded site. The mycorrhizal potential of the soils was however not significantly different. Mystroxylon aethiopicum seedling shoot height, canopy diameter and shoot biomass showed a significant increase when inoculated with AM fungi while S. myrtina seedlings showed increased shoot height when inoculated with both AM fungi and Enterobacter sp. Aloe ferox seedlings did not respond to microbial inoculation. The concentration of soil P and Na increased in treatments with Enterobacter sp. alone and in combination with AM fungi. Mystroxylon aethiopicum and S. myrtina seedlings showed a dependency on microbial inoculants indicating the importance of inoculation in the nursery before planting out into the field. Overall AM fungal inoculants applied to seedlings can be used to compensate for nutrient deficiency in soils. Although the Enterobacter isolate used was known to have various plant growth promoting capabilities. It is recommended that other rhizobacterial isolates be investigated. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2017
- Full Text:
- Date Issued: 2017
Comparative fish ecology in three periodically connected rivers in the upper Zambezi and Okavango ecoregions
- Authors: Taylor, Geraldine Claire
- Date: 2017
- Subjects: Fishes Ecology Zambia Zambezi District , Floodplain ecology Zambia Zambezi District , Stable isotopes , Fishes Food Zambia Zambezi District , Fishes Mortality Zambia Zambezi District , Fish populations Zambia Zambezi District , Fishes Growth
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/65055 , vital:28660 , DOI https://doi.org/10.21504/10962/65055
- Description: The Upper Zambezi, Kavango and Kwando rivers are three periodically interlinked floodplain rivers which share the same Upper Zambezian floodplain ichthyofauna. The aim of this thesis was to compare the biology and ecology of the fish communities in these three rivers. The objective was to test the hypothesis that fish community composition and assemblage structure, fish diets, food web structure and trophic dynamics, fish growth rates and total mortality are influenced by the differing flood magnitudes of the three rivers, in support of the flood pulse concept. To understand the abiotic characteristics of each river, water temperature, flood regime, total dissolved inorganic nitrogen concentrations and water quality parameters were measured and compared between rivers. Water temperatures varied seasonally, and seven day moving averages peaked above 30 °C in January, and fell to between 16 and 19 °C in June. The Zambezi River had the largest flood (6.14 m), followed by the Kavango River (3.80 m), while the Kwando River had the smallest flood (0.65 m). Total dissolved inorganic nitrogen concentrations were low in the Kavango and Zambezi Rivers (0.2 - 0.6 mg/l), and slightly higher in the Kwando River (<1 mg/l). Conductivity, total dissolved solids and total dissolved inorganic nitrogen concentrations decreased with the flood (dilution effect). Using biomass catch per unit effort data from experimental gillnets, fish community composition and assemblage structure was described, and differed between rivers in all hydrological seasons. In the Zambezi and Kavango rivers, fish assemblages varied with hydrological season as a result of the homogenising influence of the flood pulse, while in the Kwando River fish assemblages did not differ seasonally as flood pulses were small and often irregular. Differences in community composition were attributed to the abundance of Hydrocynus vittatus, a large bodied open water predator, in the Zambezi and Kavango rivers, and its relative absence in the Kwando River. Based on the results of the community composition, six focus species were chosen that were abundant and representative of the various feeding modes and life history strategies of the fish community. These were the striped robber Brycinus lateralis, sharptooth catfish Clarias gariepinus, blunttooth catfish Clarias ngamensis, African pike Hepsetus cuvieri, silver catfish Schilbe intermedius and purpleface largemouth Serranochromis macrocephalus. Stomach contents analysis was then used to compare the feeding ecology of the six example species between rivers. Clarias gariepinus, C. ngamensis and S. intermedius were piscivorous in the Zambezi and Kavango rivers, and preyed upon more invertebrates in the Kwando River, while Hepsetus cuvieri and S. macrocephalus were piscivorous in all three rivers. Differences in diets were attributed to seasonal prey abundance, with prey fishes abundant during falling and low water when the Zambezi and Kavango rivers were sampled, while invertebrates were abundant during rising and high water when the Kwando River was sampled. Prey mastication by B. lateralis made prey identification difficult. For other predators, the usefulness of stomach contents analysis for dietary descriptions was restricted by the high proportion of empty stomachs. As a result, whole ecosystem stable isotope analysis was used to gain a holistic understanding of the food web structure and fish feeding ecology of the three rivers. The Zambezi and Kavango river food webs were supported by C enriched resources such as C4 and C3 riparian vegetation from the floodplain, while the Kwando River food web was based on C depleted resources such as filamentous algae and aquatic macrophytes. The Zambezi River food web had a restricted nitrogen range, with reduced food chain length and the predators in this river did not occupy such elevated trophic positions compared to in the Kavango and Kwando river food webs. This was attributed to the overfishing of the primary and tertiary consumers in the Zambezi River, a phenomenon known to reduce food chain length. Focussing on predator communities, in the Zambezi and Kavango rivers, H. vittatus isotopic niche width was large and overlapped significantly with most other predators, while in the Kwando River predator niches were more distinct. This supported previously proposed hypotheses by describing H. vittatus as a dominant predator which excludes all other fishes by predation or competition. Despite the dominance of H. vittatus, C. gariepinus occupied the position of top predator in all three rivers, and information on the habitat use, feeding habits and trophic niches of the serranochromine cichlids added understanding of their ecology. Lastly, age was determined using sectioned sagittal otoliths for C. gariepinus, C. ngamensis, S. intermedius and S. macrocephalus and using whole asteriscus otoliths for B. lateralis and H. cuvieri, and growth was modelled using the von Bertalanffy growth equation. Growth performance was high in the Zambezi and Kavango rivers, and lower in the Kwando River, most likely in response to the varying flood magnitudes. Total mortality rates, estimated using Hoenig’s maximum-age based equation, were high in the Zambezi River as a result of the high fishing pressure on this river. Overall floodplain fish ecology in the Zambezi, Kavango and Kwando rivers was influenced by the flood pulse, as was predicted by the flood pulse concept. Periodic and equilibrium life history strategists were found to adapt either to the pulsing environments of the Zambezi and Kavango rivers, or to the more stable environment of the Kwando River, and large bodied, long lived periodic strategists such as C. gariepinus tended to be highly plastic and able to thrive in most conditions. Data also suggested that Zambezi River food web structure and fish mortality rates have been impacted by overfishing, for which more information is needed to conserve and manage this system. , Thesis (PhD) -- Faculty of Science, Ichthyology and Fisheries Science, 2018
- Full Text:
- Date Issued: 2017
- Authors: Taylor, Geraldine Claire
- Date: 2017
- Subjects: Fishes Ecology Zambia Zambezi District , Floodplain ecology Zambia Zambezi District , Stable isotopes , Fishes Food Zambia Zambezi District , Fishes Mortality Zambia Zambezi District , Fish populations Zambia Zambezi District , Fishes Growth
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/65055 , vital:28660 , DOI https://doi.org/10.21504/10962/65055
- Description: The Upper Zambezi, Kavango and Kwando rivers are three periodically interlinked floodplain rivers which share the same Upper Zambezian floodplain ichthyofauna. The aim of this thesis was to compare the biology and ecology of the fish communities in these three rivers. The objective was to test the hypothesis that fish community composition and assemblage structure, fish diets, food web structure and trophic dynamics, fish growth rates and total mortality are influenced by the differing flood magnitudes of the three rivers, in support of the flood pulse concept. To understand the abiotic characteristics of each river, water temperature, flood regime, total dissolved inorganic nitrogen concentrations and water quality parameters were measured and compared between rivers. Water temperatures varied seasonally, and seven day moving averages peaked above 30 °C in January, and fell to between 16 and 19 °C in June. The Zambezi River had the largest flood (6.14 m), followed by the Kavango River (3.80 m), while the Kwando River had the smallest flood (0.65 m). Total dissolved inorganic nitrogen concentrations were low in the Kavango and Zambezi Rivers (0.2 - 0.6 mg/l), and slightly higher in the Kwando River (<1 mg/l). Conductivity, total dissolved solids and total dissolved inorganic nitrogen concentrations decreased with the flood (dilution effect). Using biomass catch per unit effort data from experimental gillnets, fish community composition and assemblage structure was described, and differed between rivers in all hydrological seasons. In the Zambezi and Kavango rivers, fish assemblages varied with hydrological season as a result of the homogenising influence of the flood pulse, while in the Kwando River fish assemblages did not differ seasonally as flood pulses were small and often irregular. Differences in community composition were attributed to the abundance of Hydrocynus vittatus, a large bodied open water predator, in the Zambezi and Kavango rivers, and its relative absence in the Kwando River. Based on the results of the community composition, six focus species were chosen that were abundant and representative of the various feeding modes and life history strategies of the fish community. These were the striped robber Brycinus lateralis, sharptooth catfish Clarias gariepinus, blunttooth catfish Clarias ngamensis, African pike Hepsetus cuvieri, silver catfish Schilbe intermedius and purpleface largemouth Serranochromis macrocephalus. Stomach contents analysis was then used to compare the feeding ecology of the six example species between rivers. Clarias gariepinus, C. ngamensis and S. intermedius were piscivorous in the Zambezi and Kavango rivers, and preyed upon more invertebrates in the Kwando River, while Hepsetus cuvieri and S. macrocephalus were piscivorous in all three rivers. Differences in diets were attributed to seasonal prey abundance, with prey fishes abundant during falling and low water when the Zambezi and Kavango rivers were sampled, while invertebrates were abundant during rising and high water when the Kwando River was sampled. Prey mastication by B. lateralis made prey identification difficult. For other predators, the usefulness of stomach contents analysis for dietary descriptions was restricted by the high proportion of empty stomachs. As a result, whole ecosystem stable isotope analysis was used to gain a holistic understanding of the food web structure and fish feeding ecology of the three rivers. The Zambezi and Kavango river food webs were supported by C enriched resources such as C4 and C3 riparian vegetation from the floodplain, while the Kwando River food web was based on C depleted resources such as filamentous algae and aquatic macrophytes. The Zambezi River food web had a restricted nitrogen range, with reduced food chain length and the predators in this river did not occupy such elevated trophic positions compared to in the Kavango and Kwando river food webs. This was attributed to the overfishing of the primary and tertiary consumers in the Zambezi River, a phenomenon known to reduce food chain length. Focussing on predator communities, in the Zambezi and Kavango rivers, H. vittatus isotopic niche width was large and overlapped significantly with most other predators, while in the Kwando River predator niches were more distinct. This supported previously proposed hypotheses by describing H. vittatus as a dominant predator which excludes all other fishes by predation or competition. Despite the dominance of H. vittatus, C. gariepinus occupied the position of top predator in all three rivers, and information on the habitat use, feeding habits and trophic niches of the serranochromine cichlids added understanding of their ecology. Lastly, age was determined using sectioned sagittal otoliths for C. gariepinus, C. ngamensis, S. intermedius and S. macrocephalus and using whole asteriscus otoliths for B. lateralis and H. cuvieri, and growth was modelled using the von Bertalanffy growth equation. Growth performance was high in the Zambezi and Kavango rivers, and lower in the Kwando River, most likely in response to the varying flood magnitudes. Total mortality rates, estimated using Hoenig’s maximum-age based equation, were high in the Zambezi River as a result of the high fishing pressure on this river. Overall floodplain fish ecology in the Zambezi, Kavango and Kwando rivers was influenced by the flood pulse, as was predicted by the flood pulse concept. Periodic and equilibrium life history strategists were found to adapt either to the pulsing environments of the Zambezi and Kavango rivers, or to the more stable environment of the Kwando River, and large bodied, long lived periodic strategists such as C. gariepinus tended to be highly plastic and able to thrive in most conditions. Data also suggested that Zambezi River food web structure and fish mortality rates have been impacted by overfishing, for which more information is needed to conserve and manage this system. , Thesis (PhD) -- Faculty of Science, Ichthyology and Fisheries Science, 2018
- Full Text:
- Date Issued: 2017