The effects of elevated atmospheric CO2 on the biological control of invasive aquatic weeds in South Africa
- Authors: Baso, Nompumelelo Catherine
- Date: 2020
- Subjects: Aquatic weeds -- Biological control -- South Africa , Plants -- Effect of atmospheric carbon dioxide on , Atmospheric carbon dioxide -- Environmental aspects
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140772 , vital:37917
- Description: There has been a rapid increase in atmospheric CO2 concentration, from pre-industrial values of 280 ppm to more than 400 ppm currently, and this is expected to more than double by the end of the 21st century. Studies have shown that plants grown above 600 ppm tend to have an increased growth rate and invest more in carbon-based defences. This has important implications for the management of invasive alien plants, especially for the field of biological control which is mostly dependent on herbivorous insects. This is because insects reared on such plants have been shown to have reduced overall fitness. Nevertheless, most of the studies on potential changes in plant-insect interactions under elevated CO2 are based on agricultural systems, with only a limited number of these types of studies conducted on alien invasive weeds. However, climate change and invasive species are two of the most prevalent features of global environmental change. Therefore, this also warrants active research and experimental studies to better understand how these systems will be affected by future climates. Thus, the aim of this study was to investigate the effects of elevated atmospheric CO2 on the biological control of four invasive aquatic weeds (Azolla filiculoides, Salvinia molesta, Pistia stratiotes, and Myriophyllum aquaticum). These species are a threat to natural resources in South Africa but are currently under successful control by their biological control agents (Stenopelmus rufinasus, Cyrtobagous salviniae, Neohydronomus affinis, and Lysathia n. sp.). To achieve this, the selected plant species were grown in a three-factor experimental design in winter (CO2 X nutrients X herbivory), and another two-factorial design in summer (CO2 X herbivory). Atmospheric CO2 concentrations were set at ambient (400 ppm) or elevated (800 ppm), as per the predictions of the IPCC. As per my hypothesis, the results suggest that these species will become more challenging in future due to increased biomass production, asexual reproduction and a higher C: N ratio which is evident under high CO2 concentrations. Although the biological control agents were in some instances able to reduce this CO2 fertilisation effect, their efficacy was significantly reduced compared with the levels of control observed at ambient CO2. These results suggest that additional biological control agents and other management methods may be needed for continued control of these invasive macrophytes, both in South Africa and further afield where they are problematic.
- Full Text:
- Authors: Baso, Nompumelelo Catherine
- Date: 2020
- Subjects: Aquatic weeds -- Biological control -- South Africa , Plants -- Effect of atmospheric carbon dioxide on , Atmospheric carbon dioxide -- Environmental aspects
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140772 , vital:37917
- Description: There has been a rapid increase in atmospheric CO2 concentration, from pre-industrial values of 280 ppm to more than 400 ppm currently, and this is expected to more than double by the end of the 21st century. Studies have shown that plants grown above 600 ppm tend to have an increased growth rate and invest more in carbon-based defences. This has important implications for the management of invasive alien plants, especially for the field of biological control which is mostly dependent on herbivorous insects. This is because insects reared on such plants have been shown to have reduced overall fitness. Nevertheless, most of the studies on potential changes in plant-insect interactions under elevated CO2 are based on agricultural systems, with only a limited number of these types of studies conducted on alien invasive weeds. However, climate change and invasive species are two of the most prevalent features of global environmental change. Therefore, this also warrants active research and experimental studies to better understand how these systems will be affected by future climates. Thus, the aim of this study was to investigate the effects of elevated atmospheric CO2 on the biological control of four invasive aquatic weeds (Azolla filiculoides, Salvinia molesta, Pistia stratiotes, and Myriophyllum aquaticum). These species are a threat to natural resources in South Africa but are currently under successful control by their biological control agents (Stenopelmus rufinasus, Cyrtobagous salviniae, Neohydronomus affinis, and Lysathia n. sp.). To achieve this, the selected plant species were grown in a three-factor experimental design in winter (CO2 X nutrients X herbivory), and another two-factorial design in summer (CO2 X herbivory). Atmospheric CO2 concentrations were set at ambient (400 ppm) or elevated (800 ppm), as per the predictions of the IPCC. As per my hypothesis, the results suggest that these species will become more challenging in future due to increased biomass production, asexual reproduction and a higher C: N ratio which is evident under high CO2 concentrations. Although the biological control agents were in some instances able to reduce this CO2 fertilisation effect, their efficacy was significantly reduced compared with the levels of control observed at ambient CO2. These results suggest that additional biological control agents and other management methods may be needed for continued control of these invasive macrophytes, both in South Africa and further afield where they are problematic.
- Full Text:
The exploration of ARF1 screening assays to determine the drug status of ARF1 in cancer and malaria
- Authors: Ntlantsana, Apelele
- Date: 2020
- Subjects: ADP ribosylation , Golgi apparatus , Guanosine triphosphatase , G proteins , Malariotherapy , Malaria -- Research , Cancer -- Chemotherapy , Malaria -- Chemotherpay
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167232 , vital:41458
- Description: ARF GTPases are key regulators of the secretory and endocytic pathways. ARF1 is involved in the secretory pathway. ARF1 has been implicated in the endoplasmic reticulum to Golgi transport, function of the Golgi apparatus and transport from the trans-Golgi network to endosomes. ARFs cycle between active GTP-bound and inactive GDP-bound conformations. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ARF GEFs facilitate the activation of ARFs by mediating the exchange of GDP for GTP, while ARF GAPs terminate ARF function by stimulating the hydrolysis of the terminal phosphate group of GTP. Based on existing evidence gained from gene manipulation and cell biological investigations, ARF1 has been shown to be fundamentally important for cancer cell proliferation and metastasis and may be a promising target for the development of anti-cancer drugs. Additionally, the conservation of ARFs in eukaryotic organisms leads to an interesting question of whether a single drug target can be used to target multiple diseases. In this case, can a human cancer drug employed for cancer therapy be used in anti-malarial drug therapies? To confirm the drug target status of ARFs using chemical validation experiments, novel inhibitory compounds are needed. This requires the development of complex in vitro protein- protein interaction assays that can be used to screen chemical libraries for ARF GTPase inhibitors. In this study, we developed a fluorescence resonance energy transfer (FRET) assay and a novel in vitro colorimetric plate-based assay to detect the activation status of truncated human and Plasmodium falciparum ARF1. In the case of the FRET assay, active (GTP-bound) and inactive (GDP-bound) ARF1 could be distinguished with Z-factor values >0.5, suggesting that further development of the assay format to identify GEF and GAP inhibitors may be feasible. In the case of the colorimetric assay, robust signals could be detected and the assay was useful for detecting the activation status of ARF1. However, although the activation of ARF1 by the Sec7 domains of the BIG1 and ARNO was detectable, signals were not robust enough to employ in screening campaigns.
- Full Text:
- Authors: Ntlantsana, Apelele
- Date: 2020
- Subjects: ADP ribosylation , Golgi apparatus , Guanosine triphosphatase , G proteins , Malariotherapy , Malaria -- Research , Cancer -- Chemotherapy , Malaria -- Chemotherpay
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167232 , vital:41458
- Description: ARF GTPases are key regulators of the secretory and endocytic pathways. ARF1 is involved in the secretory pathway. ARF1 has been implicated in the endoplasmic reticulum to Golgi transport, function of the Golgi apparatus and transport from the trans-Golgi network to endosomes. ARFs cycle between active GTP-bound and inactive GDP-bound conformations. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ARF GEFs facilitate the activation of ARFs by mediating the exchange of GDP for GTP, while ARF GAPs terminate ARF function by stimulating the hydrolysis of the terminal phosphate group of GTP. Based on existing evidence gained from gene manipulation and cell biological investigations, ARF1 has been shown to be fundamentally important for cancer cell proliferation and metastasis and may be a promising target for the development of anti-cancer drugs. Additionally, the conservation of ARFs in eukaryotic organisms leads to an interesting question of whether a single drug target can be used to target multiple diseases. In this case, can a human cancer drug employed for cancer therapy be used in anti-malarial drug therapies? To confirm the drug target status of ARFs using chemical validation experiments, novel inhibitory compounds are needed. This requires the development of complex in vitro protein- protein interaction assays that can be used to screen chemical libraries for ARF GTPase inhibitors. In this study, we developed a fluorescence resonance energy transfer (FRET) assay and a novel in vitro colorimetric plate-based assay to detect the activation status of truncated human and Plasmodium falciparum ARF1. In the case of the FRET assay, active (GTP-bound) and inactive (GDP-bound) ARF1 could be distinguished with Z-factor values >0.5, suggesting that further development of the assay format to identify GEF and GAP inhibitors may be feasible. In the case of the colorimetric assay, robust signals could be detected and the assay was useful for detecting the activation status of ARF1. However, although the activation of ARF1 by the Sec7 domains of the BIG1 and ARNO was detectable, signals were not robust enough to employ in screening campaigns.
- Full Text:
The taxonomy of the topminnows (Teleostei: Cyprinodontiformes) (Procatopodidae: Procatopodinae) of southern Africa
- Authors: Van Zeeventer, Ryan Matthew
- Date: 2020
- Subjects: Cyprinodontiformes -- Africa, Southern , Killifishes -- Africa, Southern
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145559 , vital:38449
- Description: Taxonomy is a tool that plays a crucial role in the implementation of effective management assessments, the ability to identify different fish species and their life stages, and create effective conservation strategies. Recent years have seen an increase in the discovery of cryptic species which could have an effect on how we implement conservation strategies, increasing the importance of accurate taxonomic assessments. The southern African topminnows of the genera Micropanchax and Lacustricola (Family Procatopodidae) include several species of egg-laying fishes that live in the marginal areas of rivers and lakes throughout the river systems of southern Africa in separated and connected populations. It was recently discovered that these populations showed subtle differences in their morphology and colouration, which led to the belief that new and cryptic species may be present. Previous studies of the Procatopodidae relied heavily on phylogenetic analyses that drew on morphological traits without reliance on molecular methods of analysis. A few of the taxonomic issues were resolved through these limited studies but ultimately the taxonomic status of the Procatopodidae remained poorly understood. Furthermore, these studies had not been updated in recent years and hence the benefits of new technology had not been brought to bear on the issues. A multi-gene analysis using standard phylogenetic methods and five molecular markers (mitochondrial cytochrome C oxidase (COI), mitochondrial cytochrome b (cytb), mitochondrial 16S ribosomal RNA (16S rRNA), nuclear 28S ribosomal RNA (28S rRNA) and mitochondrial Tyrosine Kinase (X-src) (TyrK)) allowed phylogenetic trees to be made which demonstrated clear relationships within Micropanchax and Lacustricola of southern Africa and the out-groups used in the analysis. The inference trees showed that Micropanchax johnstoni was represented by three clades and was shown to be polyphyletic while Micropanchax hutereaui, which was represented by two clades, and Micropanchax katangae, which was represented by one clade, were monophyletic. The two species of Lacustricola, L. myaposae and L. macrurus showed very close relationships to Micropanchax and it is suggested that these two species be transferred to Micropanchax, leaving Lacustricola to only be found in central Africa.
- Full Text:
- Authors: Van Zeeventer, Ryan Matthew
- Date: 2020
- Subjects: Cyprinodontiformes -- Africa, Southern , Killifishes -- Africa, Southern
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145559 , vital:38449
- Description: Taxonomy is a tool that plays a crucial role in the implementation of effective management assessments, the ability to identify different fish species and their life stages, and create effective conservation strategies. Recent years have seen an increase in the discovery of cryptic species which could have an effect on how we implement conservation strategies, increasing the importance of accurate taxonomic assessments. The southern African topminnows of the genera Micropanchax and Lacustricola (Family Procatopodidae) include several species of egg-laying fishes that live in the marginal areas of rivers and lakes throughout the river systems of southern Africa in separated and connected populations. It was recently discovered that these populations showed subtle differences in their morphology and colouration, which led to the belief that new and cryptic species may be present. Previous studies of the Procatopodidae relied heavily on phylogenetic analyses that drew on morphological traits without reliance on molecular methods of analysis. A few of the taxonomic issues were resolved through these limited studies but ultimately the taxonomic status of the Procatopodidae remained poorly understood. Furthermore, these studies had not been updated in recent years and hence the benefits of new technology had not been brought to bear on the issues. A multi-gene analysis using standard phylogenetic methods and five molecular markers (mitochondrial cytochrome C oxidase (COI), mitochondrial cytochrome b (cytb), mitochondrial 16S ribosomal RNA (16S rRNA), nuclear 28S ribosomal RNA (28S rRNA) and mitochondrial Tyrosine Kinase (X-src) (TyrK)) allowed phylogenetic trees to be made which demonstrated clear relationships within Micropanchax and Lacustricola of southern Africa and the out-groups used in the analysis. The inference trees showed that Micropanchax johnstoni was represented by three clades and was shown to be polyphyletic while Micropanchax hutereaui, which was represented by two clades, and Micropanchax katangae, which was represented by one clade, were monophyletic. The two species of Lacustricola, L. myaposae and L. macrurus showed very close relationships to Micropanchax and it is suggested that these two species be transferred to Micropanchax, leaving Lacustricola to only be found in central Africa.
- Full Text:
The workload of flight attendants during short-haul flight operations: a system analysis
- Authors: Bennett, Chloe Kayla
- Date: 2020
- Subjects: Flight attendants -- Health and hygiene , Employees -- South Africa -- Workload
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/123431 , vital:35437
- Description: Background and aim: Flight attendants forms a significant part in 24-hour aviation industry. Flight attendant fatigue is a significant problem in the aviation industry as it continues to jeopardize the ability to fulfil important safety and security roles which is critical in performance duty of a flight attendant. However, little attention has been accomplished to the workload, working conditions and fatigue of flight attendants crew in transport aircraft. In addition, there is currently less research that have also embraced the problematic of smaller regional/commercial operation (short-haul flight operations) inducing fatigue among short-haul flight attendants as the nature of this operation are often characterised with high productivity expectations in a demanding environment with high time pressures resulting in high workloads and fatigue. Thus, flight attendant fatigue and workload is a worldwide challenge in this operational environment and less attention has been given to the determining factors. Therefore, the aim of the study was to determine the workload factors contributing to flight attendant fatigue during short-haul flight operations. Methods (System analysis): To achieve the research aim, the work system analysis, based on the Smith and Carayon-Sainfort model was chosen as the main research approach which was conducted in two ways; based on existing literature and secondly based on expert interviews. This method provided a systemic aspect to understand the whole work system of flight attendants work during short-haul operations in order to identify all the contributing factors to flight attendant fatigue and workload. Results: The literature analysis and the data from the expert interviews highlighted significant findings to flight attendant fatigue and workload. The reasons for flight attendant fatigue operating short-haul flights can be found at organizational, task, individual, environmental levels and tools and technologies and due to the interaction of the factors. The main factors of flight attendants’ fatigue are thought primarily as a function of scheduling due to irregular, mixed schedules with early starts and late finishes, extended duty days (long working hours), as well as high workload, due to the short turnaround flights, the number of sectors flown in a single duty period and duty length and high jobs demands. In addition, flight duty and rest regulations, confined work space in the cabin, vibrations, noise and lighting, sleeping in an unfamiliar environment, family responsibilities all add to additional stress placed on the body which can influence workload and sleep and consequently influencing fatigue. Conclusion: Overall the study determined that flight attendant fatigue is a significant problem in modern industry of short-haul operations. Using this systematic approach (work system analysis based on the framework of the work system model developed by Smith and Carayon-Sainfort (1989) allowed for an accurate representation of the complexity of flight attendant work environment in short-haul aviation industries, thus contributed to an increased understanding of fatigue and risk factors that span the entire work system and aid in identifying the patterns in combination of work system variables that are associated with increased risk to flight attendant fatigue. Overall flight attendant fatigue is a product of interactions with the short-haul environment. It can have a negative impact on safety, performance and well-being. Therefore, it needs to be managed and dealt with in the near future.
- Full Text:
- Authors: Bennett, Chloe Kayla
- Date: 2020
- Subjects: Flight attendants -- Health and hygiene , Employees -- South Africa -- Workload
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/123431 , vital:35437
- Description: Background and aim: Flight attendants forms a significant part in 24-hour aviation industry. Flight attendant fatigue is a significant problem in the aviation industry as it continues to jeopardize the ability to fulfil important safety and security roles which is critical in performance duty of a flight attendant. However, little attention has been accomplished to the workload, working conditions and fatigue of flight attendants crew in transport aircraft. In addition, there is currently less research that have also embraced the problematic of smaller regional/commercial operation (short-haul flight operations) inducing fatigue among short-haul flight attendants as the nature of this operation are often characterised with high productivity expectations in a demanding environment with high time pressures resulting in high workloads and fatigue. Thus, flight attendant fatigue and workload is a worldwide challenge in this operational environment and less attention has been given to the determining factors. Therefore, the aim of the study was to determine the workload factors contributing to flight attendant fatigue during short-haul flight operations. Methods (System analysis): To achieve the research aim, the work system analysis, based on the Smith and Carayon-Sainfort model was chosen as the main research approach which was conducted in two ways; based on existing literature and secondly based on expert interviews. This method provided a systemic aspect to understand the whole work system of flight attendants work during short-haul operations in order to identify all the contributing factors to flight attendant fatigue and workload. Results: The literature analysis and the data from the expert interviews highlighted significant findings to flight attendant fatigue and workload. The reasons for flight attendant fatigue operating short-haul flights can be found at organizational, task, individual, environmental levels and tools and technologies and due to the interaction of the factors. The main factors of flight attendants’ fatigue are thought primarily as a function of scheduling due to irregular, mixed schedules with early starts and late finishes, extended duty days (long working hours), as well as high workload, due to the short turnaround flights, the number of sectors flown in a single duty period and duty length and high jobs demands. In addition, flight duty and rest regulations, confined work space in the cabin, vibrations, noise and lighting, sleeping in an unfamiliar environment, family responsibilities all add to additional stress placed on the body which can influence workload and sleep and consequently influencing fatigue. Conclusion: Overall the study determined that flight attendant fatigue is a significant problem in modern industry of short-haul operations. Using this systematic approach (work system analysis based on the framework of the work system model developed by Smith and Carayon-Sainfort (1989) allowed for an accurate representation of the complexity of flight attendant work environment in short-haul aviation industries, thus contributed to an increased understanding of fatigue and risk factors that span the entire work system and aid in identifying the patterns in combination of work system variables that are associated with increased risk to flight attendant fatigue. Overall flight attendant fatigue is a product of interactions with the short-haul environment. It can have a negative impact on safety, performance and well-being. Therefore, it needs to be managed and dealt with in the near future.
- Full Text:
Towards a capability maturity model for a cyber range
- Authors: Aschmann, Michael Joseph
- Date: 2020
- Subjects: Computer software -- Development , Computer security
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163142 , vital:41013
- Description: This work describes research undertaken towards the development of a Capability Maturity Model (CMM) for Cyber Ranges (CRs) focused on cyber security. Global cyber security needs are on the rise, and the need for attribution within the cyber domain is of particular concern. This has prompted major efforts to enhance cyber capabilities within organisations to increase their total cyber resilience posture. These efforts include, but are not limited to, the testing of computational devices, networks, and applications, and cyber skills training focused on prevention, detection and cyber attack response. A cyber range allows for the testing of the computational environment. By developing cyber events within a confined virtual or sand-boxed cyber environment, a cyber range can prepare the next generation of cyber security specialists to handle a variety of potential cyber attacks. Cyber ranges have different purposes, each designed to fulfil a different computational testing and cyber training goal; consequently, cyber ranges can vary greatly in the level of variety, capability, maturity and complexity. As cyber ranges proliferate and become more and more valued as tools for cyber security, a method to classify or rate them becomes essential. Yet while a universal criteria for measuring cyber ranges in terms of their capability maturity levels becomes more critical, there are currently very limited resources for researchers aiming to perform this kind of work. For this reason, this work proposes and describes a CMM, designed to give organisations the ability to benchmark the capability maturity of a given cyber range. This research adopted a synthesised approach to the development of a CMM, grounded in prior research and focused on the production of a conceptual model that provides a useful level of abstraction. In order to achieve this goal, the core capability elements of a cyber range are defined with their relative importance, allowing for the development of a proposed classification cyber range levels. An analysis of data gathered during the course of an expert review, together with other research, further supported the development of the conceptual model. In the context of cyber range capability, classification will include the ability of the cyber range to perform its functions optimally with different core capability elements, focusing on the Measurement of Capability (MoC) with its elements, namely effect, performance and threat ability. Cyber range maturity can evolve over time and can be defined through the Measurement of Maturity (MoM) with its elements, namely people, processes, technology. The combination of these measurements utilising the CMM for a CR determines the capability maturity level of a CR. The primary outcome of this research is the proposed level-based CMM framework for a cyber range, developed using adopted and synthesised CMMs, the analysis of an expert review, and the mapping of the results.
- Full Text:
- Authors: Aschmann, Michael Joseph
- Date: 2020
- Subjects: Computer software -- Development , Computer security
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163142 , vital:41013
- Description: This work describes research undertaken towards the development of a Capability Maturity Model (CMM) for Cyber Ranges (CRs) focused on cyber security. Global cyber security needs are on the rise, and the need for attribution within the cyber domain is of particular concern. This has prompted major efforts to enhance cyber capabilities within organisations to increase their total cyber resilience posture. These efforts include, but are not limited to, the testing of computational devices, networks, and applications, and cyber skills training focused on prevention, detection and cyber attack response. A cyber range allows for the testing of the computational environment. By developing cyber events within a confined virtual or sand-boxed cyber environment, a cyber range can prepare the next generation of cyber security specialists to handle a variety of potential cyber attacks. Cyber ranges have different purposes, each designed to fulfil a different computational testing and cyber training goal; consequently, cyber ranges can vary greatly in the level of variety, capability, maturity and complexity. As cyber ranges proliferate and become more and more valued as tools for cyber security, a method to classify or rate them becomes essential. Yet while a universal criteria for measuring cyber ranges in terms of their capability maturity levels becomes more critical, there are currently very limited resources for researchers aiming to perform this kind of work. For this reason, this work proposes and describes a CMM, designed to give organisations the ability to benchmark the capability maturity of a given cyber range. This research adopted a synthesised approach to the development of a CMM, grounded in prior research and focused on the production of a conceptual model that provides a useful level of abstraction. In order to achieve this goal, the core capability elements of a cyber range are defined with their relative importance, allowing for the development of a proposed classification cyber range levels. An analysis of data gathered during the course of an expert review, together with other research, further supported the development of the conceptual model. In the context of cyber range capability, classification will include the ability of the cyber range to perform its functions optimally with different core capability elements, focusing on the Measurement of Capability (MoC) with its elements, namely effect, performance and threat ability. Cyber range maturity can evolve over time and can be defined through the Measurement of Maturity (MoM) with its elements, namely people, processes, technology. The combination of these measurements utilising the CMM for a CR determines the capability maturity level of a CR. The primary outcome of this research is the proposed level-based CMM framework for a cyber range, developed using adopted and synthesised CMMs, the analysis of an expert review, and the mapping of the results.
- Full Text:
Towards the development of a bio-fertiliser using mixed liquor from high rate algal oxidation ponds
- Authors: Masudi, Wiya Léon
- Date: 2020
- Subjects: Biofertilizers , Microalgae - Biotechnology , Algae -- Culture
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142969 , vital:38181
- Description: Mixed liquor includes consortia of microalgae and bacteria produced in high rate algal oxidation ponds (HRAOPs). The consortia of microalgae and bacteria occur as flocs called microalgae-bacterial flocs (MaB-flocs). This study aimed to source bacteria from MaB-flocs generated in HRAOPs and, after isolation and identification, evaluate their potential as plant growth promoting (PGP) microorganisms. Twelve bacterial strains namely ECCN 1b, ECCN 2b, ECCN 3b, ECCN 4b, ECCN 5b, ECCN 6b, ECCN 7b, ECCN 8b, ECCN 9b, ECCN 10b, ECCN 11b, and ECCN 12b were successfully isolated and their molecular identity established using amplified 16S rRNA gene sequence analysis that was compared to sequences deposited in the NCBI gene database. Blast analysis identified these isolates at the genus level as Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter strain ECCN 7b, Exiguobacterium strain ECCN 8b, Microbacterium strain ECCN 9b, Pseudomonas ECCN strain 10b, Ancylobacter strain ECCN 11b and Microbacterium strain ECCN 12b. These isolates were able to grow in nutrient broth in a pH range between 6 and 10, with the best growth achieved at pH 8 to 9. The results on the use of carbon substrate revealed that 5 strains including Arthrobacter strain ECCN 6b, Aeromonas strain ECCN 4b, Pseudomonas strain ECCN 10b, Enterobacter strain ECCN 7b and Bacillus strain ECCN 3b were capable of using glucose, sucrose and mannitol. No faecal coliforms were found. However, of the 12 isolates screened for bio-fertilisation potential, Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter ECCN strain 7b, Exiguobacterium strain ECCN 8b and Pseudomonas strain ECCN 10b showed multifunctional plant growth promoting (PGP) potential. The potential for PGP included the production of ammonium-N, solubilisation of phosphate-P and potassium-K, oxidation of Mn and production of auxin, indole-3-acetic acid (IAA). Results are discussed in terms of the amount or concentration (mg L-1) of plant essential nutrient and growth regulator produced by these isolated bacteria. Even so, further studies are needed to test and confirm the bio-fertiliser and plant growth promoting activity of these strains in pot trials and field experiments, or both.
- Full Text:
- Authors: Masudi, Wiya Léon
- Date: 2020
- Subjects: Biofertilizers , Microalgae - Biotechnology , Algae -- Culture
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142969 , vital:38181
- Description: Mixed liquor includes consortia of microalgae and bacteria produced in high rate algal oxidation ponds (HRAOPs). The consortia of microalgae and bacteria occur as flocs called microalgae-bacterial flocs (MaB-flocs). This study aimed to source bacteria from MaB-flocs generated in HRAOPs and, after isolation and identification, evaluate their potential as plant growth promoting (PGP) microorganisms. Twelve bacterial strains namely ECCN 1b, ECCN 2b, ECCN 3b, ECCN 4b, ECCN 5b, ECCN 6b, ECCN 7b, ECCN 8b, ECCN 9b, ECCN 10b, ECCN 11b, and ECCN 12b were successfully isolated and their molecular identity established using amplified 16S rRNA gene sequence analysis that was compared to sequences deposited in the NCBI gene database. Blast analysis identified these isolates at the genus level as Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter strain ECCN 7b, Exiguobacterium strain ECCN 8b, Microbacterium strain ECCN 9b, Pseudomonas ECCN strain 10b, Ancylobacter strain ECCN 11b and Microbacterium strain ECCN 12b. These isolates were able to grow in nutrient broth in a pH range between 6 and 10, with the best growth achieved at pH 8 to 9. The results on the use of carbon substrate revealed that 5 strains including Arthrobacter strain ECCN 6b, Aeromonas strain ECCN 4b, Pseudomonas strain ECCN 10b, Enterobacter strain ECCN 7b and Bacillus strain ECCN 3b were capable of using glucose, sucrose and mannitol. No faecal coliforms were found. However, of the 12 isolates screened for bio-fertilisation potential, Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter ECCN strain 7b, Exiguobacterium strain ECCN 8b and Pseudomonas strain ECCN 10b showed multifunctional plant growth promoting (PGP) potential. The potential for PGP included the production of ammonium-N, solubilisation of phosphate-P and potassium-K, oxidation of Mn and production of auxin, indole-3-acetic acid (IAA). Results are discussed in terms of the amount or concentration (mg L-1) of plant essential nutrient and growth regulator produced by these isolated bacteria. Even so, further studies are needed to test and confirm the bio-fertiliser and plant growth promoting activity of these strains in pot trials and field experiments, or both.
- Full Text:
Towards understanding how exploitation influences the wild energetic response of marine fish to temperature variability
- Authors: Skeeles, Michael Richard
- Date: 2020
- Subjects: Chrysoblepus laticeps -- Climatic factors , Sparidae -- Genetics , Sparidae -- South Africa -- Climatic factors
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145133 , vital:38411
- Description: Exploitation of fish populations can exacerbate the effects of climate change, yet our understanding of their synergistic effects remains limited. As fish are increasingly exposed to temperatures on the edges of their optimal thermal performance window, their physiological response is expected to shape their future performance. It is therefore concerning that exploitation can select for specific physiological phenotypes, as this may affect fished populations’ physiological response to temperature change. A recent laboratory study revealed fewer high-performance metabolic-scope phenotypes in an exploited population of the marine Sparid Chrysoblepus laticeps across a range of experimental temperatures in comparison to an unexploited population. This suggested that individuals in exploited populations may have less available energy for aerobic performance at thermal extremes, which may reduce the resilience of the population to changes in temperature. However, since laboratory experiments exclude numerous other variables that fish encounter in the wild, it was necessary to test this finding in a natural setting. This thesis aimed to further develop the laboratory study by assessing whether exploitation effects the wild energetic response of C. laticeps to thermal variability. To achieve this, the field metabolic rate of C. laticeps, a resident and endemic South African fish, from a near-pristine population (Tsitsikamma National Park) and a heavily exploited population (Port Elizabeth) was compared using acoustic accelerometry. A laboratory-based study using a swim-tunnel respirometer and accelerometer transmitters was conducted to develop a model to predict metabolic rate from acceleration data at temperatures from 10 to 22⁰C. Acceleration, temperature, mass and population (exploited/unexploited) were found to be the best predictors of the metabolic rate of C. laticeps and were incorporated into the model to estimate the field metabolic rate of fish tagged with acoustic accelerometers in the wild. To examine the combined effects of temperature and exploitation on the field metabolic rate of C. laticeps in their natural state, two fine-scale telemetry arrays with temperature loggers were used to assess the acceleration of the fish across different temperatures in the wild for three months during a period of high thermal variability. Ten fish from the exploited and unexploited populations were caught, surgically implanted with accelerometer transmitters and released back into the wild. Close to 500 000 and 400 000 acceleration estimates were recorded from wild exploited and unexploited fish, respectively. The field metabolic rate of both populations was estimated by combining the field acceleration and temperature data with the laboratory calibration model. The field metabolic rate of C. laticeps from the exploited population was constrained near cold and warm extremes compared to no constraints observed in the unexploited population. This was attributed to reduced inter-individual variability in the field metabolic rate-temperature relationship within the exploited population. There appeared to be a greater proportion of individuals that maintained a high field metabolic rate at extreme temperatures in the unexploited population. In contrast, all but one fish from the exploited population did not maintain a high field metabolic rate at extreme temperatures. These findings aligned with the laboratory-based metabolic-scope study on both populations of C. laticeps and demonstrate that passive-fishing may be removing thermally tolerant individuals and rendering exploited populations less resilient to thermal change. These findings are discussed in the context of fisheries management and particularly on the role that marine protected areas could play in maintaining physiological diversity, and therefore the resilience of fish in the Anthropocene. This study highlights the importance of applied conservation physiology in understanding the consequences of fisheries-induced evolution in an increasingly variable climate.
- Full Text:
- Authors: Skeeles, Michael Richard
- Date: 2020
- Subjects: Chrysoblepus laticeps -- Climatic factors , Sparidae -- Genetics , Sparidae -- South Africa -- Climatic factors
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145133 , vital:38411
- Description: Exploitation of fish populations can exacerbate the effects of climate change, yet our understanding of their synergistic effects remains limited. As fish are increasingly exposed to temperatures on the edges of their optimal thermal performance window, their physiological response is expected to shape their future performance. It is therefore concerning that exploitation can select for specific physiological phenotypes, as this may affect fished populations’ physiological response to temperature change. A recent laboratory study revealed fewer high-performance metabolic-scope phenotypes in an exploited population of the marine Sparid Chrysoblepus laticeps across a range of experimental temperatures in comparison to an unexploited population. This suggested that individuals in exploited populations may have less available energy for aerobic performance at thermal extremes, which may reduce the resilience of the population to changes in temperature. However, since laboratory experiments exclude numerous other variables that fish encounter in the wild, it was necessary to test this finding in a natural setting. This thesis aimed to further develop the laboratory study by assessing whether exploitation effects the wild energetic response of C. laticeps to thermal variability. To achieve this, the field metabolic rate of C. laticeps, a resident and endemic South African fish, from a near-pristine population (Tsitsikamma National Park) and a heavily exploited population (Port Elizabeth) was compared using acoustic accelerometry. A laboratory-based study using a swim-tunnel respirometer and accelerometer transmitters was conducted to develop a model to predict metabolic rate from acceleration data at temperatures from 10 to 22⁰C. Acceleration, temperature, mass and population (exploited/unexploited) were found to be the best predictors of the metabolic rate of C. laticeps and were incorporated into the model to estimate the field metabolic rate of fish tagged with acoustic accelerometers in the wild. To examine the combined effects of temperature and exploitation on the field metabolic rate of C. laticeps in their natural state, two fine-scale telemetry arrays with temperature loggers were used to assess the acceleration of the fish across different temperatures in the wild for three months during a period of high thermal variability. Ten fish from the exploited and unexploited populations were caught, surgically implanted with accelerometer transmitters and released back into the wild. Close to 500 000 and 400 000 acceleration estimates were recorded from wild exploited and unexploited fish, respectively. The field metabolic rate of both populations was estimated by combining the field acceleration and temperature data with the laboratory calibration model. The field metabolic rate of C. laticeps from the exploited population was constrained near cold and warm extremes compared to no constraints observed in the unexploited population. This was attributed to reduced inter-individual variability in the field metabolic rate-temperature relationship within the exploited population. There appeared to be a greater proportion of individuals that maintained a high field metabolic rate at extreme temperatures in the unexploited population. In contrast, all but one fish from the exploited population did not maintain a high field metabolic rate at extreme temperatures. These findings aligned with the laboratory-based metabolic-scope study on both populations of C. laticeps and demonstrate that passive-fishing may be removing thermally tolerant individuals and rendering exploited populations less resilient to thermal change. These findings are discussed in the context of fisheries management and particularly on the role that marine protected areas could play in maintaining physiological diversity, and therefore the resilience of fish in the Anthropocene. This study highlights the importance of applied conservation physiology in understanding the consequences of fisheries-induced evolution in an increasingly variable climate.
- Full Text:
Understanding of the underlying resistance mechanism of the Kat-G protein against isoniazid in Mycobacterium tuberculosis using bioinformatics approaches
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
Unravelling the replication biology of Providence virus in a cell culturebased model system
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
Using a spatial resilience lens to understand alignments and misalignments in South Africa’s marine governance system
- Authors: Hardisty, Shannon
- Date: 2020
- Subjects: Marine parks and reserves -- South Africa , Marine parks and reserves -- South Africa -- Management , Marine parks and reserves -- Government policy -- South Africa , Table Mountain National Park (South Africa) , Spatial ecology -- South Africa , Coastal zone management -- South Africa
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164649 , vital:41152
- Description: In marine protected areas (MPAs), the radically open nature of marine ecosystems, the seemingly contradictory short-term goals of fisheries management (resource use) and conservation efforts (biodiversity protection), the variety of stakeholders, and the inherently political nature of space ha ve contributed to a disconnect between policy and practice. Many groups operate in an MPA at various spatial and socio-political scales, from national government bodies to local community groups, as such an MPA can be viewed as a complex, multi-scale and multi-level social-ecological system (SES). A greater understanding of the multi-scale processes driving the spatial resilience of SES can help address scale mismatches between continuous ecological change and the long-term governance of a seascape. South Africa has a long history of spaces and natural resources being politicised and faces high levels of unemployment and political frustration. Marine governance has been characterised as being siloed, lacking transparency regarding government processes and even between departments, and not properly addressing the needs of local communities; resulting in scale and level mismatches throughout the multi-scale processes (from national policies to local regulations)therefore, it is pertinent to understand the interactions between various national policies, their implementation, and their impacts. Using a spatial resilience lens, I set out to understand scale alignment in South Africa’s marine governance systems as it relates to MPAs. A spatial resilience lens emphasises analysis of both spatial and temporal scales, thus highlighting the multi-scale variables and interactions that occur within a system. I first sought to understand the policy and management across national and regional scales by conducting a series of semi-structured interviews with experts which were analysed using thematic analysis. I then approached the question of alignment from a local to national scale by using the Table Mountain National Park Marine Protected Area as a case study, here I carried out a series of workshops and interviews with line fishers, women’s groups, and eco-tourism operators from three different communities. Experts identified six themes when describing the structure of gove rnance in South African MPAs; they consisted of: government departments and marine legislation, management level factors, communication and information sharing, local level factors, biophysical factors, and scale. Communication and information sharing was the most mentioned referenced theme (40.26 %). The relationships between the communities and other actors (i.e. how communities are impacted, or impact on, the Department of Environmental Affairs , the Department of Agriculture Forestry and Fisheries, and Park Authorities) contributed 67.27 % to the total number of mentions, 91 % of which were from the following themes: communication and information sharing, local level factors, biophysical factors, and scale. My results indicate that weak, top-down relationships dominate the governance system in South Africa. During the Cape Town workshops I applied the spatial resilience lens in three ways. Firstly, the participants collaborated on a mapping exercise discussing the existing zone types within the MPA, as well as developing their own. There was an 87.87 % overlap of community designed zones on the existing MPA zones, however the types of zones designed by the community were different. Secondly, I carried out a document analysis and used information from the workshops to analyse the status and rights designation of important species for different user groups. The results show that the various stakeholder groups, while all active in the marine environment, interact with the ecosystem at different scales and with differing species. The differing interactions between groups result in challenges such as representation and complicated power dynamics between user groups. Finally, I asked participants to discuss factors that affect their ability to gain a livelihood from the sea. The external elements and their effects differed between the user groups, identifying important socio-political interactions. Whilst the external elements were all geographically distinct and localised, the spatial resilience lens allowed for a cross-scale, and to a certain extent cross-sector, understanding of these elements thus producing a more holistic understanding. My thesis shows that user groups operate on different spatial scales within the marine environment as a result of social, economic, and political influences leading to several scale-based challenges. The identified scale-based challenges have contributed to misalignments in marine governance in South Africa. My study shows that scale mismatches have important implications for understanding how marine policy influences user groups and identified pathways that affected policy implementation , and identifies future research application of a spatial resilience lens within social-ecological systems, for example it’s potential use in understanding the gendered nature of MPAs
- Full Text:
- Authors: Hardisty, Shannon
- Date: 2020
- Subjects: Marine parks and reserves -- South Africa , Marine parks and reserves -- South Africa -- Management , Marine parks and reserves -- Government policy -- South Africa , Table Mountain National Park (South Africa) , Spatial ecology -- South Africa , Coastal zone management -- South Africa
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164649 , vital:41152
- Description: In marine protected areas (MPAs), the radically open nature of marine ecosystems, the seemingly contradictory short-term goals of fisheries management (resource use) and conservation efforts (biodiversity protection), the variety of stakeholders, and the inherently political nature of space ha ve contributed to a disconnect between policy and practice. Many groups operate in an MPA at various spatial and socio-political scales, from national government bodies to local community groups, as such an MPA can be viewed as a complex, multi-scale and multi-level social-ecological system (SES). A greater understanding of the multi-scale processes driving the spatial resilience of SES can help address scale mismatches between continuous ecological change and the long-term governance of a seascape. South Africa has a long history of spaces and natural resources being politicised and faces high levels of unemployment and political frustration. Marine governance has been characterised as being siloed, lacking transparency regarding government processes and even between departments, and not properly addressing the needs of local communities; resulting in scale and level mismatches throughout the multi-scale processes (from national policies to local regulations)therefore, it is pertinent to understand the interactions between various national policies, their implementation, and their impacts. Using a spatial resilience lens, I set out to understand scale alignment in South Africa’s marine governance systems as it relates to MPAs. A spatial resilience lens emphasises analysis of both spatial and temporal scales, thus highlighting the multi-scale variables and interactions that occur within a system. I first sought to understand the policy and management across national and regional scales by conducting a series of semi-structured interviews with experts which were analysed using thematic analysis. I then approached the question of alignment from a local to national scale by using the Table Mountain National Park Marine Protected Area as a case study, here I carried out a series of workshops and interviews with line fishers, women’s groups, and eco-tourism operators from three different communities. Experts identified six themes when describing the structure of gove rnance in South African MPAs; they consisted of: government departments and marine legislation, management level factors, communication and information sharing, local level factors, biophysical factors, and scale. Communication and information sharing was the most mentioned referenced theme (40.26 %). The relationships between the communities and other actors (i.e. how communities are impacted, or impact on, the Department of Environmental Affairs , the Department of Agriculture Forestry and Fisheries, and Park Authorities) contributed 67.27 % to the total number of mentions, 91 % of which were from the following themes: communication and information sharing, local level factors, biophysical factors, and scale. My results indicate that weak, top-down relationships dominate the governance system in South Africa. During the Cape Town workshops I applied the spatial resilience lens in three ways. Firstly, the participants collaborated on a mapping exercise discussing the existing zone types within the MPA, as well as developing their own. There was an 87.87 % overlap of community designed zones on the existing MPA zones, however the types of zones designed by the community were different. Secondly, I carried out a document analysis and used information from the workshops to analyse the status and rights designation of important species for different user groups. The results show that the various stakeholder groups, while all active in the marine environment, interact with the ecosystem at different scales and with differing species. The differing interactions between groups result in challenges such as representation and complicated power dynamics between user groups. Finally, I asked participants to discuss factors that affect their ability to gain a livelihood from the sea. The external elements and their effects differed between the user groups, identifying important socio-political interactions. Whilst the external elements were all geographically distinct and localised, the spatial resilience lens allowed for a cross-scale, and to a certain extent cross-sector, understanding of these elements thus producing a more holistic understanding. My thesis shows that user groups operate on different spatial scales within the marine environment as a result of social, economic, and political influences leading to several scale-based challenges. The identified scale-based challenges have contributed to misalignments in marine governance in South Africa. My study shows that scale mismatches have important implications for understanding how marine policy influences user groups and identified pathways that affected policy implementation , and identifies future research application of a spatial resilience lens within social-ecological systems, for example it’s potential use in understanding the gendered nature of MPAs
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