Symmetry and asymmetry in electrocatalysis: enhancing the electrocatalytic activity of phthalocyanines through synergy with doped graphene quantum dots
- Nkhahle, Reitumetse Precious
- Authors: Nkhahle, Reitumetse Precious
- Date: 2020
- Subjects: Phthalocyanines , Quantum dots , Graphene
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/117585 , vital:34529
- Description: An exploration on the enhancement of the electrocatalytic activity of phthalocyanines (Pcs) through coupling with a series of graphene quantum dots (GQDs) is undertaken. The preliminary studies using symmetrical Pcs, a cobalt and an iron chloride tetra substituted diethylaminophenoxy Pc (complexes 1 and 2), for the electro-oxidation of nitrite revealed through the various sequential modifications that doped GQDs fare better than their pristine counterparts with respect to improving the electrocatalytic behaviour of Pcs, in particular, the nitrogen-doped GQDs (NGQDs). Following up on this, a series of asymmetric Pc complexes; 2,9,16-tris-(4-tert-butylphenoxy) mono carboxyphenoxy phthalocyanato cobalt (II) (3), 2,9,16-tris-(4-tert-butylphenoxy) mono aminophenoxy phthalocyanato cobalt (II) (4), 2,9,16-tris-(3-diethylamino)phenoxy) mono carboxyphenoxy phthalocyanato cobalt (II) (5) and 2,9,16-tris-(3-diethylamino)phenoxy) mono aminophenoxy phthalocyanato cobalt (II) (6) was prepared in which push-pull systems were compared to other asymmetric complexes that lack this effect towards the electrocatalytic sensing of hydrazine. All asymmetric complexes (3-6) were π-stacked to the NGQDs while those with an NH2 group (4 and 6), were also covalently linked to the NGQDs. These complexes and their corresponding conjugates were characterized accordingly and applied as electrocatalysts in the oxidation of hydrazine. The electrochemical studies revealed that π π stacking yields better responses (higher sensitivities and lower limits of detection) than covalent linking because there are less forces acting on the graphene network. Covalent linking introduces both tensile and compressive forces which in turn results in an increase in the ID/IG ratio and that is unfavourable for electrocatalysis. In comparing the electrodes composed of the π-stacked conjugates to those altered through sequential modifications, despite the conditions not being the same, it can be inferred that the magnitude of the electrostatic forces between the Pcs and the GQDs also plays a significant role in electrocatalysis. The π-stacked conjugates, owing to the manner in which they were prepared, have stronger electrostatic forces acting between the Pc and GQDs hence they were able to elicit a better electrochemical response than the sequentially modified electrodes. In addition to that, it appears that asymmetric Pcs are better electrocatalysts in comparison to the symmetric Pcs.
- Full Text:
- Authors: Nkhahle, Reitumetse Precious
- Date: 2020
- Subjects: Phthalocyanines , Quantum dots , Graphene
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/117585 , vital:34529
- Description: An exploration on the enhancement of the electrocatalytic activity of phthalocyanines (Pcs) through coupling with a series of graphene quantum dots (GQDs) is undertaken. The preliminary studies using symmetrical Pcs, a cobalt and an iron chloride tetra substituted diethylaminophenoxy Pc (complexes 1 and 2), for the electro-oxidation of nitrite revealed through the various sequential modifications that doped GQDs fare better than their pristine counterparts with respect to improving the electrocatalytic behaviour of Pcs, in particular, the nitrogen-doped GQDs (NGQDs). Following up on this, a series of asymmetric Pc complexes; 2,9,16-tris-(4-tert-butylphenoxy) mono carboxyphenoxy phthalocyanato cobalt (II) (3), 2,9,16-tris-(4-tert-butylphenoxy) mono aminophenoxy phthalocyanato cobalt (II) (4), 2,9,16-tris-(3-diethylamino)phenoxy) mono carboxyphenoxy phthalocyanato cobalt (II) (5) and 2,9,16-tris-(3-diethylamino)phenoxy) mono aminophenoxy phthalocyanato cobalt (II) (6) was prepared in which push-pull systems were compared to other asymmetric complexes that lack this effect towards the electrocatalytic sensing of hydrazine. All asymmetric complexes (3-6) were π-stacked to the NGQDs while those with an NH2 group (4 and 6), were also covalently linked to the NGQDs. These complexes and their corresponding conjugates were characterized accordingly and applied as electrocatalysts in the oxidation of hydrazine. The electrochemical studies revealed that π π stacking yields better responses (higher sensitivities and lower limits of detection) than covalent linking because there are less forces acting on the graphene network. Covalent linking introduces both tensile and compressive forces which in turn results in an increase in the ID/IG ratio and that is unfavourable for electrocatalysis. In comparing the electrodes composed of the π-stacked conjugates to those altered through sequential modifications, despite the conditions not being the same, it can be inferred that the magnitude of the electrostatic forces between the Pcs and the GQDs also plays a significant role in electrocatalysis. The π-stacked conjugates, owing to the manner in which they were prepared, have stronger electrostatic forces acting between the Pc and GQDs hence they were able to elicit a better electrochemical response than the sequentially modified electrodes. In addition to that, it appears that asymmetric Pcs are better electrocatalysts in comparison to the symmetric Pcs.
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Synthesis, photophysicochemical properties and photodynamic therapy activities of indium and zinc phthalocyanines when incorporated into Pluronic polymer micelles
- Authors: Motloung, Banele Mike
- Date: 2020
- Subjects: Indium , Zinc , Phthalocyanines , Polymers , Photochemotherapy , Micelles
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167529 , vital:41489
- Description: This thesis reports on the syntheses, photophysicochemical properties and photodynamic therapy activities of symmetrical metallophthalocyanines (MPcs) when alone or when incorporated into Pluronic polymer micelles. The Pcs contain either zinc or indium as central metals and have phenyldiazenylphenoxy, pyridine-2-yloxy and benzo[d]thiazol-2-ylthio as ring substituents. Spectroscopic and microscopic techniques were used to confirm the formation MPcs with micelles. The photophysics and photochemistry of the Pcs were assessed when alone and with micelles. All the studied Pcs showed good photophysicochemical behavior with relatively high triplet and singlet oxygen quantum yields corresponding to their low fluorescence quantum yields. The Pcs with indium in their central cavity exhibited higher triplet and singlet oxygen quantum yields in comparison to their zinc counterparts due to the heavy atom effect obtained from the former. The in vitro dark cytotoxicity and photodynamic therapy of the Pc complexes and conjugates against MCF7 cells was tested. All studied Pc complexes alone and with micelles showed minimum dark toxicity making them applicable for PDT. All complexes displayed good phototoxicity < 50% cell viability (except for complex 2 > 50% cell viability) at concentrations ≤100 μg/mL, however the conjugates showed < 45% cell viability at concentrations ≤ 100 μg/mL, probably due to the small micellar size and EPR effect. The findings from this work show the importance of incorporating photosensitizers such as phthalocyanines into Pluronic polymers micelles and making them water soluble and ultimately improving their photodynamic effect.
- Full Text:
- Authors: Motloung, Banele Mike
- Date: 2020
- Subjects: Indium , Zinc , Phthalocyanines , Polymers , Photochemotherapy , Micelles
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167529 , vital:41489
- Description: This thesis reports on the syntheses, photophysicochemical properties and photodynamic therapy activities of symmetrical metallophthalocyanines (MPcs) when alone or when incorporated into Pluronic polymer micelles. The Pcs contain either zinc or indium as central metals and have phenyldiazenylphenoxy, pyridine-2-yloxy and benzo[d]thiazol-2-ylthio as ring substituents. Spectroscopic and microscopic techniques were used to confirm the formation MPcs with micelles. The photophysics and photochemistry of the Pcs were assessed when alone and with micelles. All the studied Pcs showed good photophysicochemical behavior with relatively high triplet and singlet oxygen quantum yields corresponding to their low fluorescence quantum yields. The Pcs with indium in their central cavity exhibited higher triplet and singlet oxygen quantum yields in comparison to their zinc counterparts due to the heavy atom effect obtained from the former. The in vitro dark cytotoxicity and photodynamic therapy of the Pc complexes and conjugates against MCF7 cells was tested. All studied Pc complexes alone and with micelles showed minimum dark toxicity making them applicable for PDT. All complexes displayed good phototoxicity < 50% cell viability (except for complex 2 > 50% cell viability) at concentrations ≤100 μg/mL, however the conjugates showed < 45% cell viability at concentrations ≤ 100 μg/mL, probably due to the small micellar size and EPR effect. The findings from this work show the importance of incorporating photosensitizers such as phthalocyanines into Pluronic polymers micelles and making them water soluble and ultimately improving their photodynamic effect.
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System analysis of fatigue in pilots and co-pilots executing short-hall flight operations
- Authors: Bennett, Cleo Taylor
- Date: 2020
- Subjects: Air pilots -- Health and hygiene , Fatigue , Work environment -- Psychological aspects , Work environment -- Physiological aspects
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146622 , vital:38543
- Description: Background: This study was conducted as part of Denel’s South African Regional Aircraft (SARA) development project. Regional aircraft have a maximum flight time of 60 minutes. Hence, the study focuses on matters pertaining to the short-haul flight context. Pilot fatigue has been recognised as a safety concern in the aviation industry. It impacts on pilot performance across the board, not least in the short-haul context. However, the specific factors that lead to pilot fatigue in short-haul operations have not been well researched. Research Aim: To identify and examine the factors which influence pilot/co-pilot fatigue in short-haul aviation contexts. Method: Fatigue is multifaceted, and has multiple definitions and descriptions. It is acknowledged as a complex phenomenon, the development of which is dynamically influenced by various factors. Thus, a systems approach based on the work system model by Smith and Carayon-Sainfort (1989) was adopted for this study. A systems analysis was conducted in two parts: 1) a literature analysis, and 2) expert interviews. Results: Both the literature analysis and the interviews indicated that pilot fatigue in short-haul flight operations represent composite system outcomes influenced by various factors. The factors identified were structured (systematised) into categories, namely organizational factors, task-related factors, environmental factors, factors linked to technology and tools, and non-work-related factors specific to the individual pilot. An example of a task-related factor would be the performance by pilots of multiple take-offs and landings; organizational factors include work time arrangements and duty scheduling (e.g. unpredictable schedule, early starts/late finishes, number of flight sectors in a shift, extended working hours, numerous consecutive work days, standby duties, flight, duty and rest limitations (regulations and guidelines); and short turnaround periods); environmental factors might include the small pressurised cockpit environment, movement restriction, very low humidity, low air pressure, vibrations, high noise levels, low light intensity light, and inclement weather); there are many examples of how tools and technology utilized by pilots might affect their fatigue levels; and finally, pilot-specific non-work-related factors would include things like the pilot’s age, health (lifestyle), family stress, work experience and sleep environment. All of these factors were identified during the literature analysis and have a significant bearing on how fatigue could present in short-haul pilots/co-pilots. Other important fatigue-related factors revealed during the expert interviews included, organizational culture, time management, health implications of fatigue, and management of fatigue. Conclusions: Pilot fatigue is a complex and multi-factorial physiological condition. There are many interacting components which contribute to pilot fatigue in short-haul operations. These should be viewed from an integrated perspective and holistic, systems-based approaches should be taken to manage these issues, particularly in the context of short-haul operations. This would optimize pilot performance and well-being and, most importantly, improve the safety of the work environment to enhance overall operation safety. Limitations: The study does not quantify the contributions made to pilot fatigue by the various factors explored. Therefore, care needs to be taken when designing and implementing interventions based on this research.
- Full Text:
- Authors: Bennett, Cleo Taylor
- Date: 2020
- Subjects: Air pilots -- Health and hygiene , Fatigue , Work environment -- Psychological aspects , Work environment -- Physiological aspects
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146622 , vital:38543
- Description: Background: This study was conducted as part of Denel’s South African Regional Aircraft (SARA) development project. Regional aircraft have a maximum flight time of 60 minutes. Hence, the study focuses on matters pertaining to the short-haul flight context. Pilot fatigue has been recognised as a safety concern in the aviation industry. It impacts on pilot performance across the board, not least in the short-haul context. However, the specific factors that lead to pilot fatigue in short-haul operations have not been well researched. Research Aim: To identify and examine the factors which influence pilot/co-pilot fatigue in short-haul aviation contexts. Method: Fatigue is multifaceted, and has multiple definitions and descriptions. It is acknowledged as a complex phenomenon, the development of which is dynamically influenced by various factors. Thus, a systems approach based on the work system model by Smith and Carayon-Sainfort (1989) was adopted for this study. A systems analysis was conducted in two parts: 1) a literature analysis, and 2) expert interviews. Results: Both the literature analysis and the interviews indicated that pilot fatigue in short-haul flight operations represent composite system outcomes influenced by various factors. The factors identified were structured (systematised) into categories, namely organizational factors, task-related factors, environmental factors, factors linked to technology and tools, and non-work-related factors specific to the individual pilot. An example of a task-related factor would be the performance by pilots of multiple take-offs and landings; organizational factors include work time arrangements and duty scheduling (e.g. unpredictable schedule, early starts/late finishes, number of flight sectors in a shift, extended working hours, numerous consecutive work days, standby duties, flight, duty and rest limitations (regulations and guidelines); and short turnaround periods); environmental factors might include the small pressurised cockpit environment, movement restriction, very low humidity, low air pressure, vibrations, high noise levels, low light intensity light, and inclement weather); there are many examples of how tools and technology utilized by pilots might affect their fatigue levels; and finally, pilot-specific non-work-related factors would include things like the pilot’s age, health (lifestyle), family stress, work experience and sleep environment. All of these factors were identified during the literature analysis and have a significant bearing on how fatigue could present in short-haul pilots/co-pilots. Other important fatigue-related factors revealed during the expert interviews included, organizational culture, time management, health implications of fatigue, and management of fatigue. Conclusions: Pilot fatigue is a complex and multi-factorial physiological condition. There are many interacting components which contribute to pilot fatigue in short-haul operations. These should be viewed from an integrated perspective and holistic, systems-based approaches should be taken to manage these issues, particularly in the context of short-haul operations. This would optimize pilot performance and well-being and, most importantly, improve the safety of the work environment to enhance overall operation safety. Limitations: The study does not quantify the contributions made to pilot fatigue by the various factors explored. Therefore, care needs to be taken when designing and implementing interventions based on this research.
- Full Text:
Taxonomic revision of the Natal mountain catfish, Amphilius natalensis (Siluriformes, Amphiliidae) in southern Africa
- Mazungula, Daniel Nkosinathi
- Authors: Mazungula, Daniel Nkosinathi
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164490 , vital:41123
- Description: Thesis (MSc)--Rhodes University, Department of Ichthyology and Fisheries Science, 2020
- Full Text:
- Authors: Mazungula, Daniel Nkosinathi
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164490 , vital:41123
- Description: Thesis (MSc)--Rhodes University, Department of Ichthyology and Fisheries Science, 2020
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Technology in conservation: towards a system for in-field drone detection of invasive vegetation
- James, Katherine Margaret Frances
- Authors: James, Katherine Margaret Frances
- Date: 2020
- Subjects: Drone aircraft in remote sensing , Neural networks (Computer science) , Drone aircraft in remote sensing -- Case studies , Machine learning , Computer vision , Environmental monitoring -- Remote sensing , Invasive plants -- Monitoring
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143408 , vital:38244
- Description: Remote sensing can assist in monitoring the spread of invasive vegetation. The adoption of camera-carrying unmanned aerial vehicles, commonly referred to as drones, as remote sensing tools has yielded images of higher spatial resolution than traditional techniques. Drones also have the potential to interact with the environment through the delivery of bio-control or herbicide, as seen with their adoption in precision agriculture. Unlike in agricultural applications, however, invasive plants do not have a predictable position relative to each other within the environment. To facilitate the adoption of drones as an environmental monitoring and management tool, drones need to be able to intelligently distinguish between invasive and non-invasive vegetation on the fly. In this thesis, we present the augmentation of a commercially available drone with a deep machine learning model to investigate the viability of differentiating between an invasive shrub and other vegetation. As a case study, this was applied to the shrub genus Hakea, originating in Australia and invasive in several countries including South Africa. However, for this research, the methodology is important, rather than the chosen target plant. A dataset was collected using the available drone and manually annotated to facilitate the supervised training of the model. Two approaches were explored, namely, classification and semantic segmentation. For each of these, several models were trained and evaluated to find the optimal one. The chosen model was then interfaced with the drone via an Android application on a mobile device and its performance was preliminarily evaluated in the field. Based on these findings, refinements were made and thereafter a thorough field evaluation was performed to determine the best conditions for model operation. Results from the classification task show that deep learning models are capable of distinguishing between target and other shrubs in ideal candidate windows. However, classification in this manner is restricted by the proposal of such candidate windows. End-to-end image segmentation using deep learning overcomes this problem, classifying the image in a pixel-wise manner. Furthermore, the use of appropriate loss functions was found to improve model performance. Field tests show that illumination and shadow pose challenges to the model, but that good recall can be achieved when the conditions are ideal. False positive detection remains an issue that could be improved. This approach shows the potential for drones as an environmental monitoring and management tool when coupled with deep machine learning techniques and outlines potential problems that may be encountered.
- Full Text:
- Authors: James, Katherine Margaret Frances
- Date: 2020
- Subjects: Drone aircraft in remote sensing , Neural networks (Computer science) , Drone aircraft in remote sensing -- Case studies , Machine learning , Computer vision , Environmental monitoring -- Remote sensing , Invasive plants -- Monitoring
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143408 , vital:38244
- Description: Remote sensing can assist in monitoring the spread of invasive vegetation. The adoption of camera-carrying unmanned aerial vehicles, commonly referred to as drones, as remote sensing tools has yielded images of higher spatial resolution than traditional techniques. Drones also have the potential to interact with the environment through the delivery of bio-control or herbicide, as seen with their adoption in precision agriculture. Unlike in agricultural applications, however, invasive plants do not have a predictable position relative to each other within the environment. To facilitate the adoption of drones as an environmental monitoring and management tool, drones need to be able to intelligently distinguish between invasive and non-invasive vegetation on the fly. In this thesis, we present the augmentation of a commercially available drone with a deep machine learning model to investigate the viability of differentiating between an invasive shrub and other vegetation. As a case study, this was applied to the shrub genus Hakea, originating in Australia and invasive in several countries including South Africa. However, for this research, the methodology is important, rather than the chosen target plant. A dataset was collected using the available drone and manually annotated to facilitate the supervised training of the model. Two approaches were explored, namely, classification and semantic segmentation. For each of these, several models were trained and evaluated to find the optimal one. The chosen model was then interfaced with the drone via an Android application on a mobile device and its performance was preliminarily evaluated in the field. Based on these findings, refinements were made and thereafter a thorough field evaluation was performed to determine the best conditions for model operation. Results from the classification task show that deep learning models are capable of distinguishing between target and other shrubs in ideal candidate windows. However, classification in this manner is restricted by the proposal of such candidate windows. End-to-end image segmentation using deep learning overcomes this problem, classifying the image in a pixel-wise manner. Furthermore, the use of appropriate loss functions was found to improve model performance. Field tests show that illumination and shadow pose challenges to the model, but that good recall can be achieved when the conditions are ideal. False positive detection remains an issue that could be improved. This approach shows the potential for drones as an environmental monitoring and management tool when coupled with deep machine learning techniques and outlines potential problems that may be encountered.
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The effect of age and maturation on anthropometric characteristics and physical abilities of youth South African footballers
- Authors: De Beer, Ashley
- Date: 2020
- Subjects: Soccer -- Physiologial aspects , Soccer players -- South Africa -- Makhanda -- Physiology , Soccer players -- South Africa -- Makhanda -- Psychophysiology , Makana Local Football Association (South Africa)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/141222 , vital:37954
- Description: Currently there is limited research investigating the football related abilities of youth South African individuals. Populations from North and South America, Europe and Asia have been extensively covered in terms of their anthropometric characteristics, physical abilities, technical competency, tactical understanding as well as various personality traits. In describing these details, and especially how each is affected during the ageing and pubertal process, a more informed understanding of the talent development systems is created. A holistic approach to talent development is required to effectively and efficiently produce elite level footballers. An important consideration is the unique socio-economic environment many youth footballers experience which may impact on the talent development process. The present study therefore sought to quantify the anthropometric characteristics and the physical ability level of youth South African footballers from Local Football Association in Makhanda in the Sarah Baartman Region of the Eastern Cape province. In order to achieve this a two-factorial design was used with age and maturity status as independent variables. Anthropometric characteristics were tested by finding the height, weight, body mass index and total fat percentage of participants. Physical ability was determined by results for aerobic capacity, power, acceleration, speed and agility. All tests were football specific and had been widely used in a variety of footballing studies. Additional demographic and socio-economic information was also recorded. A total of 136 participants were placed in their respective Under 11, 13, 15 or Under 19 age groups, while the maturity status of 96 participants were stratified into five distinct categories. Statistical analyses was conducted using p-value significance, with appropriate Tukey post-hoc tests administered when necessary, while effect sizes were calculated using Cohen’s d. Significant changes and practical effect sizes were present for nearly all dependent variables when either stratified by age or by maturity. Total fat percentage was the only dependent variable which was not influenced by either age or maturity. Age and maturity effect sizes for total body fat percentage indicated practical changes present which did not occur during statistical analysis. The present results indicate poor values when compared to normative data or that of other football playing study populations. Further longitudinal research is required to better understand individual growth rates for youth South African footballers, particularly from the Makana region. Implications of the thesis may determine long term development pathways, while the delayed maturity rate is a critical finding which needs to be further researched.
- Full Text:
- Authors: De Beer, Ashley
- Date: 2020
- Subjects: Soccer -- Physiologial aspects , Soccer players -- South Africa -- Makhanda -- Physiology , Soccer players -- South Africa -- Makhanda -- Psychophysiology , Makana Local Football Association (South Africa)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/141222 , vital:37954
- Description: Currently there is limited research investigating the football related abilities of youth South African individuals. Populations from North and South America, Europe and Asia have been extensively covered in terms of their anthropometric characteristics, physical abilities, technical competency, tactical understanding as well as various personality traits. In describing these details, and especially how each is affected during the ageing and pubertal process, a more informed understanding of the talent development systems is created. A holistic approach to talent development is required to effectively and efficiently produce elite level footballers. An important consideration is the unique socio-economic environment many youth footballers experience which may impact on the talent development process. The present study therefore sought to quantify the anthropometric characteristics and the physical ability level of youth South African footballers from Local Football Association in Makhanda in the Sarah Baartman Region of the Eastern Cape province. In order to achieve this a two-factorial design was used with age and maturity status as independent variables. Anthropometric characteristics were tested by finding the height, weight, body mass index and total fat percentage of participants. Physical ability was determined by results for aerobic capacity, power, acceleration, speed and agility. All tests were football specific and had been widely used in a variety of footballing studies. Additional demographic and socio-economic information was also recorded. A total of 136 participants were placed in their respective Under 11, 13, 15 or Under 19 age groups, while the maturity status of 96 participants were stratified into five distinct categories. Statistical analyses was conducted using p-value significance, with appropriate Tukey post-hoc tests administered when necessary, while effect sizes were calculated using Cohen’s d. Significant changes and practical effect sizes were present for nearly all dependent variables when either stratified by age or by maturity. Total fat percentage was the only dependent variable which was not influenced by either age or maturity. Age and maturity effect sizes for total body fat percentage indicated practical changes present which did not occur during statistical analysis. The present results indicate poor values when compared to normative data or that of other football playing study populations. Further longitudinal research is required to better understand individual growth rates for youth South African footballers, particularly from the Makana region. Implications of the thesis may determine long term development pathways, while the delayed maturity rate is a critical finding which needs to be further researched.
- Full Text:
The effect of alginate supplementation on the growth, feed utilization, digestive enzyme activity levels, and intestinal morphology of juvenile South African abalone (Haliotis midae) fed formulated feeds
- Authors: Tlakedi, Thato
- Date: 2020
- Subjects: Abalones -- Feeding and feeds -- South Africa , Haliotis midae , Abalones -- Digestive organs , Abalones -- Morphology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145279 , vital:38424
- Description: Alginate occurs abundantly in kelp and while a few studies have quantified the effect of kelp inclusion in formulated Haliotis midae feeds, none have researched the effect of alginate inclusion on the growth of abalone fed this feed. Feeding kelp to cultured abalone affects gut structure and modulates gut bacteria, aiding digestion by upregulating enzyme activity. This farm-based experiment compared the growth, feed utilization, digestive enzyme activity levels and intestinal villi structure between abalone (1-year-old, 10 - 20 mm shell length) fed kelp-supplemented (BK) or alginate-supplemented (BA) diets and abalone fed basal (B) and fresh kelp (K) diets for a period of eight months from June 2017 to February 2018. Growth and enzyme activities were also compared between abalone that experienced dietary changes and abalone that remained on their initial diets. The tested diets were: base diet (B, 34% protein), the same base diet supplemented with fresh minced kelp (0.90% kelp [dry mass, equivalent to 0.20% alginate]; BK), base diet supplemented with sodium alginate (0.20% alginate; BA) and a fresh kelp diet (K). Alginate or kelp inclusion in a formulated feed had no significant effect on the specific growth rates (SGR), linear shell growth rates (LGR), daily percentage feeding rates (PFR), feed conversion ratio (FCR), percentage mass gain (MG) and condition factor (CF) over the eight month trial period, compared to abalone fed the base diet. In October 2017, four months since the start of the trial in June, all the groups fed formulated feeds showed significantly higher average weight gain (8.42 ± 0.72 g to 8.86 ± 0.63 g) compared to the kelp-fed group (6.68 ± 0.43 g). However, no significant differences in the average weight gain were observed between abalone fed kelp and formulated feeds at the end of this study. Furthermore, there were no significant differences in the FCR and PFR between abalone fed the base or alginate diets and abalone fed the fresh kelp diet. However, the SW diet produced significantly lower FCR (1.00 ± 0.13) and PFR (0.78 ± 0.10%) compared to the kelp diet (10.6 ± 1.40% and 8.12 ± 1.01%, respectively). All formulated diets produced non-significant LGR, MG and CF compared to the kelp diet at the end of this study. Enzyme activity levels were compared between abalone fed the test diets in July and August 2017 and February 2018. There were no significant differences in the average alginate lyase specific activity levels between abalone fed formulated diets in July and August 2017. At the end of the trial, however, the alginate diet induced significantly higher alginate lyase specific activity levels (4.89 ± 1.64 mg.mg-1 protein) compared to the base diet (1.57 ± 0.98 mg.mg-1 protein), but showed no significant differences compared to the SW (3.78 ± 0.41 mg.mg-1 protein) and fresh kelp-only diets (4.00 ± 0.62 mg.mg-1 protein). The SW diet showed no significant differences in the alginate lyase specific activity levels compared to the base diet. There were no significant differences in the chymotrypsin activity levels between the test diets throughout this study. Alginate or kelp inclusion also had no significant effect on the intestinal structure compared to the base or fresh kelp diets. This was despite a higher degree of variability in microvilli height and width being observed in abalone fed the kelp-only diet, with microvilli visibly wider and longer than in abalone fed formulated feeds. Switching abalone between some diets significantly affected the average weight gain, MG, FCR and PFR, alginate lyase, chymotrypsin and cellulase activity levels compared to abalone kept on their initial diets. The inclusion levels of kelp and alginate used in this study were likely too low to affect abalone growth as higher inclusion levels, particularly of alginate, have been found to improve fish growth. This study concluded that minced kelp and alginate inclusion in a formulated H. midae diet did not affect the SGR, FCR and intestinal morphology, but significantly affected alginate lyase and chymotrypsin activity levels. Furthermore, changes in growth and digestive enzyme activity levels coincided with dietary changes. Consequently, higher inclusion levels, particularly of sodium alginate, and the effect of dietary changes on abalone growth and digestive enzymes must be investigated in future studies.
- Full Text:
- Authors: Tlakedi, Thato
- Date: 2020
- Subjects: Abalones -- Feeding and feeds -- South Africa , Haliotis midae , Abalones -- Digestive organs , Abalones -- Morphology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145279 , vital:38424
- Description: Alginate occurs abundantly in kelp and while a few studies have quantified the effect of kelp inclusion in formulated Haliotis midae feeds, none have researched the effect of alginate inclusion on the growth of abalone fed this feed. Feeding kelp to cultured abalone affects gut structure and modulates gut bacteria, aiding digestion by upregulating enzyme activity. This farm-based experiment compared the growth, feed utilization, digestive enzyme activity levels and intestinal villi structure between abalone (1-year-old, 10 - 20 mm shell length) fed kelp-supplemented (BK) or alginate-supplemented (BA) diets and abalone fed basal (B) and fresh kelp (K) diets for a period of eight months from June 2017 to February 2018. Growth and enzyme activities were also compared between abalone that experienced dietary changes and abalone that remained on their initial diets. The tested diets were: base diet (B, 34% protein), the same base diet supplemented with fresh minced kelp (0.90% kelp [dry mass, equivalent to 0.20% alginate]; BK), base diet supplemented with sodium alginate (0.20% alginate; BA) and a fresh kelp diet (K). Alginate or kelp inclusion in a formulated feed had no significant effect on the specific growth rates (SGR), linear shell growth rates (LGR), daily percentage feeding rates (PFR), feed conversion ratio (FCR), percentage mass gain (MG) and condition factor (CF) over the eight month trial period, compared to abalone fed the base diet. In October 2017, four months since the start of the trial in June, all the groups fed formulated feeds showed significantly higher average weight gain (8.42 ± 0.72 g to 8.86 ± 0.63 g) compared to the kelp-fed group (6.68 ± 0.43 g). However, no significant differences in the average weight gain were observed between abalone fed kelp and formulated feeds at the end of this study. Furthermore, there were no significant differences in the FCR and PFR between abalone fed the base or alginate diets and abalone fed the fresh kelp diet. However, the SW diet produced significantly lower FCR (1.00 ± 0.13) and PFR (0.78 ± 0.10%) compared to the kelp diet (10.6 ± 1.40% and 8.12 ± 1.01%, respectively). All formulated diets produced non-significant LGR, MG and CF compared to the kelp diet at the end of this study. Enzyme activity levels were compared between abalone fed the test diets in July and August 2017 and February 2018. There were no significant differences in the average alginate lyase specific activity levels between abalone fed formulated diets in July and August 2017. At the end of the trial, however, the alginate diet induced significantly higher alginate lyase specific activity levels (4.89 ± 1.64 mg.mg-1 protein) compared to the base diet (1.57 ± 0.98 mg.mg-1 protein), but showed no significant differences compared to the SW (3.78 ± 0.41 mg.mg-1 protein) and fresh kelp-only diets (4.00 ± 0.62 mg.mg-1 protein). The SW diet showed no significant differences in the alginate lyase specific activity levels compared to the base diet. There were no significant differences in the chymotrypsin activity levels between the test diets throughout this study. Alginate or kelp inclusion also had no significant effect on the intestinal structure compared to the base or fresh kelp diets. This was despite a higher degree of variability in microvilli height and width being observed in abalone fed the kelp-only diet, with microvilli visibly wider and longer than in abalone fed formulated feeds. Switching abalone between some diets significantly affected the average weight gain, MG, FCR and PFR, alginate lyase, chymotrypsin and cellulase activity levels compared to abalone kept on their initial diets. The inclusion levels of kelp and alginate used in this study were likely too low to affect abalone growth as higher inclusion levels, particularly of alginate, have been found to improve fish growth. This study concluded that minced kelp and alginate inclusion in a formulated H. midae diet did not affect the SGR, FCR and intestinal morphology, but significantly affected alginate lyase and chymotrypsin activity levels. Furthermore, changes in growth and digestive enzyme activity levels coincided with dietary changes. Consequently, higher inclusion levels, particularly of sodium alginate, and the effect of dietary changes on abalone growth and digestive enzymes must be investigated in future studies.
- Full Text:
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.
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The influence of the integration of local knowledge during chemistry hands-on practical activities on grade 8/9 learners’ attitude towards science
- Authors: Ngqinambi, Akhona
- Date: 2020
- Subjects: Science -- Study and teaching (Secondary) -- South Africa , Science teachers -- South Africa , High school students -- Attitudes , Communities of practice -- South Africa , Science clubs -- South Africa , Science projects -- South Africa , Constructivism (Education) -- South Africa , Educational counseling -- South Africa , Local knowledge
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/144724 , vital:38373
- Description: With every passing year, there is a decrease in the number of learners pursuing science degrees after completion of grade 12. One of the main reasons for this is the negative attitude that high school learners have towards science learning and science as an entity. The Natural Sciences curriculum suggests the use of local knowledge to introduce the usefulness and the relatability of science to learners. This study is an intervention that seeks to ascertain the influence that local knowledge has on the attitudes of grade 8/9 learners when integrated into their chemistry hands-on practical activities. The study was informed by Vygotsky’s social constructivist theory and Wenger’s community of practice theory. Grade 8/9 learners from four high school science clubs did three local knowledge integrated experiments based on acids and bases. The learners completed pre- and post- surveys and were interviewed about their experiences. Volunteers at the science clubs were also interviewed. Excel was used to deductively analyse quantitative data. On the other hand, an inductive-deductive thematic approach was used to analyse the qualitative data. The findings of the study showed a divergence of qualitative and quantitative methods. The quantitative data showed that there was a slight decrease (but not statistically significant) in the attitude of the learners after engaging in chemistry hands-on practical activities into which local knowledge had been integrated. On the other hand, the qualitative data showed that there was a positive shift in the learners’ attitudes after they had been engaged in the chemistry hands-on practical activities. Additionally, the findings of the study revealed that the integration of local knowledge promotes conceptual understanding and improves the performance of the learners. This study thus recommends that local knowledge should be integrated into the grade 8 and 9 learners’ curriculum to enrich and promote contextualized science for learners. Additionally, the integration of local knowledge would promote conceptual understanding and spark interest and motivation in learners to pursue science-related careers.
- Full Text:
- Authors: Ngqinambi, Akhona
- Date: 2020
- Subjects: Science -- Study and teaching (Secondary) -- South Africa , Science teachers -- South Africa , High school students -- Attitudes , Communities of practice -- South Africa , Science clubs -- South Africa , Science projects -- South Africa , Constructivism (Education) -- South Africa , Educational counseling -- South Africa , Local knowledge
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/144724 , vital:38373
- Description: With every passing year, there is a decrease in the number of learners pursuing science degrees after completion of grade 12. One of the main reasons for this is the negative attitude that high school learners have towards science learning and science as an entity. The Natural Sciences curriculum suggests the use of local knowledge to introduce the usefulness and the relatability of science to learners. This study is an intervention that seeks to ascertain the influence that local knowledge has on the attitudes of grade 8/9 learners when integrated into their chemistry hands-on practical activities. The study was informed by Vygotsky’s social constructivist theory and Wenger’s community of practice theory. Grade 8/9 learners from four high school science clubs did three local knowledge integrated experiments based on acids and bases. The learners completed pre- and post- surveys and were interviewed about their experiences. Volunteers at the science clubs were also interviewed. Excel was used to deductively analyse quantitative data. On the other hand, an inductive-deductive thematic approach was used to analyse the qualitative data. The findings of the study showed a divergence of qualitative and quantitative methods. The quantitative data showed that there was a slight decrease (but not statistically significant) in the attitude of the learners after engaging in chemistry hands-on practical activities into which local knowledge had been integrated. On the other hand, the qualitative data showed that there was a positive shift in the learners’ attitudes after they had been engaged in the chemistry hands-on practical activities. Additionally, the findings of the study revealed that the integration of local knowledge promotes conceptual understanding and improves the performance of the learners. This study thus recommends that local knowledge should be integrated into the grade 8 and 9 learners’ curriculum to enrich and promote contextualized science for learners. Additionally, the integration of local knowledge would promote conceptual understanding and spark interest and motivation in learners to pursue science-related careers.
- Full Text:
The past meets the present: exploring the biogeography of extant plecoptera of South Africa with reference to ancient middle permian fossil forms from the Onder Karoo locality near Sutherland
- Authors: Kirkaldy, Benjamin Puleng
- Date: 2020
- Subjects: Stoneflies, Fossil -- South Africa -- Northern Cape , Palaeopteron -- South Africa -- Northern Cape , Stoneflies -- Evolution , Insects, Fossil -- South Africa -- Northern Cape , Paleoentomology , Paleoentomology -- South Africa -- Northern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/165755 , vital:41278
- Description: The Onder Karoo fossil locality near Sutherland, Northern Cape, recently yielded an unprecedented diversity of middle Permian insects, which were preserved in deposits of an aquatic lake margin system, in the Lowermost Abrahamskraal Formation. A large number of Plecoptera (stonefly) specimens were found, and this study represents an in-depth analysis of these species in the context of plecopteran evolution. A significant contribution to current knowledge of the Plecoptera from the middle Permian to the present has been made, utilizing a combination of fossil and phylogenetic evidence to better understand the origins, evolution, diversity and biogeography of the stoneflies. Three new plecopteran species have been described from the Onder Karoo locality, which is recognized here as the first Lagerstätte of its kind in Gondwana. Possible ecological links and relationships with the plecopterans described here have been suggested, using extant Plecoptera as analogues. An in-depth, fossilcalibrated, dated phylogeny of the Plecoptera was completed, using representatives from all three dominant lineages, the Antarctoperlaria, Systellognatha and the Euholognatha. This provides strong support for the monophyly of the Notonemouridae and the Antarctoperlaria, however the monophyly of Systellognatha and Euholognatha was not supported. The Notonemouridae were found to represent an earlier divergence than previously believed, forming a sister group to the remaining Plecoptera. Through a combination of fossil and molecular evidence, strong support was found for the current distribution of the Plecoptera worldwide being attributable to vicariance caused by the rifting of Pangea, and subsequently Gondwana and Laurasia, and long range dispersal. The focussed effort to include Southern Hemisphere stoneflies throughout this study has been a valuable step in reducing the Northern Hemisphere bias which currently dominates plecopteran research and has assisted in opening the way for future research into this important group on a global scale.
- Full Text:
- Authors: Kirkaldy, Benjamin Puleng
- Date: 2020
- Subjects: Stoneflies, Fossil -- South Africa -- Northern Cape , Palaeopteron -- South Africa -- Northern Cape , Stoneflies -- Evolution , Insects, Fossil -- South Africa -- Northern Cape , Paleoentomology , Paleoentomology -- South Africa -- Northern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/165755 , vital:41278
- Description: The Onder Karoo fossil locality near Sutherland, Northern Cape, recently yielded an unprecedented diversity of middle Permian insects, which were preserved in deposits of an aquatic lake margin system, in the Lowermost Abrahamskraal Formation. A large number of Plecoptera (stonefly) specimens were found, and this study represents an in-depth analysis of these species in the context of plecopteran evolution. A significant contribution to current knowledge of the Plecoptera from the middle Permian to the present has been made, utilizing a combination of fossil and phylogenetic evidence to better understand the origins, evolution, diversity and biogeography of the stoneflies. Three new plecopteran species have been described from the Onder Karoo locality, which is recognized here as the first Lagerstätte of its kind in Gondwana. Possible ecological links and relationships with the plecopterans described here have been suggested, using extant Plecoptera as analogues. An in-depth, fossilcalibrated, dated phylogeny of the Plecoptera was completed, using representatives from all three dominant lineages, the Antarctoperlaria, Systellognatha and the Euholognatha. This provides strong support for the monophyly of the Notonemouridae and the Antarctoperlaria, however the monophyly of Systellognatha and Euholognatha was not supported. The Notonemouridae were found to represent an earlier divergence than previously believed, forming a sister group to the remaining Plecoptera. Through a combination of fossil and molecular evidence, strong support was found for the current distribution of the Plecoptera worldwide being attributable to vicariance caused by the rifting of Pangea, and subsequently Gondwana and Laurasia, and long range dispersal. The focussed effort to include Southern Hemisphere stoneflies throughout this study has been a valuable step in reducing the Northern Hemisphere bias which currently dominates plecopteran research and has assisted in opening the way for future research into this important group on a global scale.
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The role of northwest striking structures in controlling highgrade ore shoots at the Syama Gold Mine, Mali, West Africa
- Authors: Soro, Ali
- Date: 2020
- Subjects: Syama Gold Mine , Gold ores -- Geology -- Mali , Veins (Geology) -- Mali
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145209 , vital:38418
- Description: This study intended to investigate the relationship between the NW striking structures and the high-grade ore shoots at the Syama gold mine in Mali, West Africa. All structural data collected since 1987 from drill core have been integrated to allow the interpretation and modelling of these NW-SE structures. The structures collected were grouped into three main groups; foliations/shears/faults, veins and joints/contacts/fractures. Micromine software was used to plot the structures, printed out on A3 paper and interpreted manually using tracing paper. Analysis and interpretation of stereographic plots has shown that the majority of the high-grade zones are generally located at the intersection of the NNE structures and the NW structures. The observed cross-cutting relationship between the NNE and the NW structures suggests two different generation of faults. It is suggested that the NW structures were active during the D4 deformation event (Standing, 2007) and have played a role in reactivating earlier (D3) NNE structures, allowing greater fluid flow and enhancing the gold grade. These zones are mainly defined by brecciation and stockwork veining. The E-W structures are believed to be the latest and are attributed to the D5 event. Although gold mineralisation is grossly controlled by the NNE structures, the NW structures need to be considered as major gold enrichment upgrading factors at Syama. It is therefore strongly recommended that ongoing exploration at Syama specifically target the intersection of the NW and NNE structures as favourable zones for high-grade mineralisation.
- Full Text:
- Authors: Soro, Ali
- Date: 2020
- Subjects: Syama Gold Mine , Gold ores -- Geology -- Mali , Veins (Geology) -- Mali
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/145209 , vital:38418
- Description: This study intended to investigate the relationship between the NW striking structures and the high-grade ore shoots at the Syama gold mine in Mali, West Africa. All structural data collected since 1987 from drill core have been integrated to allow the interpretation and modelling of these NW-SE structures. The structures collected were grouped into three main groups; foliations/shears/faults, veins and joints/contacts/fractures. Micromine software was used to plot the structures, printed out on A3 paper and interpreted manually using tracing paper. Analysis and interpretation of stereographic plots has shown that the majority of the high-grade zones are generally located at the intersection of the NNE structures and the NW structures. The observed cross-cutting relationship between the NNE and the NW structures suggests two different generation of faults. It is suggested that the NW structures were active during the D4 deformation event (Standing, 2007) and have played a role in reactivating earlier (D3) NNE structures, allowing greater fluid flow and enhancing the gold grade. These zones are mainly defined by brecciation and stockwork veining. The E-W structures are believed to be the latest and are attributed to the D5 event. Although gold mineralisation is grossly controlled by the NNE structures, the NW structures need to be considered as major gold enrichment upgrading factors at Syama. It is therefore strongly recommended that ongoing exploration at Syama specifically target the intersection of the NW and NNE structures as favourable zones for high-grade mineralisation.
- 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: