The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
A statistical study of travelling ionospheric disturbances over the African-European and American sectors
- Authors: Thaganyana, Golekamang Piet
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422541 , vital:71956 , DOI 10.21504/10962/422543
- Description: This research presents a long-term statistical study of travelling ionospheric disturbances (TIDs) of low- and high-latitude origin over the American and African-European sectors between 2010 and 2018. The TIDs of low latitude origin (hereafter known as poleward TIDs) were studied in both quiet and disturbed conditions, whereas the equatorward TIDs were only studied during quiet conditions. The Kp > 4 and Dst_ -50 nT was used as a criterion for geomagnetic disturbed conditions, while the four geomagnetically quiet days were selected each month based on Kp < 3. Observations of TIDs are made using Global Navigational Satellite Systems (GNSS) total electron content derived data. During quiet conditions, seven and two transhemispheric TIDs were identified over the African-European and American sectors, respectively. The observed TIDs originated from the wintertime hemisphere and propagated into the summertime hemisphere. The horizontal velocity, periods, and horizontal wavelengths of TIDs are in range of cH = 120-274 m/s, 48-80 min and _H = 379-1104 km, respectively. These quiet-time equatorward TIDs have been associated with tertiary gravity waves (GWs) from the dissipation of secondary GWs which are in turn generated from the dissipation of mountain waves (MWs) as a result of excited orographic forcing. The poleward TIDs during geomagnetically quiet conditions over the African and American sectors occur mainly during local daytime. Poleward TIDs were observed mostly in the African-European sector than the American sector. Their horizontal propagation velocities and periods range between 129-280 m/s and 39-70 min over African-European and American sectors. Although the mechanisms responsible for launching quiet-time poleward TIDs have not been established in this study, lower atmospheric processes such as convection systems, sudden stratospheric warming and cold weather fronts may have a role in their generation. During geomagnetic storms in the African sector, almost all poleward TIDs (with the exception of two cases) during the main phase were large-scale with horizontal velocities and periods ranging from 250-503 m/s and 30 min to 2 hours. During recovery phase, poleward TIDs fall under the category of medium scale. In the American sector, the majority of poleward TIDs occurred during the storm's main phase, as opposed to the African-European sector, which experienced a significant number of poleward TIDs during the recovery phase. The periods and horizontal velocities of TIDs range from 45 min-1.5 h and 180-296 m/s during main phase. During the recovery phase, the horizontal velocity and period range from 177-271 m/s and 40-1.5 h, respectively. Overall, it has been shown that statistically, changes in equatorial electrodynamics related to enhanced eastward electric _eld and hence increased equatorial electrojet (vertical E_B drift) correlates highly with the reported poleward TIDs. , Thesis (PhD) -- Faculty of Science, Physics and Electronics, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Thaganyana, Golekamang Piet
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422541 , vital:71956 , DOI 10.21504/10962/422543
- Description: This research presents a long-term statistical study of travelling ionospheric disturbances (TIDs) of low- and high-latitude origin over the American and African-European sectors between 2010 and 2018. The TIDs of low latitude origin (hereafter known as poleward TIDs) were studied in both quiet and disturbed conditions, whereas the equatorward TIDs were only studied during quiet conditions. The Kp > 4 and Dst_ -50 nT was used as a criterion for geomagnetic disturbed conditions, while the four geomagnetically quiet days were selected each month based on Kp < 3. Observations of TIDs are made using Global Navigational Satellite Systems (GNSS) total electron content derived data. During quiet conditions, seven and two transhemispheric TIDs were identified over the African-European and American sectors, respectively. The observed TIDs originated from the wintertime hemisphere and propagated into the summertime hemisphere. The horizontal velocity, periods, and horizontal wavelengths of TIDs are in range of cH = 120-274 m/s, 48-80 min and _H = 379-1104 km, respectively. These quiet-time equatorward TIDs have been associated with tertiary gravity waves (GWs) from the dissipation of secondary GWs which are in turn generated from the dissipation of mountain waves (MWs) as a result of excited orographic forcing. The poleward TIDs during geomagnetically quiet conditions over the African and American sectors occur mainly during local daytime. Poleward TIDs were observed mostly in the African-European sector than the American sector. Their horizontal propagation velocities and periods range between 129-280 m/s and 39-70 min over African-European and American sectors. Although the mechanisms responsible for launching quiet-time poleward TIDs have not been established in this study, lower atmospheric processes such as convection systems, sudden stratospheric warming and cold weather fronts may have a role in their generation. During geomagnetic storms in the African sector, almost all poleward TIDs (with the exception of two cases) during the main phase were large-scale with horizontal velocities and periods ranging from 250-503 m/s and 30 min to 2 hours. During recovery phase, poleward TIDs fall under the category of medium scale. In the American sector, the majority of poleward TIDs occurred during the storm's main phase, as opposed to the African-European sector, which experienced a significant number of poleward TIDs during the recovery phase. The periods and horizontal velocities of TIDs range from 45 min-1.5 h and 180-296 m/s during main phase. During the recovery phase, the horizontal velocity and period range from 177-271 m/s and 40-1.5 h, respectively. Overall, it has been shown that statistically, changes in equatorial electrodynamics related to enhanced eastward electric _eld and hence increased equatorial electrojet (vertical E_B drift) correlates highly with the reported poleward TIDs. , Thesis (PhD) -- Faculty of Science, Physics and Electronics, 2023
- Full Text:
- Date Issued: 2023-03-31
An in-depth investigation of an early literacy intervention in Grade R in the Eastern Cape Province, South Africa
- Authors: Hodgskiss, Jennifer Adelé
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/366222 , vital:65844
- Description: Thesis embargoed. Expected release date early 2025. , Thesis (PhD) -- Faculty of Education, Education, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Hodgskiss, Jennifer Adelé
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/366222 , vital:65844
- Description: Thesis embargoed. Expected release date early 2025. , Thesis (PhD) -- Faculty of Education, Education, 2023
- Full Text:
- Date Issued: 2023-03-29
Optimization of an intranasal levodopa nanocrystalline formulation for delivery to the brain
- Authors: Kakono, Chiedza
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/420709 , vital:71771
- Description: Thesis embargoed. Probable release date in 2025. , Thesis (MSc (Pharm)) -- Faculty of Pharmacy, Pharmacy, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Kakono, Chiedza
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/420709 , vital:71771
- Description: Thesis embargoed. Probable release date in 2025. , Thesis (MSc (Pharm)) -- Faculty of Pharmacy, Pharmacy, 2023
- Full Text:
- Date Issued: 2023-03-29
Quantifying feed intake and feeding intensity using two experimental conditions and the effect of different feeding strategies on the production parameters of farmed South African abalone, haliotis midae
- Authors: Wortley, Ross Michael
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/${Handle} , vital:71932
- Description: As abalone are slow-growing animals there is a high investment of capital, running costs and labour in commercial abalone farming. Revenue needs to be maximised by achieving fast growth rates. A foremost driver of abalone growth is feed intake and feeding intensity of the abalone as well as the feeding regimen a farm utilises. While feed intake is well-documented in H. midae, there is a paucity of information regarding this aspect in abalone above 70 g and the relationship between feeding intensity, feed conversion rate (FCR) and daily growth rate needs to be documented. Similarly, there are a limited number of studies dealing with the effects of different pellet types of the same formulation, that differ only in shape and size, on feed intake and production in H. midae. This study made use of both a controlled laboratory experiment and commercial-scale farm experiment with the aim of testing whether feed intake information produced under laboratory conditions can provide good estimates of feed intake under farming conditions, which can further be implemented into feeding strategies on abalone farms. This study quantified daily feed intake (F) in 10 – 20 g, 100 – 110 g and 150 – 160 g abalone weight classes using two different pellet types and determined the relationship between the duration of feed availability and feed intake under laboratory conditions. The effect of three different pellet type strategies (fed daily: a leaf-only strategy = L d-1, a short-pellet only strategy = SP d-1, and a strategy that used both = B d-1) on abalone production parameters under farming conditions such as growth rate, feed conversion ratio (FCR), feeding intensity (FI) and weight variation of 30 – 40 g H. midae was also investigated. A commercial-scale farm study was designed to test the effect of four commercially relevant feeding regimens (three size-specific regimens and one commercially practiced feeding method) on the growth, FCR, feeding intensity and weight variation of these three abalone weight classes while taking into account an economic model to assess the profitability of each feeding regimen. Daily feed intake as a percentage of body mass (% BM d-1) was a function of abalone weight class between the 10 – 20 g and two larger weight classes 100 – 110 g and 150 – 160 g p < 0.0001), however feed intake was not different between the 100 – 110 g and 150 – 160 g weight classes. Pellet type did not affect feed intake in all abalone weight classes (p = 0.15). Feed intake (F) was positively linearly correlated with duration of feed availability (h) in the 10 – 20 g weight class (r2 = 0.76, p <0.00001) and logarithmically correlated in the 100 – 110 g (r2 = 0.25, p < 0.02) and 150 – 160 g (r2 = 0.52, p < 0.0001) weight classes (10 – 20 g abalone-1: 3 F (% BM) = 0.02(h) + 0.1976, 100 – 110 g abalone-1: F (% BM) = log10(h)*0.17 + 0.17, 150 – 160 g abalone-1: F (% BM) = log10(h)*0.36 + 0.07). There was no significant difference in monthly average abalone weight, daily growth rate (G), FCR and feeding intensity between each pellet type strategy (G: p = 0.60, FCR: p = 0.62, FI: p = 0.54 ). However, abalone grew well over the 112-day growth period with average abalone weight increasing significantly between each monthly sample time (pooled pellet type strategy: Huynh-Feldt correction; p < 0.00001). Furthermore, abalone fed the leaf-only pellet type strategy (L d-1) fell into higher weight classes after a 112-day growth period (Z test: 50 – 70 g: 72%) which was 16.1 % higher compared to the SP d-1 and B d-1 strategies. Daily growth rate (r2 = 0.34, p < 0.01) and FCR (r2 = 0.42, p < 0.004) was negatively correlated and FCR was positively correlated with daily feeding intensity, respectively (G = - 2.59 (FI) + 1.526, FCR = 8.8082 (FI) – 2.7108). Feeding regimen affected the production parameters of three abalone weight classes. The method practiced on the farm resulted in the best growth in the 10 – 20 g abalone weight class. The farm feeding method resulted in slower yet more efficient growth rates (lowest FCR values) in the abalone weight classes, 100 – 110 g and 150 – 160 g abalone- 1. However, size-specific feeding regimens resulted in the fastest growth rates but resulted in higher FCR values (less efficient). The relationship between feeding intensity, daily growth rate and FCR all showed that an increase in feeding intensity results in increased daily growth rates and FCR values. The economic model suggests that the higher FCR values associated with size-specific regimens, which have higher associated costs to producing abalone, were greatly outweighed by the growth attained by the abalone in the 100 – 110 g and 150 – 160 g weight classes. The size-specific regimens generated a higher potential monetary value of abalone after a 112-day period, which would consequently result in higher income for abalone farms. For abalone ranging from 100 – 110 and 150 – 160 grams, the economic model suggested that in a quarterly grading schedule (112 days) that abalone be fed the size specific daily rations, which is a function of body mass, at 0.35 % BM d-1 and 0.352 % BM d-1, respectively. The two experimental conditions used in this study produced feed intake and production parameter information that is beneficial to South African abalone farmers. The small-scale laboratory study produced information on feed intake that can be used as reference values as to what abalone in these weight classes can consume on a daily basis. The laboratory study can provide estimates of feed intake under farming conditions but should only be used as minimum 4 values when determining size-specific feeding regimens. To maximise abalone growth, farmers should utilise size-specific feeding regimens for abalone above 30 g. Daily growth rate and FCR can be predicted as a function of the abalone’s feeding intensity. Further studies are needed to determine the effects of abalone weight class on production parameters when testing different pellet types as well as an exploration into behavioural studies focusing on diet preferences. Additionally, future studies need to take into consideration abalone above the weight of 100 g with additional focus of research on behavioural, genetic and environmental aspects on abalone feed intake. , Thesis (MSc) -- Faculty of Science, Ichthyology and Fisheries Science, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Wortley, Ross Michael
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/${Handle} , vital:71932
- Description: As abalone are slow-growing animals there is a high investment of capital, running costs and labour in commercial abalone farming. Revenue needs to be maximised by achieving fast growth rates. A foremost driver of abalone growth is feed intake and feeding intensity of the abalone as well as the feeding regimen a farm utilises. While feed intake is well-documented in H. midae, there is a paucity of information regarding this aspect in abalone above 70 g and the relationship between feeding intensity, feed conversion rate (FCR) and daily growth rate needs to be documented. Similarly, there are a limited number of studies dealing with the effects of different pellet types of the same formulation, that differ only in shape and size, on feed intake and production in H. midae. This study made use of both a controlled laboratory experiment and commercial-scale farm experiment with the aim of testing whether feed intake information produced under laboratory conditions can provide good estimates of feed intake under farming conditions, which can further be implemented into feeding strategies on abalone farms. This study quantified daily feed intake (F) in 10 – 20 g, 100 – 110 g and 150 – 160 g abalone weight classes using two different pellet types and determined the relationship between the duration of feed availability and feed intake under laboratory conditions. The effect of three different pellet type strategies (fed daily: a leaf-only strategy = L d-1, a short-pellet only strategy = SP d-1, and a strategy that used both = B d-1) on abalone production parameters under farming conditions such as growth rate, feed conversion ratio (FCR), feeding intensity (FI) and weight variation of 30 – 40 g H. midae was also investigated. A commercial-scale farm study was designed to test the effect of four commercially relevant feeding regimens (three size-specific regimens and one commercially practiced feeding method) on the growth, FCR, feeding intensity and weight variation of these three abalone weight classes while taking into account an economic model to assess the profitability of each feeding regimen. Daily feed intake as a percentage of body mass (% BM d-1) was a function of abalone weight class between the 10 – 20 g and two larger weight classes 100 – 110 g and 150 – 160 g p < 0.0001), however feed intake was not different between the 100 – 110 g and 150 – 160 g weight classes. Pellet type did not affect feed intake in all abalone weight classes (p = 0.15). Feed intake (F) was positively linearly correlated with duration of feed availability (h) in the 10 – 20 g weight class (r2 = 0.76, p <0.00001) and logarithmically correlated in the 100 – 110 g (r2 = 0.25, p < 0.02) and 150 – 160 g (r2 = 0.52, p < 0.0001) weight classes (10 – 20 g abalone-1: 3 F (% BM) = 0.02(h) + 0.1976, 100 – 110 g abalone-1: F (% BM) = log10(h)*0.17 + 0.17, 150 – 160 g abalone-1: F (% BM) = log10(h)*0.36 + 0.07). There was no significant difference in monthly average abalone weight, daily growth rate (G), FCR and feeding intensity between each pellet type strategy (G: p = 0.60, FCR: p = 0.62, FI: p = 0.54 ). However, abalone grew well over the 112-day growth period with average abalone weight increasing significantly between each monthly sample time (pooled pellet type strategy: Huynh-Feldt correction; p < 0.00001). Furthermore, abalone fed the leaf-only pellet type strategy (L d-1) fell into higher weight classes after a 112-day growth period (Z test: 50 – 70 g: 72%) which was 16.1 % higher compared to the SP d-1 and B d-1 strategies. Daily growth rate (r2 = 0.34, p < 0.01) and FCR (r2 = 0.42, p < 0.004) was negatively correlated and FCR was positively correlated with daily feeding intensity, respectively (G = - 2.59 (FI) + 1.526, FCR = 8.8082 (FI) – 2.7108). Feeding regimen affected the production parameters of three abalone weight classes. The method practiced on the farm resulted in the best growth in the 10 – 20 g abalone weight class. The farm feeding method resulted in slower yet more efficient growth rates (lowest FCR values) in the abalone weight classes, 100 – 110 g and 150 – 160 g abalone- 1. However, size-specific feeding regimens resulted in the fastest growth rates but resulted in higher FCR values (less efficient). The relationship between feeding intensity, daily growth rate and FCR all showed that an increase in feeding intensity results in increased daily growth rates and FCR values. The economic model suggests that the higher FCR values associated with size-specific regimens, which have higher associated costs to producing abalone, were greatly outweighed by the growth attained by the abalone in the 100 – 110 g and 150 – 160 g weight classes. The size-specific regimens generated a higher potential monetary value of abalone after a 112-day period, which would consequently result in higher income for abalone farms. For abalone ranging from 100 – 110 and 150 – 160 grams, the economic model suggested that in a quarterly grading schedule (112 days) that abalone be fed the size specific daily rations, which is a function of body mass, at 0.35 % BM d-1 and 0.352 % BM d-1, respectively. The two experimental conditions used in this study produced feed intake and production parameter information that is beneficial to South African abalone farmers. The small-scale laboratory study produced information on feed intake that can be used as reference values as to what abalone in these weight classes can consume on a daily basis. The laboratory study can provide estimates of feed intake under farming conditions but should only be used as minimum 4 values when determining size-specific feeding regimens. To maximise abalone growth, farmers should utilise size-specific feeding regimens for abalone above 30 g. Daily growth rate and FCR can be predicted as a function of the abalone’s feeding intensity. Further studies are needed to determine the effects of abalone weight class on production parameters when testing different pellet types as well as an exploration into behavioural studies focusing on diet preferences. Additionally, future studies need to take into consideration abalone above the weight of 100 g with additional focus of research on behavioural, genetic and environmental aspects on abalone feed intake. , Thesis (MSc) -- Faculty of Science, Ichthyology and Fisheries Science, 2023
- Full Text:
- Date Issued: 2023-03-29
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