Towards a biological profile for South African perinatal remains: osteological and genetic perspectives
- Authors: Thornton, Roxanne
- Date: 2019
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198
- Description: Expected release date-April 2021
- Full Text: false
- Authors: Thornton, Roxanne
- Date: 2019
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/68102 , vital:29198
- Description: Expected release date-April 2021
- Full Text: false
An in-silico investigation of Morita-Baylis-Hillman accessible heterocyclic analogues for applications as novel HIV-1 C protease inhibitors
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
- Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
- Full Text:
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
- Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
- Full Text:
Development of a high-throughput bioassay to determine the rate of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
Vachellia erioloba (camel thorn) and microbial interactions
- Date: 2018
- Subjects: Uncatalogued
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63716 , http://vital.seals.ac.za8080/10962/81755 , vital:28475
- Description: Expected release date-April 2019
- Full Text:
- Date: 2018
- Subjects: Uncatalogued
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63716 , http://vital.seals.ac.za8080/10962/81755 , vital:28475
- Description: Expected release date-April 2019
- Full Text:
Investigating the use of Arbuscular Mycorrhizas and Plant Growth Promoting Bacteria to improve the drought tolerance of maize (Zea mays L.)
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
In silico characterization of plasmodial transketolases as potential malaria drug target
- Authors: Boateng, Rita Afriyie
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63540 , vital:28433
- Description: Expected release date-April 2019
- Full Text:
- Authors: Boateng, Rita Afriyie
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63540 , vital:28433
- Description: Expected release date-April 2019
- Full Text:
Using bioinformatics tools to screen for trypanosomal cathepsin B cysteine protease inhibitors from the SANCDB as a novel therapeutic modality against Human African Trypanosomiasis (HAT)
- Authors: Mokhawa, Gaone
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3304 , vital:20470
- Description: Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a fatal chronic disease that is caused by flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. HAT is spread by a bite from an infected tsetse fly of the Glosina genus. Up to 60 million people in 36 countries in sub-Saharan Africa are at a risk of infection from HAT with up to 30 000 deaths reported every year. Current chemotherapy for HAT is insufficient since the available drugs exhibit unacceptable side effects (toxicity) and parasite resistance. Novel treatments and approaches for development of specific and more potent drugs for HAT are therefore required. One approach is to target vital proteins that are essential to the life cycle of the parasite. The main interest of this study is to explore Trypanosoma brucei cathepsin B-like protease (TbCatB) structural and functional properties with the primary goal of discovering non peptide small molecule inhibitors of TbCatB using bioinformatics approaches. TbCatB is a papain family C1 cysteine protease which belongs to clan CA group and it has emerged as a potential HAT drug target. Papain family cysteine proteases of Clan CA group of Trypanosoma brucei (rhodesain and TbCatB) have demonstrated potential as chemotherapeutic targets using synthetic protease inhibitors like Z-Phe-Ala-CHN2 to kill the parasite in vitro and in vivo. TbCatB has been identified as the essential cysteine protease of T. brucei since mRNA silencing of TbCatB killed the parasite and resulted in a cure in mice infected with T. brucei while mRNA silencing of rhodesain only extended mice life. TbCatB is therefore a promising drug target against HAT and the discovery and development of compounds that can selectively inhibit TbCatB without posing any danger to the human host represent a great therapeutic solution for treatment of HAT. To understand protein-inhibitor interactions, useful information can be obtained from high resolution protease-inhibitor crystal structure complexes. This study aims to use bioinformatics approaches to carry out comparative sequence, structural and functional analysis of TbCatB protease and its homologs from T. congolense, T, cruzi, T. vivax and H. sapien as well as to identify non-peptide small molecule inhibitors of TbCatB cysteine proteases from natural compounds of South African origin. Sequences of TbCatB (PDB ID: 3HHI) homologs were retrieved by a BLAST search. Human cathepsin B (PDB ID: 3CBJ) was selected from a list of templates for homology modelling found by HHpred. MODELLER version 9.10 program was used to generate a hundred models for T. congolense, T, cruzi and T. vivax cathepsin B like proteases using 3HHI and 3CBJ as templates. The best models were chosen based on their low DOPE Z scores before validation using MetaMQAPII, ANOLEA, PROCHECK and QMEAN6. The DOPE Z scores and the RMSD (RMS) values of the calculated models indicate that the models are of acceptable energy (stability) and fold (conformation). Results from the different MQAPs indicate the models are of acceptable quality and they can be used for docking studies. High throughput screening of SANCDB using AutoDock Vina revealed nine compounds, SANC00 478, 479, 480, 481, 482, 488, 489, 490 and 491, having a strong affinity for Trypanosoma spp. cathepsin B proteases than HsCatB. SANC00488 has the strongest binding to Trypanosoma spp. cathepsin B proteases and the weakest binding to HsCatB protease. Molecular dynamics (MD) simulations show that the complexes between SANC00488 and TbCatB, TcCatB, TcrCatB and TvCatB are stable and do not come apart during simulation. The complex between this compound and HsCatB however is unstable and comes apart during simulation. Residues that are important for the stability of SANC00488-TbCatB complex are Gly328 of the S2 subsite, Phe208, and Ala256. In conclusion SANC00488 is a good candidate for development of a drug against HAT.
- Full Text:
- Authors: Mokhawa, Gaone
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3304 , vital:20470
- Description: Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a fatal chronic disease that is caused by flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. HAT is spread by a bite from an infected tsetse fly of the Glosina genus. Up to 60 million people in 36 countries in sub-Saharan Africa are at a risk of infection from HAT with up to 30 000 deaths reported every year. Current chemotherapy for HAT is insufficient since the available drugs exhibit unacceptable side effects (toxicity) and parasite resistance. Novel treatments and approaches for development of specific and more potent drugs for HAT are therefore required. One approach is to target vital proteins that are essential to the life cycle of the parasite. The main interest of this study is to explore Trypanosoma brucei cathepsin B-like protease (TbCatB) structural and functional properties with the primary goal of discovering non peptide small molecule inhibitors of TbCatB using bioinformatics approaches. TbCatB is a papain family C1 cysteine protease which belongs to clan CA group and it has emerged as a potential HAT drug target. Papain family cysteine proteases of Clan CA group of Trypanosoma brucei (rhodesain and TbCatB) have demonstrated potential as chemotherapeutic targets using synthetic protease inhibitors like Z-Phe-Ala-CHN2 to kill the parasite in vitro and in vivo. TbCatB has been identified as the essential cysteine protease of T. brucei since mRNA silencing of TbCatB killed the parasite and resulted in a cure in mice infected with T. brucei while mRNA silencing of rhodesain only extended mice life. TbCatB is therefore a promising drug target against HAT and the discovery and development of compounds that can selectively inhibit TbCatB without posing any danger to the human host represent a great therapeutic solution for treatment of HAT. To understand protein-inhibitor interactions, useful information can be obtained from high resolution protease-inhibitor crystal structure complexes. This study aims to use bioinformatics approaches to carry out comparative sequence, structural and functional analysis of TbCatB protease and its homologs from T. congolense, T, cruzi, T. vivax and H. sapien as well as to identify non-peptide small molecule inhibitors of TbCatB cysteine proteases from natural compounds of South African origin. Sequences of TbCatB (PDB ID: 3HHI) homologs were retrieved by a BLAST search. Human cathepsin B (PDB ID: 3CBJ) was selected from a list of templates for homology modelling found by HHpred. MODELLER version 9.10 program was used to generate a hundred models for T. congolense, T, cruzi and T. vivax cathepsin B like proteases using 3HHI and 3CBJ as templates. The best models were chosen based on their low DOPE Z scores before validation using MetaMQAPII, ANOLEA, PROCHECK and QMEAN6. The DOPE Z scores and the RMSD (RMS) values of the calculated models indicate that the models are of acceptable energy (stability) and fold (conformation). Results from the different MQAPs indicate the models are of acceptable quality and they can be used for docking studies. High throughput screening of SANCDB using AutoDock Vina revealed nine compounds, SANC00 478, 479, 480, 481, 482, 488, 489, 490 and 491, having a strong affinity for Trypanosoma spp. cathepsin B proteases than HsCatB. SANC00488 has the strongest binding to Trypanosoma spp. cathepsin B proteases and the weakest binding to HsCatB protease. Molecular dynamics (MD) simulations show that the complexes between SANC00488 and TbCatB, TcCatB, TcrCatB and TvCatB are stable and do not come apart during simulation. The complex between this compound and HsCatB however is unstable and comes apart during simulation. Residues that are important for the stability of SANC00488-TbCatB complex are Gly328 of the S2 subsite, Phe208, and Ala256. In conclusion SANC00488 is a good candidate for development of a drug against HAT.
- Full Text:
Evaluation of SNPs of G6PD, with regard to the 3D conformational, structural and stability alterations, in order to investigate the clinical implications and potential applications
- Authors: Sanabria, Natasha Mary-Anne
- Date: 2019
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/76500 , vital:30574
- Description: Expected release date-April 2020
- Full Text: false
- Authors: Sanabria, Natasha Mary-Anne
- Date: 2019
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/76500 , vital:30574
- Description: Expected release date-April 2020
- Full Text: false
Molecular simulations of potential agents and targets of Alzheimer’s disease
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
- Full Text:
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
- Full Text:
Localizing selected endocytosis protein candidates in Plasmodium falciparum using GFP-tagged fusion constructs
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
Production, purification, and characterisation of proteases from an ericoid mycorrhizal fungus, Oidiodendron maius
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: Expected release date-April 2019
- Full Text:
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: Expected release date-April 2019
- Full Text:
The development of biodegradable aerogel scaffolds for the generation of vascularised 3D adipose tissue models
- Authors: Makhene, Lebohang
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59245 , vital:27492
- Description: Expected release date-April 2019
- Full Text:
- Authors: Makhene, Lebohang
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59245 , vital:27492
- Description: Expected release date-April 2019
- Full Text:
Application of machine learning, molecular modelling and structural data mining against antiretroviral drug resistance in HIV-1
- Sheik Amamuddy, Olivier Serge André
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
Identification of potential novel roles for Hsp70/Hsp90 organising protein (Hop) using proteomic analysis in human cells
- Authors: Wingate, Ianthe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64758 , vital:28598
- Description: Expected release date-May 2018
- Full Text:
- Authors: Wingate, Ianthe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64758 , vital:28598
- Description: Expected release date-May 2018
- Full Text:
The relationship between OCT4 and an aggressive phenotype in triple negative breast cancer (TNBC)
- Authors: Jackson, Hayley Claire
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59209 , vital:27477
- Description: Expected release date-April 2019
- Full Text:
- Authors: Jackson, Hayley Claire
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59209 , vital:27477
- Description: Expected release date-April 2019
- Full Text:
Molecular cloning and expression of equine CYP1A2 in Escherichia coli
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
Biological properties and interactions of Kalaharituber pfeilii
- Authors: Krele, Viwe
- Date: 2019
- Subjects: Kalaharituber pfeilii , Pezizales -- South Africa , Desert plants -- South Africa , Truffle culture -- South Africa , Plant biochemical genetics , Enzymes -- Analysis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72257 , vital:30022
- Description: Dessert truffles are seasonal macro fungi and have been identified in several parts of the world including South Africa. The first part of the present study dealt with the assessment of the biologically active compounds of the Kalahari truffles found in the Northern Cape of South Africa. Truffles extracts (methanol, ethanol, aqueous) were investigated for their antimicrobial properties towards Gram-positive and Gram-negative bacteria. The results demonstrated that the truffle extracts tested had no inhibitory effects against the bacterial isolates. The truffle mycelial growth was also noted to be ineffective against the selected bacteria. The bacteria tested in the present study showed some antagonistic effects against the fungus. Cultures of K. pfeilii were also screened for enzyme production including amylase, protease, cellulose, and laccase. Evaluation of the potential of K. pfeilii mycelia to produce these industrially and economically important enzymes demonstrated both amylase and protease activity. However, for laccase and cellulose, no activity was detected. The second part of the present study aimed at optimizing biomass production by K. pfeilii in liquid culture media. FF Microplate containing 95 discreet carbon sources were employed to test for substrate utilization. Blanked readings above 0.1 were regarded as positive for utilization, and 4 substrates were selected as potential substrates and were included in liquid media. Media was evaluated for mycelial biomass production. Of the carbon sources tested sucrose proved to be the most suitable for supporting mycelial growth. The third part of the current study included investigating the diversity of microbial communities colonizing the rhizosheath of Stipagrostis ciliata var. capensis (the host plant of K. pfeilii) and these were identified by means of next-generation sequencing using Illumina Miseq. Bioinformatics tools were utilized in analyzing the data. Actinobacteria were found to be the most dominant bacterial phylum, followed by unclassified bacteria, Proteobacteria, and Acidobacteria. The top 25 sequences were selected and clustered into bacterial OTUs (at 97% threshold) which were assigned into 1 phylum (Actinobacteria), 1 family (Geodermatophilaceae) and 23 genera. This phylum is well known for its secondary metabolites. Streptomyces sp. was the most frequently encountered genus. The results from this study necessitate further investigations with regards to the function and evolution of fungal-bacterial associations. Wheather these bacteria have a contribution towards the truffle development, it is still not confirmed.
- Full Text:
- Authors: Krele, Viwe
- Date: 2019
- Subjects: Kalaharituber pfeilii , Pezizales -- South Africa , Desert plants -- South Africa , Truffle culture -- South Africa , Plant biochemical genetics , Enzymes -- Analysis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72257 , vital:30022
- Description: Dessert truffles are seasonal macro fungi and have been identified in several parts of the world including South Africa. The first part of the present study dealt with the assessment of the biologically active compounds of the Kalahari truffles found in the Northern Cape of South Africa. Truffles extracts (methanol, ethanol, aqueous) were investigated for their antimicrobial properties towards Gram-positive and Gram-negative bacteria. The results demonstrated that the truffle extracts tested had no inhibitory effects against the bacterial isolates. The truffle mycelial growth was also noted to be ineffective against the selected bacteria. The bacteria tested in the present study showed some antagonistic effects against the fungus. Cultures of K. pfeilii were also screened for enzyme production including amylase, protease, cellulose, and laccase. Evaluation of the potential of K. pfeilii mycelia to produce these industrially and economically important enzymes demonstrated both amylase and protease activity. However, for laccase and cellulose, no activity was detected. The second part of the present study aimed at optimizing biomass production by K. pfeilii in liquid culture media. FF Microplate containing 95 discreet carbon sources were employed to test for substrate utilization. Blanked readings above 0.1 were regarded as positive for utilization, and 4 substrates were selected as potential substrates and were included in liquid media. Media was evaluated for mycelial biomass production. Of the carbon sources tested sucrose proved to be the most suitable for supporting mycelial growth. The third part of the current study included investigating the diversity of microbial communities colonizing the rhizosheath of Stipagrostis ciliata var. capensis (the host plant of K. pfeilii) and these were identified by means of next-generation sequencing using Illumina Miseq. Bioinformatics tools were utilized in analyzing the data. Actinobacteria were found to be the most dominant bacterial phylum, followed by unclassified bacteria, Proteobacteria, and Acidobacteria. The top 25 sequences were selected and clustered into bacterial OTUs (at 97% threshold) which were assigned into 1 phylum (Actinobacteria), 1 family (Geodermatophilaceae) and 23 genera. This phylum is well known for its secondary metabolites. Streptomyces sp. was the most frequently encountered genus. The results from this study necessitate further investigations with regards to the function and evolution of fungal-bacterial associations. Wheather these bacteria have a contribution towards the truffle development, it is still not confirmed.
- Full Text:
Preliminary investigation of the molecular pathogenicity determinants of Xanthomonas campestris pv. zeae
- Authors: Downing, T G
- Date: 1998
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4066 , http://hdl.handle.net/10962/d1004922
- Description: Xanthomonas campestris pv. zeae was shown to posses a vast range of potential plant cell wall degrading enzymes including at least one protease, a carboxymethylcellulase, pectin lyase, polygalacturonase and a B-endoglucanase. Replicon stability and transfer system efficiencies were determined for a range of oriC, and oriT -tra combinations, and suitable vectors were constructed and identified for insertional inactivation by homologous recombination. Ideal suicide replicons were found to be pACYC184 and p15A, while P, Wand Q group replicons were supported by X campestris pv. zeae. Group P and group N transfer systems were shown to be highly efficient in intra-genus matings between Escherichia coli and X campestris pv. zeae, with the exception of P-tra systems in trans for the delivery of Tn5. Various cloning vectors were tested for stability and mobility. Tn5 was shown to transpose at high frequencies into the genome of the bacterial plant pathogen, and insertion was relatively random. Suitable screening assays were established to allow rapid isolation of mutants with potential virulence or pathogenic deviations, after mutagenesis. Two non-pathogenic mutants were identified, one of which was a putative hrp·, while the other was a leaky virulence. A single mutant showing 40% reduced protease activity was also shown to exhibit reduced virulence indicating a minor role for the protease in pathogenicity. The majority of virulence mutants showed altered growth in different levels of nutritional availability and complexity. Nutritional viability (the ability to acquire and use nutrients at a sufficient rate to grow fast enough to overcome host defences) was shown to be essential for virulence and possibly pathogenicity. Wild-type in-planta behaviour was analysed and growth and spread patterns typical for pathogenic response identified. Chief amongst these was the requirement for a threshold level of cells per leaf area or length, before symptoms could develop. Occlusion of vascular bundles was shown not to be the primary factor in the pathogenicity of X campestris pv. zeae. Threshold levels for lesion development indicate the absence of a diffusable lesion forming element, and possibly the requirement of cell density for induction of certain functions. , KMBT_363
- Full Text:
- Authors: Downing, T G
- Date: 1998
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4066 , http://hdl.handle.net/10962/d1004922
- Description: Xanthomonas campestris pv. zeae was shown to posses a vast range of potential plant cell wall degrading enzymes including at least one protease, a carboxymethylcellulase, pectin lyase, polygalacturonase and a B-endoglucanase. Replicon stability and transfer system efficiencies were determined for a range of oriC, and oriT -tra combinations, and suitable vectors were constructed and identified for insertional inactivation by homologous recombination. Ideal suicide replicons were found to be pACYC184 and p15A, while P, Wand Q group replicons were supported by X campestris pv. zeae. Group P and group N transfer systems were shown to be highly efficient in intra-genus matings between Escherichia coli and X campestris pv. zeae, with the exception of P-tra systems in trans for the delivery of Tn5. Various cloning vectors were tested for stability and mobility. Tn5 was shown to transpose at high frequencies into the genome of the bacterial plant pathogen, and insertion was relatively random. Suitable screening assays were established to allow rapid isolation of mutants with potential virulence or pathogenic deviations, after mutagenesis. Two non-pathogenic mutants were identified, one of which was a putative hrp·, while the other was a leaky virulence. A single mutant showing 40% reduced protease activity was also shown to exhibit reduced virulence indicating a minor role for the protease in pathogenicity. The majority of virulence mutants showed altered growth in different levels of nutritional availability and complexity. Nutritional viability (the ability to acquire and use nutrients at a sufficient rate to grow fast enough to overcome host defences) was shown to be essential for virulence and possibly pathogenicity. Wild-type in-planta behaviour was analysed and growth and spread patterns typical for pathogenic response identified. Chief amongst these was the requirement for a threshold level of cells per leaf area or length, before symptoms could develop. Occlusion of vascular bundles was shown not to be the primary factor in the pathogenicity of X campestris pv. zeae. Threshold levels for lesion development indicate the absence of a diffusable lesion forming element, and possibly the requirement of cell density for induction of certain functions. , KMBT_363
- Full Text: