A medicinal chemistry study in nitrogen containing heterocycles
- Authors: Lunga, Mayibongwe Junior
- Date: 2018
- Subjects: Indole , Tetrazoles , Antimalarials , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63521 , vital:28430
- Description: Heterocyclic structures have found extensive utility in the field of medicinal chemistry, as prominent regions of pharmacophores resulting in numerous drug treatments for many diseases. Accordingly, in this project we explored the respective antimalarial and anticancer activity exhibited by compounds featuring nitrogen containing indole and tetrazole heterocycles respectively. This thesis therefore comprises of two distinct parts. Part 1. Following the development of resistance towards traditional antimalarial therapy such as chloroquine and emerging resistance towards artemisinin combination therapies, the WHO reported the urgent need for new, effective drugs and identification of new drug targets to combat the Plasmodium falciparum parasite. In 2015 the parasite was the cause of 429 000 deaths, the majority occurring in the sub-Saharan region of Africa. This highlights the failing effectiveness of vector control strategies, reiterating the need to develop alternative control and treatment strategies. In response to this need we wanted to expand and further describe the SAR of the indole based series, indolyl-3-ethanone-α- thioethers, previously synthesized in our laboratory. These compounds were found to exhibit antimalarial activity with compounds 2.26 and 2.27 exhibiting activity against P. falciparum 3D7 in the nanomolar range. Based on these compounds we synthesized compounds 3.21 and 3.24 – 3.32 following a three step reaction pathway. Our results in this study, indicate that compound 3.28, a pnitrothiophenol analogue of 2.27 was the most active of the compounds we synthesized and furthermore was superior in activity against Plasmodium compared to 2.27. This result indicated that the presence of p-NO2 is important in enhancing anti-plasmodial activity. Comparing compounds 3.25 and 3.26 with an oxygen on the ether bridge to compounds 3.29 and 3.30 with a sulfur, we observed an increase in hydrophilicity coupled to a decrease in anti-plasmodial activity in the compounds, thus, highlighting the importance of sulfur for enhanced activity. Furthermore, we investigated bioisosteric replacement of the 5-chloro substituent present in hit compounds 2.27 and 3.28, with an electron withdrawing nitrile (3.27) and electron donating methyl (3.29) and methoxy (3.31) substituents. These substituents decreased anti-plasmodial activity, confirming that a chlorine substituent is optimal for biological activity. This study furthered our understanding of the SAR of indolyl-3-ethanone-α- thioethers for the development of potent anti-plasmodial lead compounds. Part 2. Triple negative breast cancer (TNBC), which disproportionately affects women of sub-Saharan Africa, is unresponsive to hormone-based therapies. This emergence presents a population of patients devoid of effective drug treatment, signaling the urgent need to develop new effective therapies with novel drug targets. Therefore, we identified our target in TNBC cells as the protein-protein interaction between the co-chaperones HOP and HSP90. We reasoned that a disruption of this interaction would ultimately result in cancer cell death via the degradation of essential oncogenic client proteins. Following a fragment screening campaign, which identified several acid and tetrazole containing hits (4.56 – 4.58) which bound to HOP, with low anticancer activity, we sought to develop synthetic methodology to elaborate our fragment hits synthesizing tetrazole containing fragments to target TNBC cell lines. We therefore proceeded to synthesize a range of multi substituted fragments (4.59 – 4.63), utilizing a nitrile (4.66) to access tetrazoles via 1,3-cycloaddition and an acid by nitrile hydrolysis. We successfully synthesized the tetrazole and acid fragments which are currently undergoing characterization for activity against TNBC. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Authors: Lunga, Mayibongwe Junior
- Date: 2018
- Subjects: Indole , Tetrazoles , Antimalarials , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63521 , vital:28430
- Description: Heterocyclic structures have found extensive utility in the field of medicinal chemistry, as prominent regions of pharmacophores resulting in numerous drug treatments for many diseases. Accordingly, in this project we explored the respective antimalarial and anticancer activity exhibited by compounds featuring nitrogen containing indole and tetrazole heterocycles respectively. This thesis therefore comprises of two distinct parts. Part 1. Following the development of resistance towards traditional antimalarial therapy such as chloroquine and emerging resistance towards artemisinin combination therapies, the WHO reported the urgent need for new, effective drugs and identification of new drug targets to combat the Plasmodium falciparum parasite. In 2015 the parasite was the cause of 429 000 deaths, the majority occurring in the sub-Saharan region of Africa. This highlights the failing effectiveness of vector control strategies, reiterating the need to develop alternative control and treatment strategies. In response to this need we wanted to expand and further describe the SAR of the indole based series, indolyl-3-ethanone-α- thioethers, previously synthesized in our laboratory. These compounds were found to exhibit antimalarial activity with compounds 2.26 and 2.27 exhibiting activity against P. falciparum 3D7 in the nanomolar range. Based on these compounds we synthesized compounds 3.21 and 3.24 – 3.32 following a three step reaction pathway. Our results in this study, indicate that compound 3.28, a pnitrothiophenol analogue of 2.27 was the most active of the compounds we synthesized and furthermore was superior in activity against Plasmodium compared to 2.27. This result indicated that the presence of p-NO2 is important in enhancing anti-plasmodial activity. Comparing compounds 3.25 and 3.26 with an oxygen on the ether bridge to compounds 3.29 and 3.30 with a sulfur, we observed an increase in hydrophilicity coupled to a decrease in anti-plasmodial activity in the compounds, thus, highlighting the importance of sulfur for enhanced activity. Furthermore, we investigated bioisosteric replacement of the 5-chloro substituent present in hit compounds 2.27 and 3.28, with an electron withdrawing nitrile (3.27) and electron donating methyl (3.29) and methoxy (3.31) substituents. These substituents decreased anti-plasmodial activity, confirming that a chlorine substituent is optimal for biological activity. This study furthered our understanding of the SAR of indolyl-3-ethanone-α- thioethers for the development of potent anti-plasmodial lead compounds. Part 2. Triple negative breast cancer (TNBC), which disproportionately affects women of sub-Saharan Africa, is unresponsive to hormone-based therapies. This emergence presents a population of patients devoid of effective drug treatment, signaling the urgent need to develop new effective therapies with novel drug targets. Therefore, we identified our target in TNBC cells as the protein-protein interaction between the co-chaperones HOP and HSP90. We reasoned that a disruption of this interaction would ultimately result in cancer cell death via the degradation of essential oncogenic client proteins. Following a fragment screening campaign, which identified several acid and tetrazole containing hits (4.56 – 4.58) which bound to HOP, with low anticancer activity, we sought to develop synthetic methodology to elaborate our fragment hits synthesizing tetrazole containing fragments to target TNBC cell lines. We therefore proceeded to synthesize a range of multi substituted fragments (4.59 – 4.63), utilizing a nitrile (4.66) to access tetrazoles via 1,3-cycloaddition and an acid by nitrile hydrolysis. We successfully synthesized the tetrazole and acid fragments which are currently undergoing characterization for activity against TNBC. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
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Aligning local government service delivery communication with digital citizen engagement: a case study of Makana Municipality
- Authors: Machiri, Mwazvita Chipo
- Date: 2018
- Subjects: Political participation South Africa Makana , Municipal services South Africa Makana , Communication in public administration South Africa Makana , Internet in public administration South Africa Makana , Information technology South Africa Makana , Electronic government information South Africa Makana , Government accountability South Africa Makana
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/194146 , vital:45423
- Description: The effective management of local municipalities in South Africa determines the quality of service delivery to citizens. Policies and other legislation have been implemented in an attempt to improve service delivery backlogs in South Africa since post-apartheid. However, these have not been effective in meeting community needs, hence leading to poor service delivery and increased community protests of frustration. These problems are partly linked to the lack of communication between government and citizens, and within internal government. Government needs to be held accountable for the services it provides, and improve on these services through practicing transparency, providing information to citizens, and allowing citizens to provide feedback on the various challenges experienced. Information access and communication is vital to the citizen engagement process, as this supports evidence-based engagement between citizens and government. Over the most recent years, innovative ICTs have emerged as a critical strategic tool in facilitating communication between the government and its citizens, commonly referred to as digital citizen engagement. The effective implementation of such initiatives at the local government level, especially in resource-constrained contexts, is not straightforward. This research aims to develop a guiding framework for aligning municipal communication processes with innovative ICTs to support ongoing social accountability and transparency through citizen engagement in local municipalities. The formulation of this framework is built on the Adaptive Structuration Theory, which is based on studying organisational change that occurs due to implementation and adoption of technology. Using a pragmatist approach and case study of Makana Municipality, the research study investigates a digital citizen engagement initiative called MobiSAM (Mobile Social Accountability Monitoring). The key findings from the qualitative empirical investigation indicates that there are significant social and political factors to consider when aligning Digital Citizen Engagement initiatives in a resource-constrained environment. The findings illustrated that the change and alignment process of DCE in local municipalities rely on a diverse set of inputs from different stakeholders, and a dynamic change process, which result in key citizen engagement outcomes of empowerment, accountability, transparency, and increased engagement depending – all depending on the success of the process. The extent of the outcomes will vary with the effectiveness of change management, idea generation, and participation, which Preliminaries are impacted by perceived usefulness, attitude towards the system, and the intentions of the user. The proposed framework provides a guideline for the implementation and introduction of innovations ICTs for citizen engagement at local government level. , Thesis (MCom) -- Faculty of Commerce, Information Systems, 2018
- Full Text:
- Authors: Machiri, Mwazvita Chipo
- Date: 2018
- Subjects: Political participation South Africa Makana , Municipal services South Africa Makana , Communication in public administration South Africa Makana , Internet in public administration South Africa Makana , Information technology South Africa Makana , Electronic government information South Africa Makana , Government accountability South Africa Makana
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/194146 , vital:45423
- Description: The effective management of local municipalities in South Africa determines the quality of service delivery to citizens. Policies and other legislation have been implemented in an attempt to improve service delivery backlogs in South Africa since post-apartheid. However, these have not been effective in meeting community needs, hence leading to poor service delivery and increased community protests of frustration. These problems are partly linked to the lack of communication between government and citizens, and within internal government. Government needs to be held accountable for the services it provides, and improve on these services through practicing transparency, providing information to citizens, and allowing citizens to provide feedback on the various challenges experienced. Information access and communication is vital to the citizen engagement process, as this supports evidence-based engagement between citizens and government. Over the most recent years, innovative ICTs have emerged as a critical strategic tool in facilitating communication between the government and its citizens, commonly referred to as digital citizen engagement. The effective implementation of such initiatives at the local government level, especially in resource-constrained contexts, is not straightforward. This research aims to develop a guiding framework for aligning municipal communication processes with innovative ICTs to support ongoing social accountability and transparency through citizen engagement in local municipalities. The formulation of this framework is built on the Adaptive Structuration Theory, which is based on studying organisational change that occurs due to implementation and adoption of technology. Using a pragmatist approach and case study of Makana Municipality, the research study investigates a digital citizen engagement initiative called MobiSAM (Mobile Social Accountability Monitoring). The key findings from the qualitative empirical investigation indicates that there are significant social and political factors to consider when aligning Digital Citizen Engagement initiatives in a resource-constrained environment. The findings illustrated that the change and alignment process of DCE in local municipalities rely on a diverse set of inputs from different stakeholders, and a dynamic change process, which result in key citizen engagement outcomes of empowerment, accountability, transparency, and increased engagement depending – all depending on the success of the process. The extent of the outcomes will vary with the effectiveness of change management, idea generation, and participation, which Preliminaries are impacted by perceived usefulness, attitude towards the system, and the intentions of the user. The proposed framework provides a guideline for the implementation and introduction of innovations ICTs for citizen engagement at local government level. , Thesis (MCom) -- Faculty of Commerce, Information Systems, 2018
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An investigation into the bacterial communities associated with pyrroloiminoquinone-producing South African latrunculid sponges
- Authors: Hilliar, Storm Hannah
- Date: 2018
- Subjects: Sponges South Africa Algoa Bay , Betaproteobacteria , Spirochaeta , Symbiosis , Bacterial communities
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62112 , vital:28128
- Description: Marine sponges belonging to the family Latrunculiidae are known for their production of cytotoxic pyrroloiminoquinone alkaloids and the South African coast provides a unique environment for the exploitation of these potent bioactive compounds. The isolation of structurally similar pyrroloiminoquinone compounds from unrelated, non poriferan sources has led to the suggestion that South African latrunculid pyrroloiminoquinones may be secondary metabolites produced by sponge associated microbial symbionts. Previous studies investigating the bacterial communities of South African latrunculid sponges have shown the conservation of distinct microbial populations with unusual bacterial taxa dominated by a novel betaproteobacterial and spirochete species. This study describes the further investigation into these associated bacterial communities, their conservation and sponge microbiome comparisons across spatial, temporal and environmental scales. The bacterial communities associated with seven latrunculid species representing three genera (Tsitsikamma, Cyclacanthia and Latrunculia) were characterized as well as a Mycale and Tethya rubra species. Latrunculid sponge microbiomes were significantly different from those associated with sympatric outlier sponge species and the surrounding environment. The bacterial communities associated with latrunculid sponges appear host specific with the conservation of two dominant bacterial symbionts which mirror the phylogeny of their host species. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Hilliar, Storm Hannah
- Date: 2018
- Subjects: Sponges South Africa Algoa Bay , Betaproteobacteria , Spirochaeta , Symbiosis , Bacterial communities
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62112 , vital:28128
- Description: Marine sponges belonging to the family Latrunculiidae are known for their production of cytotoxic pyrroloiminoquinone alkaloids and the South African coast provides a unique environment for the exploitation of these potent bioactive compounds. The isolation of structurally similar pyrroloiminoquinone compounds from unrelated, non poriferan sources has led to the suggestion that South African latrunculid pyrroloiminoquinones may be secondary metabolites produced by sponge associated microbial symbionts. Previous studies investigating the bacterial communities of South African latrunculid sponges have shown the conservation of distinct microbial populations with unusual bacterial taxa dominated by a novel betaproteobacterial and spirochete species. This study describes the further investigation into these associated bacterial communities, their conservation and sponge microbiome comparisons across spatial, temporal and environmental scales. The bacterial communities associated with seven latrunculid species representing three genera (Tsitsikamma, Cyclacanthia and Latrunculia) were characterized as well as a Mycale and Tethya rubra species. Latrunculid sponge microbiomes were significantly different from those associated with sympatric outlier sponge species and the surrounding environment. The bacterial communities associated with latrunculid sponges appear host specific with the conservation of two dominant bacterial symbionts which mirror the phylogeny of their host species. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
Chipangano: vigilantism and community responses in Mbare District, Zimbabwe, c.2000-2013
- Authors: Munyarari, Tinashe
- Date: 2018
- Subjects: Vigilantism Zimbabwe Mbare , ZANU-PF (Organization : Zimbabwe) , Zimbabwe History 1980- , Vigilantism Zimbabwe Mbare Public opinion , Collective memory Zimbabwe Mbare
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62126 , vital:28130
- Description: This study examines an aspect of Zimbabwe’s political history, namely the emergence and operations of Chipangano vigilante group in Mbare (2000-2013) and how the community responded to the scourges of this vigilante group. This study shows that Chipangano, which means a pact, was formed with the intention to extend ZANU-PF patronage system, canvassing support and regain control of Mbare district from the MDC. The group consolidated itself as a shadow militia group for ZANU-PF that intimidated the opposition parties and coerced the general populace to attend ZANU-PF functions. It also shows that the vigilante group seized Harare City Council’s duties, such as collecting gate-takings from local and long distance public transport at Mbare Bus Terminal; controlling allocations of market stalls and collecting rents from market stalls, for personal benefits. The group also politicised the access to local state property. Chipangano’s collusion with ZANU-PF and state structures licensed it to engage in criminal activities with impunity, thus this thesis seeks to understand the relationship between the vigilantes, ZANU-PF and state structures. The activities of Chipangano such as abduction, intimidation, beatings, killings and displacement of people resulted in social trauma. This thesis will also explore how this phase of violence is remembered today by the research informants. , Thesis (MA) -- Faculty of Humanities, History, 2018
- Full Text:
- Authors: Munyarari, Tinashe
- Date: 2018
- Subjects: Vigilantism Zimbabwe Mbare , ZANU-PF (Organization : Zimbabwe) , Zimbabwe History 1980- , Vigilantism Zimbabwe Mbare Public opinion , Collective memory Zimbabwe Mbare
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62126 , vital:28130
- Description: This study examines an aspect of Zimbabwe’s political history, namely the emergence and operations of Chipangano vigilante group in Mbare (2000-2013) and how the community responded to the scourges of this vigilante group. This study shows that Chipangano, which means a pact, was formed with the intention to extend ZANU-PF patronage system, canvassing support and regain control of Mbare district from the MDC. The group consolidated itself as a shadow militia group for ZANU-PF that intimidated the opposition parties and coerced the general populace to attend ZANU-PF functions. It also shows that the vigilante group seized Harare City Council’s duties, such as collecting gate-takings from local and long distance public transport at Mbare Bus Terminal; controlling allocations of market stalls and collecting rents from market stalls, for personal benefits. The group also politicised the access to local state property. Chipangano’s collusion with ZANU-PF and state structures licensed it to engage in criminal activities with impunity, thus this thesis seeks to understand the relationship between the vigilantes, ZANU-PF and state structures. The activities of Chipangano such as abduction, intimidation, beatings, killings and displacement of people resulted in social trauma. This thesis will also explore how this phase of violence is remembered today by the research informants. , Thesis (MA) -- Faculty of Humanities, History, 2018
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Evaluating metabolism-induced toxicity using a non-hepatic cell line
- Authors: Weyers, Carli
- Date: 2018
- Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/61950 , vital:28087
- Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Authors: Weyers, Carli
- Date: 2018
- Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/61950 , vital:28087
- Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
Exploring para-thiophenols to expand the SAR of antimalarial 3-indolylethanones
- Authors: Chisango, Ruramai Lissa
- Date: 2018
- Subjects: Antimalarials , Malaria Chemotherapy , Thiols , Plasmodium falciparum , Blood-brain barrier
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63515 , vital:28428
- Description: According to the WHO, malaria is responsible for over half a million deaths annually especially in populations from disadvantaged settings. Although there has been a documented improvement in the mortality rates, malaria has proved to be a global emergency. Mostly affecting the poor population, this disease is perpetuating a vicious cycle of poverty in the developing world as current preventive measures are not adequate unless adopted in addition to effective treatment. However, there has been a worldwide increase in resistance to available treatment which presents a need for novel, affordable treatment. A study conducted in our laboratory identified two hit thiophenol containing compounds 2.24 and 2.25. These molecules provided initial insight into the SAR and potential pharmacophore of this class of compounds. We decided to further explore these compounds by making bioisosteric replacements to optimize the structure as we monitor the effect of these modifications on the anti-plasmodial activity. The synthetic pathway to form the target compounds of our study comprised of three steps which were initiated by the Friedel-Crafts acetylation of the indoles resulting in compounds 3.5 - 3.7. A bromination step followed which yielded the -bromo ketones (3.8 - 3.11). Some of the thiophenols (3.14 and 3.16) were not readily available in our laboratory and so were synthesized for the final synthetic step. This step involved the nucleophilic displacement of the -bromine to generate the -aryl substituted 3-indolylethanones (3.17 - 3.27). The thioethers displayed improved antimalarial activity from 2.24 and 2.25 against the chloroquine sensitive 3D7 Plasmodium falciparum strain. In addition, these compounds were non-toxic against HeLa cells which indicated this potential novel class of antimalarials is selective for the malaria parasite as hypothesized in the previous study conducted in our laboratory. In an attempt to predict the bioavailability of some of our compounds, in silico studies were conducted revealing that these compounds could be passively absorbed by the gastrointestinal tract, a positive result for bioavailability purposes. However, results from these studies indicate that modifications of these compounds would be necessary to allow for permeation through the blood brain barrier (BBB) for instances when the patient has cerebral malaria. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Authors: Chisango, Ruramai Lissa
- Date: 2018
- Subjects: Antimalarials , Malaria Chemotherapy , Thiols , Plasmodium falciparum , Blood-brain barrier
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63515 , vital:28428
- Description: According to the WHO, malaria is responsible for over half a million deaths annually especially in populations from disadvantaged settings. Although there has been a documented improvement in the mortality rates, malaria has proved to be a global emergency. Mostly affecting the poor population, this disease is perpetuating a vicious cycle of poverty in the developing world as current preventive measures are not adequate unless adopted in addition to effective treatment. However, there has been a worldwide increase in resistance to available treatment which presents a need for novel, affordable treatment. A study conducted in our laboratory identified two hit thiophenol containing compounds 2.24 and 2.25. These molecules provided initial insight into the SAR and potential pharmacophore of this class of compounds. We decided to further explore these compounds by making bioisosteric replacements to optimize the structure as we monitor the effect of these modifications on the anti-plasmodial activity. The synthetic pathway to form the target compounds of our study comprised of three steps which were initiated by the Friedel-Crafts acetylation of the indoles resulting in compounds 3.5 - 3.7. A bromination step followed which yielded the -bromo ketones (3.8 - 3.11). Some of the thiophenols (3.14 and 3.16) were not readily available in our laboratory and so were synthesized for the final synthetic step. This step involved the nucleophilic displacement of the -bromine to generate the -aryl substituted 3-indolylethanones (3.17 - 3.27). The thioethers displayed improved antimalarial activity from 2.24 and 2.25 against the chloroquine sensitive 3D7 Plasmodium falciparum strain. In addition, these compounds were non-toxic against HeLa cells which indicated this potential novel class of antimalarials is selective for the malaria parasite as hypothesized in the previous study conducted in our laboratory. In an attempt to predict the bioavailability of some of our compounds, in silico studies were conducted revealing that these compounds could be passively absorbed by the gastrointestinal tract, a positive result for bioavailability purposes. However, results from these studies indicate that modifications of these compounds would be necessary to allow for permeation through the blood brain barrier (BBB) for instances when the patient has cerebral malaria. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
Production, purification, and characterisation of proteases from an ericoid mycorrhizal fungus, Oidiodendron maius
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
Vachellia erioloba (camel thorn) and microbial interactions
- Authors: Van Aswegen, Sunet
- Date: 2018
- Subjects: Vesicular-arbuscular mycorrhizas , Cadmium , Rhizobacteria , Plant growth-promoting rhizobacteria , Acacia erioloba
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63716 , vital:28475
- Description: Vachellia erioloba (camel thorn) is one of South Africa’s economically important tree species and therefore requires further investigation to improve its health and growth. Beneficial soil microbes have positive effects on plants through various mechanisms such as nitrogen fixation, phosphate solubilisation, indole acetic acid and siderophore production and biofilm formation. These traits enhance plant growth and protect the host plant against parasitic organisms that are present in soil. The arbuscular mycorrhizal (AM) fungi are well known for their beneficial symbiotic effects on host plants. The objective of this study was to determine the role of AM fungi and associated beneficial rhizobacteria in improving the growth of V. erioloba seedlings. Soil and root samples were collected from a farm in the Northern Cape, South Africa. Fifty-seven bacterial cultures were isolated from the soil and tested for plant growth promoting characteristics. Fourteen isolates showing at least four beneficial traits were molecularly identified using the GenBank database. The AM fungal and bacterial populations in the soil samples were assessed using Illumina sequencing. Sequences were identified using the MaarJAM and GenBank databases, respectively. Three separate pot trials were conducted to determine; 1) the effects of cadmium (Cd) on seedling growth; 2) the individual effects of three selected bacterial isolates and AM fungi alone and combined on seedling growth, and 3) the combined effects of the selected bacteria on AM fungal inoculated and uninoculated seedlings. Of the fourteen isolates the Enterobacter genera was the dominant species identified, with Acinetobacter, Pantoea and Bacillus each having one isolate. All were described as plant growth promoting rhizobacteria. One isolate from each genus, excluding Pantoea, was used in the pot trials. Three genera were identified in the AM fungal population that was assessed, namely Ambispora, Paraglomus and Glomus with Ambispora being the dominant genus. The bacterial population assessed showed a high diversity of bacteria from the Actinobacteria phylum being the dominant group. The results of the heavy metal pot trial showed that the symbiotic relationship between the seedlings and AM fungi increased the seedlings’ health and growth during heavy metal stress. The combination of bacteria and AM fungi increased growth parameters in all the inoculated seedlings, but not when compared to uninoculated seedlings indicating a possible competition for nutrients. The results were influenced by the presence of a nematode, which was suspected to have been seed borne. Further investigations on these interactions are required. Inoculation of AM fungi and selected PGPR is recommended for V. erioloba seedling production. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Van Aswegen, Sunet
- Date: 2018
- Subjects: Vesicular-arbuscular mycorrhizas , Cadmium , Rhizobacteria , Plant growth-promoting rhizobacteria , Acacia erioloba
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63716 , vital:28475
- Description: Vachellia erioloba (camel thorn) is one of South Africa’s economically important tree species and therefore requires further investigation to improve its health and growth. Beneficial soil microbes have positive effects on plants through various mechanisms such as nitrogen fixation, phosphate solubilisation, indole acetic acid and siderophore production and biofilm formation. These traits enhance plant growth and protect the host plant against parasitic organisms that are present in soil. The arbuscular mycorrhizal (AM) fungi are well known for their beneficial symbiotic effects on host plants. The objective of this study was to determine the role of AM fungi and associated beneficial rhizobacteria in improving the growth of V. erioloba seedlings. Soil and root samples were collected from a farm in the Northern Cape, South Africa. Fifty-seven bacterial cultures were isolated from the soil and tested for plant growth promoting characteristics. Fourteen isolates showing at least four beneficial traits were molecularly identified using the GenBank database. The AM fungal and bacterial populations in the soil samples were assessed using Illumina sequencing. Sequences were identified using the MaarJAM and GenBank databases, respectively. Three separate pot trials were conducted to determine; 1) the effects of cadmium (Cd) on seedling growth; 2) the individual effects of three selected bacterial isolates and AM fungi alone and combined on seedling growth, and 3) the combined effects of the selected bacteria on AM fungal inoculated and uninoculated seedlings. Of the fourteen isolates the Enterobacter genera was the dominant species identified, with Acinetobacter, Pantoea and Bacillus each having one isolate. All were described as plant growth promoting rhizobacteria. One isolate from each genus, excluding Pantoea, was used in the pot trials. Three genera were identified in the AM fungal population that was assessed, namely Ambispora, Paraglomus and Glomus with Ambispora being the dominant genus. The bacterial population assessed showed a high diversity of bacteria from the Actinobacteria phylum being the dominant group. The results of the heavy metal pot trial showed that the symbiotic relationship between the seedlings and AM fungi increased the seedlings’ health and growth during heavy metal stress. The combination of bacteria and AM fungi increased growth parameters in all the inoculated seedlings, but not when compared to uninoculated seedlings indicating a possible competition for nutrients. The results were influenced by the presence of a nematode, which was suspected to have been seed borne. Further investigations on these interactions are required. Inoculation of AM fungi and selected PGPR is recommended for V. erioloba seedling production. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta
- Authors: Van der Merwe, Marcel
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62963 , vital:28347
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Van der Merwe, Marcel
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62963 , vital:28347
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
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