Application of web design techniques and best practices in implementing web development, maintenance and enhancement of RUBi websites and web application systems
- Authors: Tshabalalala, Thulani
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424688 , vital:72175
- Description: The popularity of the web has seen various fields, such as the sciences taking advantage of this resource to further their scientific endeavours. This has seen science groups moving into developing websites and web applications, and such a group is the Research Unit in Bioinformative (RUBi). With the use of the web, the development and maintenance of whatever web-related tools become inevitable, given the continuous changes in the web space. This continuous evolution of web development and maintenance will come with techniques, principles and standards which will not only enable faster development of web entities but also ensure that modern hardware, fulfilment of the requirements to use such hardware and modern concepts are incorporated into forming web tools that enable such progression. Furthermore, introducing the previously mentioned progress of the web becomes an essential part of its development and maintenance. This paper did implement the processes of progressing the web using the technique of documentation and version control systems. The web development for the COVIDRUG website was done for the Covidrug-Africa Consortium (COVIDRUG) using the Django webdevelopment framework. The RUBi website and the MDM-Task we band the Job Management System (JMS) web applications were maintained for the maintenance aspect. Archives brought value regarding the traceability it provides of the various web-related aspects. The development showed a website’s potential value, particularly for research groups. The maintenance carried out showed how different techniques and approaches could be used in different maintenance prospects to achieve set objectives. The development and maintenance resulted in websites and web applications that have the features stated in their respective maintenance plans. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Tshabalalala, Thulani
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424688 , vital:72175
- Description: The popularity of the web has seen various fields, such as the sciences taking advantage of this resource to further their scientific endeavours. This has seen science groups moving into developing websites and web applications, and such a group is the Research Unit in Bioinformative (RUBi). With the use of the web, the development and maintenance of whatever web-related tools become inevitable, given the continuous changes in the web space. This continuous evolution of web development and maintenance will come with techniques, principles and standards which will not only enable faster development of web entities but also ensure that modern hardware, fulfilment of the requirements to use such hardware and modern concepts are incorporated into forming web tools that enable such progression. Furthermore, introducing the previously mentioned progress of the web becomes an essential part of its development and maintenance. This paper did implement the processes of progressing the web using the technique of documentation and version control systems. The web development for the COVIDRUG website was done for the Covidrug-Africa Consortium (COVIDRUG) using the Django webdevelopment framework. The RUBi website and the MDM-Task we band the Job Management System (JMS) web applications were maintained for the maintenance aspect. Archives brought value regarding the traceability it provides of the various web-related aspects. The development showed a website’s potential value, particularly for research groups. The maintenance carried out showed how different techniques and approaches could be used in different maintenance prospects to achieve set objectives. The development and maintenance resulted in websites and web applications that have the features stated in their respective maintenance plans. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
Identification of novel Arf1 GTPase inhibitors for cancer target validation
- Authors: Mqwathi, Nomxolisi Vuyokasi
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424666 , vital:72173
- Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Mqwathi, Nomxolisi Vuyokasi
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424666 , vital:72173
- Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
The development and op timisation of a Theiler’s murine encephalomyelitis virus antiviral assay
- Authors: Naidoo, Urisha Tirah
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424677 , vital:72174
- Description: Picornaviruses belong to the Picornaviridae family which are one of the largest and most diverse family of RNA viruses that cause a broad spectrum of infections in both humans and animals. These diseases range from severe infections such as poliomyelitis, meningitis, myocarditis to mild illnesses such as the common cold. Picornavirus outbreaks are a worldwide threat as they are continuously occurring. A recent outbreak of foot-and-mouth disease caused by a picornavirus occurred in South Africa, resulting in a temporary ban on the movement of cattle. Currently, the FDA has not approved any antiviral drugs against this virus, increasing the urgency for identifying effective antivirals. Picornaviruses have similar genomes and capsid organisation as such, those that are non-hazardous to humans can be used as a model system. A Theiler’s murine encephalomyelitis virus (TMEV) strain GDVII and Baby Hamster Kidney fibroblasts (BHK-21 cells) was used as a replication system to develop and optimise a medium-throughput antiviral screening assay. The TMEV GDVII replication system in BHK-21 cells was validated, and preliminary experiments were performed that were necessary for the development of the TMEV GDVII antiviral assay. This was achieved by conducting a CPE assay to visually monitor the onset and development of CPE induced by TMEV GDVII. Plaque assays accurately quantified the number of infectious virus particles required for calculating the MOI in downstream experiments. Lastly, indirect immunofluorescence and Western blot analysis detected the expression of viral proteins using previously generated antibodies against the TMEV GDVII VP1 capsid and 2C protein, thereby confirming infection in BHK-21 cells. The development of robust and reproducible assays is an essential component in antiviral drug discovery. Therefore, the confirmed replication system was then used as a foundation to develop a medium-throughput CPE-based TMEV GDVII antiviral assay whereby the parameters were optimised to produce one of high quality. Firstly, the quantitation of viral-induced CPE was examined and confirmed in a 96-well plate using resazurin as a cell viability indicator. Each parameter was tested at varying conditions, and the optimal was concluded as 2 % FBS in the assay media, a 15 000 cells/well seeding density, infecting the cells with TMEV GDVII at an MOI of 0.00625 and measuring resazurin at an endpoint of 72 hpi. Furthermore, the parameters were ii validated by calculating the Z’- factor, which consistently produced scores above 0.5, indicative of a reliable, robust, reproducible antiviral assay. Currently, there are no inhibitors against TMEV GDVII that have been reported or confirmed in cell lines, animal models or clinical trials. Therefore, once the optimal assay parameters were selected, it presented an opportunity to assess whether potential compounds, including itraconazole (ITZ) and dipyridamole (DIP), possessed antiviral activity that could firstly, be utilised as a control inhibitor when screening compounds against TMEV GDVII and secondly, contribute to research on this virus. Additionally, the previously produced anti-TMEV GDVII capsid antibody was shown to neutralise viral infection and was also included as a potential control. The sensitivity of the cells towards DMSO, a solution in which the compounds were solubilised, was first investigated. It was found that concentrations above 1 % are toxic to the cells; as such, the final DMSO concentrations were always kept below 1 % when screening compounds. Lastly, the generation of dose-response curves aided in the conclusion that the antibody was the most suitable control inhibitor as it displayed potent antiviral activity and no cytotoxicity towards the cells. In contrast, ITZ and DIP did not possess effective antiviral action and were toxic to cells at high concentrations. Finally, after all the components of the medium-throughput TMEV GDVII antiviral assay were identified, it was possible to screen 24 compounds from a coumarin and marine natural product library for cell cytotoxicity and antiviral activity. After generating dose-response curves, it was concluded that no compound effectively inhibited virus-induced CPE, and most were toxic to cells at relatively high concentrations. In conclusion, this is the first study that describes the development and optimisation of a robust medium-throughput CPE-based antiviral assay that has immense potential to screen other libraries of compounds for antiviral activity against TMEV GDVII. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Naidoo, Urisha Tirah
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424677 , vital:72174
- Description: Picornaviruses belong to the Picornaviridae family which are one of the largest and most diverse family of RNA viruses that cause a broad spectrum of infections in both humans and animals. These diseases range from severe infections such as poliomyelitis, meningitis, myocarditis to mild illnesses such as the common cold. Picornavirus outbreaks are a worldwide threat as they are continuously occurring. A recent outbreak of foot-and-mouth disease caused by a picornavirus occurred in South Africa, resulting in a temporary ban on the movement of cattle. Currently, the FDA has not approved any antiviral drugs against this virus, increasing the urgency for identifying effective antivirals. Picornaviruses have similar genomes and capsid organisation as such, those that are non-hazardous to humans can be used as a model system. A Theiler’s murine encephalomyelitis virus (TMEV) strain GDVII and Baby Hamster Kidney fibroblasts (BHK-21 cells) was used as a replication system to develop and optimise a medium-throughput antiviral screening assay. The TMEV GDVII replication system in BHK-21 cells was validated, and preliminary experiments were performed that were necessary for the development of the TMEV GDVII antiviral assay. This was achieved by conducting a CPE assay to visually monitor the onset and development of CPE induced by TMEV GDVII. Plaque assays accurately quantified the number of infectious virus particles required for calculating the MOI in downstream experiments. Lastly, indirect immunofluorescence and Western blot analysis detected the expression of viral proteins using previously generated antibodies against the TMEV GDVII VP1 capsid and 2C protein, thereby confirming infection in BHK-21 cells. The development of robust and reproducible assays is an essential component in antiviral drug discovery. Therefore, the confirmed replication system was then used as a foundation to develop a medium-throughput CPE-based TMEV GDVII antiviral assay whereby the parameters were optimised to produce one of high quality. Firstly, the quantitation of viral-induced CPE was examined and confirmed in a 96-well plate using resazurin as a cell viability indicator. Each parameter was tested at varying conditions, and the optimal was concluded as 2 % FBS in the assay media, a 15 000 cells/well seeding density, infecting the cells with TMEV GDVII at an MOI of 0.00625 and measuring resazurin at an endpoint of 72 hpi. Furthermore, the parameters were ii validated by calculating the Z’- factor, which consistently produced scores above 0.5, indicative of a reliable, robust, reproducible antiviral assay. Currently, there are no inhibitors against TMEV GDVII that have been reported or confirmed in cell lines, animal models or clinical trials. Therefore, once the optimal assay parameters were selected, it presented an opportunity to assess whether potential compounds, including itraconazole (ITZ) and dipyridamole (DIP), possessed antiviral activity that could firstly, be utilised as a control inhibitor when screening compounds against TMEV GDVII and secondly, contribute to research on this virus. Additionally, the previously produced anti-TMEV GDVII capsid antibody was shown to neutralise viral infection and was also included as a potential control. The sensitivity of the cells towards DMSO, a solution in which the compounds were solubilised, was first investigated. It was found that concentrations above 1 % are toxic to the cells; as such, the final DMSO concentrations were always kept below 1 % when screening compounds. Lastly, the generation of dose-response curves aided in the conclusion that the antibody was the most suitable control inhibitor as it displayed potent antiviral activity and no cytotoxicity towards the cells. In contrast, ITZ and DIP did not possess effective antiviral action and were toxic to cells at high concentrations. Finally, after all the components of the medium-throughput TMEV GDVII antiviral assay were identified, it was possible to screen 24 compounds from a coumarin and marine natural product library for cell cytotoxicity and antiviral activity. After generating dose-response curves, it was concluded that no compound effectively inhibited virus-induced CPE, and most were toxic to cells at relatively high concentrations. In conclusion, this is the first study that describes the development and optimisation of a robust medium-throughput CPE-based antiviral assay that has immense potential to screen other libraries of compounds for antiviral activity against TMEV GDVII. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
Phytoplankton communities provide insight into ecosystem functioning of the Agulhas Current system
- Authors: Gibb, Ross-Lynne Alida
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422646 , vital:71965
- Description: Access restricted. Embargoed until 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Gibb, Ross-Lynne Alida
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422646 , vital:71965
- Description: Access restricted. Embargoed until 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-31
South African supratidal microbialites: prokaryote communities, metabolic capabilities, and biogeochemical processes
- Authors: Isemonger, Eric William
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422641 , vital:71964
- Description: Access restricted. Embargoed until 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Isemonger, Eric William
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422641 , vital:71964
- Description: Access restricted. Embargoed until 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-31
Biochemical and genetic analysis of the Mycobacterium smegmatis CnoX Chaperedoxin
- Authors: Watkins, Ariana Heloise Jo
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422403 , vital:71939
- Description: Mycobacterium (M.) tuberculosis (Mtb) encounters numerous physical and chemical stresses associated with host immunity during infection. These include exposure to reactive oxygen, chlorine and nitrogen species, low pH, hypoxia, nutrient starvation, and metal toxicity. Cellular proteins are particularly susceptible to damage by these stresses, and the ability to prevent their irreversible damage is consequently crucial for bacterial growth and survival. Mtb employs a network of proteins that includes chaperones, disaggregases, and proteases to maintain the integrity of its proteome. The chaperedoxin, CnoX, is a recently identified stress-inducible chaperone that combines redox and holdase activities to prevent the over-oxidation and aggregation of proteins in E. coli and other proteobacterial species. In this study, we identified orthologs of the E. coli CnoX (EcCnoX) in Mtb and M. smegmatis (Msm). Bioinformatics analysis of the Mtb and Msm CnoX orthologs (MtCnoX and MsCnoX, respectively) revealed that they possess similar domains, domain architectures and predicted tertiary structures as previously characterised CnoX enzymes, i.e. an N-terminal thioredoxin (Trx) domain fused to a C-terminal TPR-motif containing domain. The EcCnoX, MsCnoX, and MtCnoX enzymes were expressed as recombinant, His-tagged proteins in E. coli and purified to near homogeneity. Biochemical analysis of the recombinant CnoX enzymes revealed that the MsCnoX and MtCnoX both lack thiol-disulphide oxidoreductase (thioredoxin) activity, as evidenced by their inability to catalyse the reduction of the disulphide bonds of insulin in vitro. Both mycobacterial CnoX enzymes displayed activity as chaperones (holdases) during thermal aggregation assays of the model substrate, malate dehydrogenase (MDH). In contrast to previously reported findings for EcCnoX, the holdase activity of the mycobacterial CnoX enzymes was constitutive and did not require exposure to hypochlorous acid (HOCl) for activation. To establish the physiological role of CnoX in Msm, cnoX knockdown (KD) and knockout (KO) mutants were generated using CRISPRi-mediated gene silencing or homologous recombination, respectively. Consistent with previous findings, CnoX activity was not essential for the growth of Msm under conventional growth conditions. Reducing or eliminating CnoX activity in the Msm KD or KO mutants, respectively, did not confer increased sensitivity to HOCl as has been observed for an E. coli cnoX mutant. Reduced CnoX activity in Msm did, however, confer sensitivity to the superoxide generator, plumbagin, and front-line antitubercular drugs rifampicin and isoniazid. The combination of biochemical and physiological data presented suggests that MsCnoX may function as a holdase for substrates following proteotoxic damage induced by certain types of oxidants, a line of investigation that will be pursued in future studies. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Watkins, Ariana Heloise Jo
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422403 , vital:71939
- Description: Mycobacterium (M.) tuberculosis (Mtb) encounters numerous physical and chemical stresses associated with host immunity during infection. These include exposure to reactive oxygen, chlorine and nitrogen species, low pH, hypoxia, nutrient starvation, and metal toxicity. Cellular proteins are particularly susceptible to damage by these stresses, and the ability to prevent their irreversible damage is consequently crucial for bacterial growth and survival. Mtb employs a network of proteins that includes chaperones, disaggregases, and proteases to maintain the integrity of its proteome. The chaperedoxin, CnoX, is a recently identified stress-inducible chaperone that combines redox and holdase activities to prevent the over-oxidation and aggregation of proteins in E. coli and other proteobacterial species. In this study, we identified orthologs of the E. coli CnoX (EcCnoX) in Mtb and M. smegmatis (Msm). Bioinformatics analysis of the Mtb and Msm CnoX orthologs (MtCnoX and MsCnoX, respectively) revealed that they possess similar domains, domain architectures and predicted tertiary structures as previously characterised CnoX enzymes, i.e. an N-terminal thioredoxin (Trx) domain fused to a C-terminal TPR-motif containing domain. The EcCnoX, MsCnoX, and MtCnoX enzymes were expressed as recombinant, His-tagged proteins in E. coli and purified to near homogeneity. Biochemical analysis of the recombinant CnoX enzymes revealed that the MsCnoX and MtCnoX both lack thiol-disulphide oxidoreductase (thioredoxin) activity, as evidenced by their inability to catalyse the reduction of the disulphide bonds of insulin in vitro. Both mycobacterial CnoX enzymes displayed activity as chaperones (holdases) during thermal aggregation assays of the model substrate, malate dehydrogenase (MDH). In contrast to previously reported findings for EcCnoX, the holdase activity of the mycobacterial CnoX enzymes was constitutive and did not require exposure to hypochlorous acid (HOCl) for activation. To establish the physiological role of CnoX in Msm, cnoX knockdown (KD) and knockout (KO) mutants were generated using CRISPRi-mediated gene silencing or homologous recombination, respectively. Consistent with previous findings, CnoX activity was not essential for the growth of Msm under conventional growth conditions. Reducing or eliminating CnoX activity in the Msm KD or KO mutants, respectively, did not confer increased sensitivity to HOCl as has been observed for an E. coli cnoX mutant. Reduced CnoX activity in Msm did, however, confer sensitivity to the superoxide generator, plumbagin, and front-line antitubercular drugs rifampicin and isoniazid. The combination of biochemical and physiological data presented suggests that MsCnoX may function as a holdase for substrates following proteotoxic damage induced by certain types of oxidants, a line of investigation that will be pursued in future studies. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
Expression, partial characterisation and utilization of a GH11 xylanase (Xyn2A) from Trichoderma viride as an additive in monogastric animal feeds
- Mzimkulu-Ncoyi, Nosabatha Happyness
- Authors: Mzimkulu-Ncoyi, Nosabatha Happyness
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422409 , vital:71940
- Description: Endo-xylanases (shortly called xylanases) are a group of glycoside hydrolase enzymes that target β-D-1,4-linkages in the xylan backbone, leading to the production of xylooligosaccharides (XOS) of varying degree of polymerization (DP). Xylan is an indigestible non-starch polysaccharide present in monogastric animal feeds which in high amounts leads to increased digesta viscosity, slow movement of digesta in the intestines, malabsorption of nutrients among other challenges. The aim of this study was to investigate the effect of xylanase 2A (Xyn2A) from Trichoderma viride on broiler chicken feeds, particularly the hydrolysis of the xylan content, reduction of feed viscosity and the effect of produced XOS on eliciting the growth of gut associated probiotic bacteria. Xyn2AE was successfully induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced in Escherichia coli BL21 (DE3) and Xyn2AC was expressed in tobacco mosaic plants. For the purification of Xyn2AE, an immobilized metal affinity chromatography (IMAC) column and diafiltration using a 3kDa cut-off Amicon filter membranes were used. Xyn2AE and Xyn2AC showed a xylanase active band at a relative weight of 21 kDa. Both enzymes showed high specificity towards soluble wheat arabinoxylan (WAX), with specific activities of 7.61 U/mg for Xyn2AE and 536.5 U/mg for Xyn2AC. Xyn2A kinetic parameters (Vmax and Km) were determined by Michaelis-Menten plots on soluble and insoluble WAX. The Vmax and Km values of Xyn2AC were 1003.01 U/mg and 9.25 mg/mL, 302.89 U/mg and 13.54 mg/mL, respectively. The Vmax and Km values of Xyn2AE for soluble and insoluble WAX were 20.45 U/mg and 12.95 mg/mL, and 8.31 U/mg and 13.15 mg/mL. Xyn2A enzymes displayed optimum activity at pH and temperature parameters of 5.0 and 50°C, respectively, and stability in temperatures ranging between 50 and 80°C and pH 4.0-9.0. Broiler chicken feeds were hydrolysed using Xyn2AE over a 24 h period and analysed using the dinitrosalicylic (DNS) assay, thin layer chromatography (TLC), viscometry and visualized using scanning electron microscope (SEM). The results showed a release of release of XOS xylotriose, xylopentose and xylohexose; enzyme’s ability to decrease the viscosity of the feeds and punched holes of feed surface, which was indicative of xylanase action. XOS produced during hydrolysis was used to study prebiotic effect on selected few bacteria and released short chain fatty acids (SCFAs) were measured. Additionally, SCFAs formation was detected in the presence of XOS as a carbon source for S. thermophilus and L. bulgaricus, whereas B. subtilis formed fewer organic acids in the presence of XOS. The results obtained from this study demonstrated that the supplementation of Xyn2A on broiler feeds has ii a positive effect in decreasing feed viscosity. Furthermore, the results of this investigation will assist the South African poultry farming sector to increase profitability in poultry farming and gain stability in the global trade as far as poultry feed is concerned. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Mzimkulu-Ncoyi, Nosabatha Happyness
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422409 , vital:71940
- Description: Endo-xylanases (shortly called xylanases) are a group of glycoside hydrolase enzymes that target β-D-1,4-linkages in the xylan backbone, leading to the production of xylooligosaccharides (XOS) of varying degree of polymerization (DP). Xylan is an indigestible non-starch polysaccharide present in monogastric animal feeds which in high amounts leads to increased digesta viscosity, slow movement of digesta in the intestines, malabsorption of nutrients among other challenges. The aim of this study was to investigate the effect of xylanase 2A (Xyn2A) from Trichoderma viride on broiler chicken feeds, particularly the hydrolysis of the xylan content, reduction of feed viscosity and the effect of produced XOS on eliciting the growth of gut associated probiotic bacteria. Xyn2AE was successfully induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced in Escherichia coli BL21 (DE3) and Xyn2AC was expressed in tobacco mosaic plants. For the purification of Xyn2AE, an immobilized metal affinity chromatography (IMAC) column and diafiltration using a 3kDa cut-off Amicon filter membranes were used. Xyn2AE and Xyn2AC showed a xylanase active band at a relative weight of 21 kDa. Both enzymes showed high specificity towards soluble wheat arabinoxylan (WAX), with specific activities of 7.61 U/mg for Xyn2AE and 536.5 U/mg for Xyn2AC. Xyn2A kinetic parameters (Vmax and Km) were determined by Michaelis-Menten plots on soluble and insoluble WAX. The Vmax and Km values of Xyn2AC were 1003.01 U/mg and 9.25 mg/mL, 302.89 U/mg and 13.54 mg/mL, respectively. The Vmax and Km values of Xyn2AE for soluble and insoluble WAX were 20.45 U/mg and 12.95 mg/mL, and 8.31 U/mg and 13.15 mg/mL. Xyn2A enzymes displayed optimum activity at pH and temperature parameters of 5.0 and 50°C, respectively, and stability in temperatures ranging between 50 and 80°C and pH 4.0-9.0. Broiler chicken feeds were hydrolysed using Xyn2AE over a 24 h period and analysed using the dinitrosalicylic (DNS) assay, thin layer chromatography (TLC), viscometry and visualized using scanning electron microscope (SEM). The results showed a release of release of XOS xylotriose, xylopentose and xylohexose; enzyme’s ability to decrease the viscosity of the feeds and punched holes of feed surface, which was indicative of xylanase action. XOS produced during hydrolysis was used to study prebiotic effect on selected few bacteria and released short chain fatty acids (SCFAs) were measured. Additionally, SCFAs formation was detected in the presence of XOS as a carbon source for S. thermophilus and L. bulgaricus, whereas B. subtilis formed fewer organic acids in the presence of XOS. The results obtained from this study demonstrated that the supplementation of Xyn2A on broiler feeds has ii a positive effect in decreasing feed viscosity. Furthermore, the results of this investigation will assist the South African poultry farming sector to increase profitability in poultry farming and gain stability in the global trade as far as poultry feed is concerned. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
An in-silico study of the type II NADH: Quinone Oxidoreductase (ndh2). A new anti-malaria drug target
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Assessment of cytotoxic artemisinin and its derivatives as DNA damaging inducing agents in triple-negative breast cancer cells
- Authors: Mkhwanazi, Ntando
- Date: 2022-10-14
- Subjects: Breast Cancer , Artemisinin , DNA damage , Antineoplastic agents , Breast Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362960 , vital:65378
- Description: In developing countries, including South Africa, breast cancer is the primary cause of cancer-related deaths among women. TNBC (triple-negative breast cancer) is an aggressive breast cancer subtype that is more prevalent in women of African descent. This subtype lacks the key receptors, namely the estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 (HER2-) that are the basis of successful targeted therapies for other subtypes of the disease. To date, there are no effective, standardized targeted therapies for TNBC. Artemisinin is an anti-malarial drug and numerous derivatives of the compound have been developed to improve the potency and solubility of the parent compound. Artemisinin and its derivatives have gained attention as potential anti-cancer agents; however, such studies have not yet progressed to clinical trials and the precise mechanism of action of these compounds is yet to be fully explained. In this study, artemisinin, and its known derivative artesunate, as well as a novel derivative, WHN11, were investigated as DNA damage-inducing agents in TNBC. WHN11 was found to be the most potent of the three compounds, displaying an IC50 of 3.20 μM against HCC70 cells, artemisinin displayed an IC50 of 214.70 μM and artesunate displayed an IC50 of 25.48 μM. The compounds were less toxic to the MCF12A non-cancerous cells, with IC50 values 298.30, 87.53, and 8.35 μM for artemisinin, artesunate, and WHN11, respectively, and displayed selectivity indices of 1.39, 3.44 and 2.61 μM for artemisinin, artesunate, and WHN11, respectively. In silico and in vitro studies revealed that the artemisinin compounds bind to DNA through the minor groove. While all three compounds were able to bind to DNA, a comet assay revealed that only artemisinin and artesunate, and not WHN11, were able to cause DNA damage compared to the vehicle control, DMSO. Finally, a topoisomerase I (TOPO I) enzyme assay demonstrated that while the compounds appeared to display a degree of inhibition of TOPO I, as evidenced by a downward shift in the plasmid band on the agarose gel, they were not able to fully inhibit the enzyme to return the plasmid to the supercoiled conformation. In addition, combination studies revealed that artemisinin, artesunate, and WHN11 acted synergistically in combination with camptothecin, but displayed either an additive (artemisinin) or antagonistic (artesunate and WHN11) relationship when used in combination with etoposide. In conclusion, artemisinin, its known derivative artesunate, and novel and highly toxic derivative WHN11, all bind to DNA via the minor groove, however only artemisinin and artesunate, and not WHN11, cause DNA damage, indicating a potentially different mechanism of action of the three artemisinins. All three compounds act synergistically with camptothecin, which suggests interference with topoisomerase activity, partially supported by slight inhibition of TOPO I activity, and could indicate either direct inhibition of the enzyme or interference with enzyme function by competitive binding to the DNA. Further studies could help explore alternate DNA damage assays, to validate these findings, and the effect of the compounds on TOPO II activity could also be assessed. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mkhwanazi, Ntando
- Date: 2022-10-14
- Subjects: Breast Cancer , Artemisinin , DNA damage , Antineoplastic agents , Breast Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362960 , vital:65378
- Description: In developing countries, including South Africa, breast cancer is the primary cause of cancer-related deaths among women. TNBC (triple-negative breast cancer) is an aggressive breast cancer subtype that is more prevalent in women of African descent. This subtype lacks the key receptors, namely the estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 (HER2-) that are the basis of successful targeted therapies for other subtypes of the disease. To date, there are no effective, standardized targeted therapies for TNBC. Artemisinin is an anti-malarial drug and numerous derivatives of the compound have been developed to improve the potency and solubility of the parent compound. Artemisinin and its derivatives have gained attention as potential anti-cancer agents; however, such studies have not yet progressed to clinical trials and the precise mechanism of action of these compounds is yet to be fully explained. In this study, artemisinin, and its known derivative artesunate, as well as a novel derivative, WHN11, were investigated as DNA damage-inducing agents in TNBC. WHN11 was found to be the most potent of the three compounds, displaying an IC50 of 3.20 μM against HCC70 cells, artemisinin displayed an IC50 of 214.70 μM and artesunate displayed an IC50 of 25.48 μM. The compounds were less toxic to the MCF12A non-cancerous cells, with IC50 values 298.30, 87.53, and 8.35 μM for artemisinin, artesunate, and WHN11, respectively, and displayed selectivity indices of 1.39, 3.44 and 2.61 μM for artemisinin, artesunate, and WHN11, respectively. In silico and in vitro studies revealed that the artemisinin compounds bind to DNA through the minor groove. While all three compounds were able to bind to DNA, a comet assay revealed that only artemisinin and artesunate, and not WHN11, were able to cause DNA damage compared to the vehicle control, DMSO. Finally, a topoisomerase I (TOPO I) enzyme assay demonstrated that while the compounds appeared to display a degree of inhibition of TOPO I, as evidenced by a downward shift in the plasmid band on the agarose gel, they were not able to fully inhibit the enzyme to return the plasmid to the supercoiled conformation. In addition, combination studies revealed that artemisinin, artesunate, and WHN11 acted synergistically in combination with camptothecin, but displayed either an additive (artemisinin) or antagonistic (artesunate and WHN11) relationship when used in combination with etoposide. In conclusion, artemisinin, its known derivative artesunate, and novel and highly toxic derivative WHN11, all bind to DNA via the minor groove, however only artemisinin and artesunate, and not WHN11, cause DNA damage, indicating a potentially different mechanism of action of the three artemisinins. All three compounds act synergistically with camptothecin, which suggests interference with topoisomerase activity, partially supported by slight inhibition of TOPO I activity, and could indicate either direct inhibition of the enzyme or interference with enzyme function by competitive binding to the DNA. Further studies could help explore alternate DNA damage assays, to validate these findings, and the effect of the compounds on TOPO II activity could also be assessed. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Bioactivity evaluation of manno-oligosaccharides produced from spent coffee grounds using a Bacillus sp. derived endo-1,4-β-mannanase
- Authors: Magengelele, Mihle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365233 , vital:65719
- Description: Thesis embargoed. Possible release date set for early 2024. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Magengelele, Mihle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365233 , vital:65719
- Description: Thesis embargoed. Possible release date set for early 2024. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Bioinformatics tool and web server development focusing on structural bioinformatics applications
- Authors: Nabatanzi, Margaret
- Date: 2022-10-14
- Subjects: Structural bioinformatics , Proteins Structure , Protein structure prediction , Proteins Conformation , Protein complex
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365700 , vital:65777 , DOI https://doi.org/10.21504/10962/365700
- Description: This thesis is divided into two main sections: Part 1 describes the design, and evaluation of the accuracy of a new web server – PRotein Interactive MOdeling (PRIMO-Complexes) for modeling protein complexes and biological assemblies. The second part describes the development of bioinformatics tools to predict HIV-1 drug resistance and support bioinformatics research and education. Recent technological advances have resulted in a tremendous increase in the number of sequences and protein structures deposited in the Universal Protein Resource Knowledgebase (UniProtKB) and the Protein Data Bank (PDB). However, the number of sequences has increased at a higher rate compared with the experimentally solved multimeric protein structures. This is partly due to advances in high-throughput sequencing technology. To fill this protein sequence-structure gap, computational approaches have been developed to predict protein structures from available sequences. Computational approaches include template-based and ab initio modeling with the former being the most reliable. Template-based modeling process can be achieved using either standalone software or automated modeling web servers. However, using standalone software requires familiarity with command-line interfaces as well as utilising other intermediate programs which could be daunting to novice users. To alleviate some of these problems, the modeling process has been automated, however, it still has numerous challenges. To date, only a few web servers that support multimeric protein modeling have been developed and even these provide little, if any user involvement in the process. To address some of these issues, a new web server – PRIMO-Complexes – was developed to model protein complexes and biological assemblies. The existing PRIMO web server could only model monomeric proteins. Part 1 of this thesis provides a detailed account of the development and evaluation of PRIMO-Complexes. The rationale for developing this new web server was based on the understanding that most proteins function as protein multimers and often the ligand-binding sites, and enzyme active sites are located at the protein-protein interfaces. It, therefore, necessitated developing capabilities for modeling multimeric proteins. PRIMO-Complexes web server was developed using the Waterfall system development life cycle model, is based on the Django web framework and makes use of high-performance computing resources to execute jobs. The accuracy of the algorithms embedded in PRIMO- Complexes was evaluated and the results were promising. Additionally, PRIMO-Complexes performs comparatively well in relation to other web servers that offer multimeric protein modeling. Another unique feature of PRIMO-Complexes is its interactivity. The webserver was developed with capabilities for allowing users to model multimeric proteins with an appreciable degree of control over the process. In the second part of the thesis several other bioinformatics tools are described, for example, a webserver for predicting HIV-1 drug resistance, the RUBi protein model repository, and a bioinformatics web portal for education and research resources. RUBi protein model repository stores verified theoretical models built using various modeling approaches. This enables users to easily access models to reproduce and/or further the research. This is described in chapter 5. Chapter 6 describes the design and development of the Human Immunodeficiency type 1 Resistance Predictor (HIV-1 ResPredictor), a web application that employs artificial neural networks (ANN) to predict drug resistance in patients infected with HIV-1 subtype B. The ANNs and subtype classifiers performed well making this web application potentially useful to both clinicians and researchers in this era of personalised medicine. Finally, chapter 7 describes a bioinformatics education web portal that equips students with information on how to use bioinformatics online resources. Being aware of these resources is not enough without a deeper understanding and guidance on how to apply bioinformatics methods to solve practical problems. This web portal was aimed at familiarising students with the basic terminology and approaches in structural bioinformatics. Students will potentially gain skills to conduct real-life bioinformatics research to obtain biological insights. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Nabatanzi, Margaret
- Date: 2022-10-14
- Subjects: Structural bioinformatics , Proteins Structure , Protein structure prediction , Proteins Conformation , Protein complex
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365700 , vital:65777 , DOI https://doi.org/10.21504/10962/365700
- Description: This thesis is divided into two main sections: Part 1 describes the design, and evaluation of the accuracy of a new web server – PRotein Interactive MOdeling (PRIMO-Complexes) for modeling protein complexes and biological assemblies. The second part describes the development of bioinformatics tools to predict HIV-1 drug resistance and support bioinformatics research and education. Recent technological advances have resulted in a tremendous increase in the number of sequences and protein structures deposited in the Universal Protein Resource Knowledgebase (UniProtKB) and the Protein Data Bank (PDB). However, the number of sequences has increased at a higher rate compared with the experimentally solved multimeric protein structures. This is partly due to advances in high-throughput sequencing technology. To fill this protein sequence-structure gap, computational approaches have been developed to predict protein structures from available sequences. Computational approaches include template-based and ab initio modeling with the former being the most reliable. Template-based modeling process can be achieved using either standalone software or automated modeling web servers. However, using standalone software requires familiarity with command-line interfaces as well as utilising other intermediate programs which could be daunting to novice users. To alleviate some of these problems, the modeling process has been automated, however, it still has numerous challenges. To date, only a few web servers that support multimeric protein modeling have been developed and even these provide little, if any user involvement in the process. To address some of these issues, a new web server – PRIMO-Complexes – was developed to model protein complexes and biological assemblies. The existing PRIMO web server could only model monomeric proteins. Part 1 of this thesis provides a detailed account of the development and evaluation of PRIMO-Complexes. The rationale for developing this new web server was based on the understanding that most proteins function as protein multimers and often the ligand-binding sites, and enzyme active sites are located at the protein-protein interfaces. It, therefore, necessitated developing capabilities for modeling multimeric proteins. PRIMO-Complexes web server was developed using the Waterfall system development life cycle model, is based on the Django web framework and makes use of high-performance computing resources to execute jobs. The accuracy of the algorithms embedded in PRIMO- Complexes was evaluated and the results were promising. Additionally, PRIMO-Complexes performs comparatively well in relation to other web servers that offer multimeric protein modeling. Another unique feature of PRIMO-Complexes is its interactivity. The webserver was developed with capabilities for allowing users to model multimeric proteins with an appreciable degree of control over the process. In the second part of the thesis several other bioinformatics tools are described, for example, a webserver for predicting HIV-1 drug resistance, the RUBi protein model repository, and a bioinformatics web portal for education and research resources. RUBi protein model repository stores verified theoretical models built using various modeling approaches. This enables users to easily access models to reproduce and/or further the research. This is described in chapter 5. Chapter 6 describes the design and development of the Human Immunodeficiency type 1 Resistance Predictor (HIV-1 ResPredictor), a web application that employs artificial neural networks (ANN) to predict drug resistance in patients infected with HIV-1 subtype B. The ANNs and subtype classifiers performed well making this web application potentially useful to both clinicians and researchers in this era of personalised medicine. Finally, chapter 7 describes a bioinformatics education web portal that equips students with information on how to use bioinformatics online resources. Being aware of these resources is not enough without a deeper understanding and guidance on how to apply bioinformatics methods to solve practical problems. This web portal was aimed at familiarising students with the basic terminology and approaches in structural bioinformatics. Students will potentially gain skills to conduct real-life bioinformatics research to obtain biological insights. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Characterisation of two novel ferrocenyl benzoxazines as in vitro triple-negative breast cancer inhibitors
- Authors: Mhlanga, Richwell
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365689 , vital:65776
- Description: Thesis access embargoed. Expected released date early 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mhlanga, Richwell
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365689 , vital:65776
- Description: Thesis access embargoed. Expected released date early 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Evaluating the role of Stress Induced Phosphoprotein 1 isoforms in Kaposi's Sarcoma-Associated Herpesvirus biology
- Authors: Ruck, Duncan Kyle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365280 , vital:65723
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Ruck, Duncan Kyle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365280 , vital:65723
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Fucoidans from South African brown seaweeds: establishing the link between their structure and biological properties (anti-diabetic and anti-cancer activities)
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Identification and characterisation of microbial communities and their metabolic potential in meltwater ponds, Western Dronning Mau Land, Antarctica
- Authors: Van Aswegen, Sunet
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365723 , vital:65779
- Description: Thesis embargoed. Expected release date early 2024. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Van Aswegen, Sunet
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365723 , vital:65779
- Description: Thesis embargoed. Expected release date early 2024. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Identification of novel compounds against Plasmodium falciparum Cytochrome bc1 Complex inhibiting the trans-membrane electron transfer pathway: an In Silico study
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Identification of selective novel hits against Mycobacterium tuberculosis KasA potential allosteric sites using bioinformatics approaches
- Authors: Hare, Fadzayi Faith
- Date: 2022-10-14
- Subjects: Tuberculosis , Docking , Molecules Models , Virtual screening , Multidrug-resistant tuberculosis , Fatty acids Synthesis , Drugs Design
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362842 , vital:65367
- Description: Tuberculosis (TB) is a global health threat that has led to approximately 1.5 million deaths annually. According to the World Health Organization (WHO), TB is among the top ten deadly diseases and is the leading cause of death due to a single infectious agent. The main challenge in the effective treatment and control of TB is the ongoing emergence of resistant strains of Mycobacterium tuberculosis (Mtb) which lead to multi-drug resistant (MDR) and extensive-drug resistant (XDR) TB. Hence, the identification and characterization of novel drug targets and drugs that modulate the activity of the pathogen are an urgent priority. The current situation even necessitates the reengineering or repurposing of drugs in order to achieve effective control. The β-ketoacyl-acyl carrier protein synthase I (KasA) of Mycobacterium tuberculosis is an essential enzyme in the mycobacterial fatty acid synthesis (FAS-II) pathway and is believed to be a promising target for drug discovery in TB. It is one of the five main proteins of the FAS-II pathway and catalyzes a key condensation reaction in the synthesis of meromycolate chains, the precursors of mycolic acids involved in cell wall formation. Although this protein has been extensively studied, little research has been devoted to the allosteric inhibition of potential drug compounds. The main aim of this research was to identify the allosteric sites on the protein that could be involved in the inhibition of substrate binding activities and novel drug compounds that bind to these sites by use of in-silico approaches. The bioinformatics approaches used in this study were divided into four main objectives namely identification of KasA homolog sequences, sequence analysis and protein characterization, allosteric site search and lastly virtual screening of DrugBank compounds via molecular docking. Fifteen homolog sequences were identified from the BLASTP analysis and were derived from bacteria, fungi and mammals. In order to discover important residues and regions within the KasA proteins, sequence alignment, motif analysis and phylogenetic studies were performed using Mtb KasA as a reference. Sequence alignment revealed conserved residues in all KasA proteins that have functional importance such as the catalytic triad residues (Cys171, His311 and His345). Motif analysis identified 18 highly conserved motifs within the KasA proteins with structural and functional roles. In addition, motifs unique to the Mtb KasA protein were also identified and explored for inhibitor drug design purposes. Phylogenetic analysis of the homolog sequences showed a distinct clustering of prokaryotes and eukaryotes. A distinctive clustering was also observed for species belonging to the same genus. Since the mechanism of action of most drugs involves the active site, allosteric site search was conducted on Mtb KasA and the human homolog protein using a combination of pocket detection algorithms with the aim of identifying sites that could be utilized in allosteric modulator drug discovery. This was followed by the virtual screening of 2089 FDA approved DrugBank compounds against the entire protein surfaces of Mtb KasA and Hsmt KasA, performed via molecular docking using AutoDock Vina. Screening of the compounds was based on the binding energies, with more focus on identifying ligands that bound exclusively to the acyl-binding tunnel of Mtb KasA. This reduced the data set to 27 promising drug compounds with a relatively high binding affinity for Mtb KasA, however, further experiments need to be performed to validate this result. Among these compounds were DB08889, DB06755, DB09270, DB11226, DB00392, DB12278, DB08936, DB00781, DB13720 and DB00392, which displayed relatively low binding energies for Mtb KasA when compared to the human homolog protein. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Hare, Fadzayi Faith
- Date: 2022-10-14
- Subjects: Tuberculosis , Docking , Molecules Models , Virtual screening , Multidrug-resistant tuberculosis , Fatty acids Synthesis , Drugs Design
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362842 , vital:65367
- Description: Tuberculosis (TB) is a global health threat that has led to approximately 1.5 million deaths annually. According to the World Health Organization (WHO), TB is among the top ten deadly diseases and is the leading cause of death due to a single infectious agent. The main challenge in the effective treatment and control of TB is the ongoing emergence of resistant strains of Mycobacterium tuberculosis (Mtb) which lead to multi-drug resistant (MDR) and extensive-drug resistant (XDR) TB. Hence, the identification and characterization of novel drug targets and drugs that modulate the activity of the pathogen are an urgent priority. The current situation even necessitates the reengineering or repurposing of drugs in order to achieve effective control. The β-ketoacyl-acyl carrier protein synthase I (KasA) of Mycobacterium tuberculosis is an essential enzyme in the mycobacterial fatty acid synthesis (FAS-II) pathway and is believed to be a promising target for drug discovery in TB. It is one of the five main proteins of the FAS-II pathway and catalyzes a key condensation reaction in the synthesis of meromycolate chains, the precursors of mycolic acids involved in cell wall formation. Although this protein has been extensively studied, little research has been devoted to the allosteric inhibition of potential drug compounds. The main aim of this research was to identify the allosteric sites on the protein that could be involved in the inhibition of substrate binding activities and novel drug compounds that bind to these sites by use of in-silico approaches. The bioinformatics approaches used in this study were divided into four main objectives namely identification of KasA homolog sequences, sequence analysis and protein characterization, allosteric site search and lastly virtual screening of DrugBank compounds via molecular docking. Fifteen homolog sequences were identified from the BLASTP analysis and were derived from bacteria, fungi and mammals. In order to discover important residues and regions within the KasA proteins, sequence alignment, motif analysis and phylogenetic studies were performed using Mtb KasA as a reference. Sequence alignment revealed conserved residues in all KasA proteins that have functional importance such as the catalytic triad residues (Cys171, His311 and His345). Motif analysis identified 18 highly conserved motifs within the KasA proteins with structural and functional roles. In addition, motifs unique to the Mtb KasA protein were also identified and explored for inhibitor drug design purposes. Phylogenetic analysis of the homolog sequences showed a distinct clustering of prokaryotes and eukaryotes. A distinctive clustering was also observed for species belonging to the same genus. Since the mechanism of action of most drugs involves the active site, allosteric site search was conducted on Mtb KasA and the human homolog protein using a combination of pocket detection algorithms with the aim of identifying sites that could be utilized in allosteric modulator drug discovery. This was followed by the virtual screening of 2089 FDA approved DrugBank compounds against the entire protein surfaces of Mtb KasA and Hsmt KasA, performed via molecular docking using AutoDock Vina. Screening of the compounds was based on the binding energies, with more focus on identifying ligands that bound exclusively to the acyl-binding tunnel of Mtb KasA. This reduced the data set to 27 promising drug compounds with a relatively high binding affinity for Mtb KasA, however, further experiments need to be performed to validate this result. Among these compounds were DB08889, DB06755, DB09270, DB11226, DB00392, DB12278, DB08936, DB00781, DB13720 and DB00392, which displayed relatively low binding energies for Mtb KasA when compared to the human homolog protein. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
In silico substrate binding profiling for SARS-COV-2 main protease (mpro) using hexapeptide substrates
- Authors: Zabo, Sophakama
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365566 , vital:65760
- Description: COVID-19, as a disease resulting from SARS-CoV-2 infection, and a pandemic has had a devastating effect on the world. There are limited effective measures that control the spread and treatment of COVID-19 illness. The homodimeric cysteine main protease (Mpro) is crucial to the life cycle of the virus, as it cleaves the large polyproteins 1a and 1ab into matured, functional non-structural proteins. The Mpro exhibits high degrees of conservation in sequence, structure and specificity across coronavirus species, making it an ideal drug target. The Mpro substrate-binding profiles remain, despite the resolution of its recognition sequence and cleavage points (Leu-Gln↓(Ser/Ala/Gly)). In this study, a series of hexapeptide sequences containing the appropriate recognition sequence and cleavage points were generated and screened against the Mpro to study these binding profiles, and to further be the basis for efficiency-driven drug design. A multi-conformer hexapeptide substrate library comprising optimised 81000 models of 810 unique sequences was generated using RDKit within the context of python. Terminal capping with ACE and NMe was effected using SMILES and SMARTS matching. Multiple hexapeptides were complexed with chain B of crystallographic Mpro (PDS ID: 6XHM), following the validation of chain B for this purpose using AutoDock Vina at high levels of exhaustiveness (480). The resulting Vina scores ranged between -8.7 and -7.0 kcal.mol-1, and the reproducibility of best poses was validated through redocking. Ligand efficiency indices were calculated to identify substrate residues with high binding efficiency at their respective positions, revealing Val (P3), Ala (P1′); and Gly and Ala (P2′ and P3′) as leading efficient binders. Binding efficiencies were lowered by molecular weight. Substrate recognition was assessed by mapping of binding subsites, and Mpro specificity was evaluated through the resolution of intermolecular interaction at the binding interface. Molecular dynamics simulations for 20 ns were performed to assess the stability and behaviour of 132 Mpro systems complexed with KLQ*** substrates. Principal component analysis (PCA), was performed to assess II protein motions and conformational changes during the simulations. A strategy was formulated to classify and evaluate relations in the Mpro PCA motions, revealing four main clades of similarity. Similarity within a clade (Group 2) and dissimilarity between clades were confirmed. Trajectory visualisation revealed complex stability, substrate unbinding and dimer dissociation for various Mpro systems. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Zabo, Sophakama
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365566 , vital:65760
- Description: COVID-19, as a disease resulting from SARS-CoV-2 infection, and a pandemic has had a devastating effect on the world. There are limited effective measures that control the spread and treatment of COVID-19 illness. The homodimeric cysteine main protease (Mpro) is crucial to the life cycle of the virus, as it cleaves the large polyproteins 1a and 1ab into matured, functional non-structural proteins. The Mpro exhibits high degrees of conservation in sequence, structure and specificity across coronavirus species, making it an ideal drug target. The Mpro substrate-binding profiles remain, despite the resolution of its recognition sequence and cleavage points (Leu-Gln↓(Ser/Ala/Gly)). In this study, a series of hexapeptide sequences containing the appropriate recognition sequence and cleavage points were generated and screened against the Mpro to study these binding profiles, and to further be the basis for efficiency-driven drug design. A multi-conformer hexapeptide substrate library comprising optimised 81000 models of 810 unique sequences was generated using RDKit within the context of python. Terminal capping with ACE and NMe was effected using SMILES and SMARTS matching. Multiple hexapeptides were complexed with chain B of crystallographic Mpro (PDS ID: 6XHM), following the validation of chain B for this purpose using AutoDock Vina at high levels of exhaustiveness (480). The resulting Vina scores ranged between -8.7 and -7.0 kcal.mol-1, and the reproducibility of best poses was validated through redocking. Ligand efficiency indices were calculated to identify substrate residues with high binding efficiency at their respective positions, revealing Val (P3), Ala (P1′); and Gly and Ala (P2′ and P3′) as leading efficient binders. Binding efficiencies were lowered by molecular weight. Substrate recognition was assessed by mapping of binding subsites, and Mpro specificity was evaluated through the resolution of intermolecular interaction at the binding interface. Molecular dynamics simulations for 20 ns were performed to assess the stability and behaviour of 132 Mpro systems complexed with KLQ*** substrates. Principal component analysis (PCA), was performed to assess II protein motions and conformational changes during the simulations. A strategy was formulated to classify and evaluate relations in the Mpro PCA motions, revealing four main clades of similarity. Similarity within a clade (Group 2) and dissimilarity between clades were confirmed. Trajectory visualisation revealed complex stability, substrate unbinding and dimer dissociation for various Mpro systems. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14