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
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
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
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
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
Investigating the roles of HOP isoforms in KSHV biology
- Matandirotya, Lorraine Tariro
- Authors: Matandirotya, Lorraine Tariro
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365257 , vital:65721
- 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: Matandirotya, Lorraine Tariro
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365257 , vital:65721
- 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
Linking Hop and LANA1 in the KSHV life cycle
- Authors: Ruck, Jamie-Lee
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365291 , vital:65724
- 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, Jamie-Lee
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365291 , vital:65724
- 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
Production of mannooligosaccharides from pineapple pulp and pine sawdust using Aspergillus niger derived Man26A and determination of their prebiotic effect
- Authors: Hlalukana, Nosipho Pretty
- Date: 2022-10-14
- Subjects: Oligosaccharides , Prebiotics , Lignocellulose , Mannans
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362853 , vital:65368
- Description: Lignocellulosic biomass is the most abundant source of renewable biomass on earth. Lignocellulosic biomass consists of cellulose, hemicelluloses and lignin. These can be used as a source of renewable fuel as well as other value-added products . Mannans are part of the hemicellulose fraction of lignocellulosic biomass and are the major hemicellulosic polysaccharide fraction in softwoods, where they are found as galactoglucomannans and as glucomannans. Mannans are also found in hardwoods in the form of glucomannans. Mannans can be enzymatically hydrolysed using endo-mannanases to produce of short chain mannooligosaccharides (MOS). MOS have received significant attention for their prebiotic properties, as they promote the growth of probiotic bacteria, which have positively affects on gut health. This study focused on the production of prebiotic MOS from lignocellulosic biomass waste (LBW) and an evaluation of the prebiotic potential of the produced MOS. An Aspergillus niger derived endo-mannanase, Man26A, was fractionated and biochemically analysed. Purified Man26A had a fold purification of 1.25 and a yield of 41.1%. SDS-PAGE analysis of the enzyme revealed that it had a molecular weight of 46 kDa. The pH and temperature optima of Man26A were determined and the pH optimum was found to be pH 4.0 (but the enzyme displayed high activity over a broad acidic pH range, with up to 90% of the activity retained between pH 3.0 and 7.0). The temperature optimum was 50℃. The enzyme was shown to have the highest specific activity on locust bean gum (52.27 U/mg) and ivory nut mannan (57.25 U/mg), compared to guar gum (29.07 U/mg), which indicated that it was affected by the substitution pattern of the mannans. Man26A produced MOS of different diversity on model mannan substrates, where the MOS produced were mannobiose, mannotriose, and mannotetraose for ivory nut mannan, mannobiose, mannotriose, mannotetraose, and mannopentaose and MOS with a higher degree of polymerisation for locust bean gum, and mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexose and MOS with a higher degree of polymerisation for guar gum, as determined by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Pretreatment and characterisation of pineapple pulp (PP) and pine sawdust (PSD) was conducted, and the impact of the pretreatment procedures was analysed using Megazyme sugar kits, thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and microscopic analysis using scanning electron microscopy (SEM) and light microscopy. Compositional analysis of the carbohydrates present in both substrates revealed that they had a glucan content of 36.41 and 50.47% for untreated PP and PSD, respectively. Their respective mannan content was 6.74 and 11.59% and was deemed sufficient for the production of MOS via enzymatic hydrolysis. TGA analysis revealed that untreated and sodium chlorite-acetic acid delignified samples decomposed at approximately the same time, and had a negligible ash content at 600℃, while delignified plus phosphoric acid swollen substrates decomposed at a faster rate, but had a residual ash content at 600℃. FTIR analysis of the substrates revealed slight changes in the structures of untreated and pretreated samples. SEM analysis of PP and PSD showed a change in the morphology of the substrates with subsequent pretreatment steps. Histochemical analysis for lignin for PP and PSD showed successful delignification upon pretreatment. Untreated and sodium chlorite delignified PP and PSD released low amounts of reducing sugars compared to delignified + phosphoric acid swollen substrates. The delignified + phosphoric acid swollen substrates were used for further experiments. MOS produced from delignified and phosphoric acid swollen (Del + PAS) PP and PSD at 0.1 mg/ml enzyme loading and 80 mg/ml (8% (w/v)) substrate concentration, ran between mannose and mannobiose and between mannobiose and manotriose on TLC, with low concentrations of MOS running between mannotetraose and mannopentaose. HPLC analysis of the MOS revealed that Del + PAS PP produced mannose to mannohexose, while Del + PAS PSD produced mannose, mannobiose, and mannotetraose. The MOS were analysed using FTIR, to determine whether the MOS produced contained any acetyl groups, which were present for Del + PAS PSD at 1706 cm-1. The MOS were stable at different pHs, and at temperatures below 200℃. The MOS were also found to be stable in a simulated gastrointestinal environment, in the presence of bile salts and digestive enzymes. The prebiotic effect of the MOS derived from Del + PAS PP and PSD was evaluated. MOS had a proliferative effect on probiotic bacteria (Lactobacillus bulgaricus, Bacillus subtilis and Streptococcus thermophilus). The production of short chain fatty acids (SCFAs) was evaluated on TLC, where no SCFAs were observed on the plate. The effect of MOS on the adhesion ability of bacteria revealed that they do not positively influence the adhesion of probiotic bacteria. The antioxidant activities of 1 mg/ml MOS produced from both substrates were determined to be approximately 15% using the ABTS radical scavenging assay, compared to a radical scavenging activity of 45% for the 0.02 mg/ml gallic acid standard. This study demonstrated that biomass waste could be used to produce prebiotic MOS, which play a positive role in gut ecology and provide health benefits. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Hlalukana, Nosipho Pretty
- Date: 2022-10-14
- Subjects: Oligosaccharides , Prebiotics , Lignocellulose , Mannans
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362853 , vital:65368
- Description: Lignocellulosic biomass is the most abundant source of renewable biomass on earth. Lignocellulosic biomass consists of cellulose, hemicelluloses and lignin. These can be used as a source of renewable fuel as well as other value-added products . Mannans are part of the hemicellulose fraction of lignocellulosic biomass and are the major hemicellulosic polysaccharide fraction in softwoods, where they are found as galactoglucomannans and as glucomannans. Mannans are also found in hardwoods in the form of glucomannans. Mannans can be enzymatically hydrolysed using endo-mannanases to produce of short chain mannooligosaccharides (MOS). MOS have received significant attention for their prebiotic properties, as they promote the growth of probiotic bacteria, which have positively affects on gut health. This study focused on the production of prebiotic MOS from lignocellulosic biomass waste (LBW) and an evaluation of the prebiotic potential of the produced MOS. An Aspergillus niger derived endo-mannanase, Man26A, was fractionated and biochemically analysed. Purified Man26A had a fold purification of 1.25 and a yield of 41.1%. SDS-PAGE analysis of the enzyme revealed that it had a molecular weight of 46 kDa. The pH and temperature optima of Man26A were determined and the pH optimum was found to be pH 4.0 (but the enzyme displayed high activity over a broad acidic pH range, with up to 90% of the activity retained between pH 3.0 and 7.0). The temperature optimum was 50℃. The enzyme was shown to have the highest specific activity on locust bean gum (52.27 U/mg) and ivory nut mannan (57.25 U/mg), compared to guar gum (29.07 U/mg), which indicated that it was affected by the substitution pattern of the mannans. Man26A produced MOS of different diversity on model mannan substrates, where the MOS produced were mannobiose, mannotriose, and mannotetraose for ivory nut mannan, mannobiose, mannotriose, mannotetraose, and mannopentaose and MOS with a higher degree of polymerisation for locust bean gum, and mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexose and MOS with a higher degree of polymerisation for guar gum, as determined by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Pretreatment and characterisation of pineapple pulp (PP) and pine sawdust (PSD) was conducted, and the impact of the pretreatment procedures was analysed using Megazyme sugar kits, thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and microscopic analysis using scanning electron microscopy (SEM) and light microscopy. Compositional analysis of the carbohydrates present in both substrates revealed that they had a glucan content of 36.41 and 50.47% for untreated PP and PSD, respectively. Their respective mannan content was 6.74 and 11.59% and was deemed sufficient for the production of MOS via enzymatic hydrolysis. TGA analysis revealed that untreated and sodium chlorite-acetic acid delignified samples decomposed at approximately the same time, and had a negligible ash content at 600℃, while delignified plus phosphoric acid swollen substrates decomposed at a faster rate, but had a residual ash content at 600℃. FTIR analysis of the substrates revealed slight changes in the structures of untreated and pretreated samples. SEM analysis of PP and PSD showed a change in the morphology of the substrates with subsequent pretreatment steps. Histochemical analysis for lignin for PP and PSD showed successful delignification upon pretreatment. Untreated and sodium chlorite delignified PP and PSD released low amounts of reducing sugars compared to delignified + phosphoric acid swollen substrates. The delignified + phosphoric acid swollen substrates were used for further experiments. MOS produced from delignified and phosphoric acid swollen (Del + PAS) PP and PSD at 0.1 mg/ml enzyme loading and 80 mg/ml (8% (w/v)) substrate concentration, ran between mannose and mannobiose and between mannobiose and manotriose on TLC, with low concentrations of MOS running between mannotetraose and mannopentaose. HPLC analysis of the MOS revealed that Del + PAS PP produced mannose to mannohexose, while Del + PAS PSD produced mannose, mannobiose, and mannotetraose. The MOS were analysed using FTIR, to determine whether the MOS produced contained any acetyl groups, which were present for Del + PAS PSD at 1706 cm-1. The MOS were stable at different pHs, and at temperatures below 200℃. The MOS were also found to be stable in a simulated gastrointestinal environment, in the presence of bile salts and digestive enzymes. The prebiotic effect of the MOS derived from Del + PAS PP and PSD was evaluated. MOS had a proliferative effect on probiotic bacteria (Lactobacillus bulgaricus, Bacillus subtilis and Streptococcus thermophilus). The production of short chain fatty acids (SCFAs) was evaluated on TLC, where no SCFAs were observed on the plate. The effect of MOS on the adhesion ability of bacteria revealed that they do not positively influence the adhesion of probiotic bacteria. The antioxidant activities of 1 mg/ml MOS produced from both substrates were determined to be approximately 15% using the ABTS radical scavenging assay, compared to a radical scavenging activity of 45% for the 0.02 mg/ml gallic acid standard. This study demonstrated that biomass waste could be used to produce prebiotic MOS, which play a positive role in gut ecology and provide health benefits. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Cloning, expression, partial characterisation and application of a recombinant GH10 xylanase, XT6, from Geobacillus stearothermophilus T6 as an additive to chicken feeds
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Effects of hydraulic fracking fluid on soil microbial composition and diversity
- Authors: Sianyuka, Nicolette
- Date: 2022-04-06
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/232366 , vital:49985
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Sianyuka, Nicolette
- Date: 2022-04-06
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/232366 , vital:49985
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Molecular characterization of microbial communities in the Sundays and Swartkops estuaries impacted by anthropogenic activities
- Kgomokhumo, Tlhoafalang Evah
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Exploring targeted metagenomics and untargeted metabolomics for characterising aquaponics bacterial ecology and phytochemistry
- Authors: Abraham, Benjamin Melakail
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192453 , vital:45227
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Abraham, Benjamin Melakail
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192453 , vital:45227
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Exploring the use of in vitro colorimetric and bioluminescence assays to distinguish between Arf GTPase isoforms and detect Arf GTPase activity
- Authors: Woolf, Alexander Robert
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192582 , vital:45240
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Woolf, Alexander Robert
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192582 , vital:45240
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29