Structural determinants of the HSP90-Fibronectin interaction and implications for fibrillogenesis
- Authors: Chakraborty, Abir
- Date: 2022-04-08
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/294572 , vital:57234
- Description: Thesis embargoed. Release date April 2024. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-08
- Authors: Chakraborty, Abir
- Date: 2022-04-08
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/294572 , vital:57234
- Description: Thesis embargoed. Release date April 2024. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-08
A novel Arf GTPase assay for antimalarial drug discovery
- Authors: Swart, Tarryn
- Date: 2021-04
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178558 , vital:42950
- Description: Access restricted until April 2022. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Swart, Tarryn
- Date: 2021-04
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178558 , vital:42950
- Description: Access restricted until April 2022. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
An evaluation of synergistic interactions between feruloyl esterases and xylanases during the hydrolysis of various pre-treated agricultural residues
- Authors: Mkabayi, Lithalethu
- Date: 2021-04
- Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
- Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Mkabayi, Lithalethu
- Date: 2021-04
- Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
- Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
The effect of various substrate pretreatment methods on the enzymatic degradability of a Eucalyptus sp. – a potential feedstock for producing fermentable sugars
- Authors: Thoresen, Mariska
- Date: 2021-04
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178580 , vital:42952
- Description: Access restricted until April 2022. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Thoresen, Mariska
- Date: 2021-04
- Subjects: To be added
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178580 , vital:42952
- Description: Access restricted until April 2022. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
A novel, improved throughput bioassay for determining the delative speed of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/139902 , vital:37810
- Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials, there has been little progress towards a proven vaccine. Pending a long-term solution, endemic countries rely heavily on the development of innovative drugs that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
- Full Text:
- Date Issued: 2020
- Authors: Laming, Dustin
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/139902 , vital:37810
- Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials, there has been little progress towards a proven vaccine. Pending a long-term solution, endemic countries rely heavily on the development of innovative drugs that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
- Full Text:
- Date Issued: 2020
An evaluation of the cytotoxic activities of novel artemisinin derivatives: towards targeted therapies for triple-negative breast cancers (TNBC)
- Authors: Kajewole, Deborah Ifeoluwa
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163329 , vital:41029 , doi:10.21504/10962/163329
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
- Full Text:
- Date Issued: 2020
- Authors: Kajewole, Deborah Ifeoluwa
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163329 , vital:41029 , doi:10.21504/10962/163329
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
- Full Text:
- Date Issued: 2020
Application of machine learning, molecular modelling and structural data mining against antiretroviral drug resistance in HIV-1
- Sheik Amamuddy, Olivier Serge André
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Date Issued: 2020
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Date Issued: 2020
Bioinformatic analysis of Aminoacyl tRNA Synthetases as potential antimalarial drug targets
- Authors: Nyamai, Dorothy Wavinya
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164579 , vital:41142 , doi:10.21504/10962/164579
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Nyamai, Dorothy Wavinya
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164579 , vital:41142 , doi:10.21504/10962/164579
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Comparative analysis of the known Hop1b and the novel Hop1a isoforms of the Hop gene
- Authors: Makhubu, Portia
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164311 , vital:41108 , doi:10.21504/10962/164311
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Makhubu, Portia
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164311 , vital:41108 , doi:10.21504/10962/164311
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Computer aided approaches against Human African Trypanosomiasis
- Authors: Kimuda, Magambo Phillip
- Date: 2020
- Subjects: African trypanosomiasis , African trypanosomiasis -- Chemotherapy , Genomics , Macrophage migration inhibitory factor , Trypanosoma brucei , Pteridines , Tetrahydrofolate dehydrogenase , Adenylic acid , Molecular dynamics , Principal components analysis , Bioinformatics , Single nucleotide polymorphisms , Single Nucleotide Variants , Candidate Gene Association Study (CGAS)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142542 , vital:38089
- Description: The thesis presented here is divided into two parts under a common theme that is the use of computer based tools, genomics, and in vitro experiments to develop innovative ways of tackling Human African Trypanosomiasis (HAT). Part I of this thesis focused on the human host genetic determinants while Part II focused on the discovery of novel chemotherapeutics against the parasite. Part I is further sub-divided into two parts: The first involves a Candidate Gene Association Study (CGAS) on an African population to identify genetic determinants associated with disease and/or susceptibility to HAT. The second involves studying the effects of missense Single Nucleotide Variants (SNVs) on protein structure, dynamics, and function using Macrophage Migration Inhibitory Factor (MIF) as a case study. Part II is also sub-divided into two parts: The first involves a computer based rational drug discovery of potential inhibitors against the Trypanosoma the folate pathway; particularly by targeting Trypanosoma brucei Pteridine Reductase (TbPTR1) which is an enzyme used by trypanosomes to overcome T. brucei Dihydrofolate Reductase (TbDHFR) inhibition. Lastly the derivation of CHARMM force-field parameters that can be used to accurately model the geometry and dynamics of the T. brucei Phosphodiesterase B1 enzyme (TbrPDEB1) bimetallic active site center. The derived parameters were then used in MD simulations to characterise protein-ligand residue interactions that are important in TbrPDEB1 inhibition with the goal of targeting the cyclic Adenosine Monophosphate (cAMP) signalling pathway. In the CGAS we were unable to detect any genetic associations in the Ugandan cohort analysed that passed correction for multiple testing in spite of the study being sufficiently powered. Additionally, our study found no association of the Apo lipoprotein 1 (APOL1) G2 allele association with protection against acute HAT that has been previously reported. Future investigations for example, Genome Wide Association Studies using larger samples sizes (>3000 cases and controls) are required. Macrophage migration inhibitory factor (MIF) is a cytokine that is important in both innate and adaptive immunity that has been shown to play a role in T. brucei pathogenicity using murine models. A total of 27 missense SNVs were modelled using homology modelling to create MIF protein mutants that were investigated using in silico effect prediction tools, molecular dynamics (MD), Principal Component Analysis (PCA), and Dynamic Residue Network (DRN) analysis. Our results demonstrate that mutations P2Q, I5M, P16Q, L23F, T24S, T31I, Y37H, H41P, M48V, P44L, G52C, S54R, I65M, I68T, S75F, N106S, and T113S caused significant conformational changes. Further, DRN analysis showed that residues P2, T31, Y37, G52, I65, I68, S75, N106, and T113S are part of a similar local residue interaction network with functional significance. These results show how polymorphisms such as missense SNVs can affect protein conformation, dynamics, and function. Trypanosomes are auxotrophic for folates and pterins but require them for survival. They scavenge them from their hosts. PTR1 is a multifunctional enzyme that is unique to trypanosomatids that reduces both pterins and folates. In the presence of DHFR inhibitors, PTR1 is over-expressed thus providing an escape from the effects of DHFR inhibition. Both TbPTR1 and TbDHFR are pharmacologically and genetically validated drug targets. In this study 5742 compounds were screened using molecular docking, and 13 promising binding modes were further analysed using MD simulations. The trajectories were analysed using RMSD, Rg, RMSF, PCA, Essential Dynamics Analysis (EDA), Molecular Mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and DRN analysis. The computational screening approach allowed us to identify five of the compounds, named RUBi004, RUBi007, RUBi014, RUBi016 and RUBi018 that exhibited antitrypanosomal growth activities against trypanosomes in culture with IC50 values of 12.5 ± 4.8 μM, 32.4 ± 4.2 μM, 5.9 ± 1.4 μM, 28.2 ± 3.3 μM, and 9.7 ± 2.1 μM, respectively. Further when used in combination with WR99210 a known TbDHFR inhibitor RUBi004, RUBi007, RUBi014 and RUBi018 showed antagonism while RUBi016 showed an additive effect. These results indicate that the four compounds might be competing with TbDHFR while RUBi016 might be more specific for TbPTR1. These compounds provide scaffolds that can be further optimised to improve their potency and specificity. Lastly, using a systematic approach we derived CHARMM force-field parameters to accurately describe the TbrPDEB1 bi-metal catalytic center. For dynamics, we employed mixed bonded and non-bonded approach. We optimised the structure using a two-layer QM/MM ONIOM (B3LYP/6-31(g): UFF). The TbrPDEB1 bi-metallic center bonds, angles, and dihedrals were parameterized by fitting the energy profiles from Potential Energy Surface (PES) scans to the CHARMM potential energy function. The parameters were validated by means of MD simulations and analysed using RMSD, Rg, RMSF, hydrogen bonding, bond/angle/dihedral evaluations, EDA, PCA, and DRN analysis. The force-field parameters were able to accurately reproduce the geometry and dynamics of the TbrPDEB1 bi-metal catalytic center during MD simulations. Molecular docking was used to identify 6 potential hits, that inhibited trypanosome growth in vitro. The derived force-field parameters were used to simulate the 6 protein-ligand complexes with the aim of elucidating crucial protein-ligand residue interactions. Using the most potent ligand RUBi022 that had an IC50 of 14.96 μM we were able to identify key residue interactions that can be of use in in silico prediction of potential TbrPDEB1 inhibitors. Overall we demonstrate how bioinformatics tools can complement current disease eradication strategies. Future work will focus on identifying variants identified in Genome Wide Association Studies and partnering with wet labs to carry out further enzyme-ligand activity relationship studies, structure determination or characterisation of appropriate protein-ligand complexes by crystallography, and site specific mutation studies
- Full Text:
- Date Issued: 2020
- Authors: Kimuda, Magambo Phillip
- Date: 2020
- Subjects: African trypanosomiasis , African trypanosomiasis -- Chemotherapy , Genomics , Macrophage migration inhibitory factor , Trypanosoma brucei , Pteridines , Tetrahydrofolate dehydrogenase , Adenylic acid , Molecular dynamics , Principal components analysis , Bioinformatics , Single nucleotide polymorphisms , Single Nucleotide Variants , Candidate Gene Association Study (CGAS)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142542 , vital:38089
- Description: The thesis presented here is divided into two parts under a common theme that is the use of computer based tools, genomics, and in vitro experiments to develop innovative ways of tackling Human African Trypanosomiasis (HAT). Part I of this thesis focused on the human host genetic determinants while Part II focused on the discovery of novel chemotherapeutics against the parasite. Part I is further sub-divided into two parts: The first involves a Candidate Gene Association Study (CGAS) on an African population to identify genetic determinants associated with disease and/or susceptibility to HAT. The second involves studying the effects of missense Single Nucleotide Variants (SNVs) on protein structure, dynamics, and function using Macrophage Migration Inhibitory Factor (MIF) as a case study. Part II is also sub-divided into two parts: The first involves a computer based rational drug discovery of potential inhibitors against the Trypanosoma the folate pathway; particularly by targeting Trypanosoma brucei Pteridine Reductase (TbPTR1) which is an enzyme used by trypanosomes to overcome T. brucei Dihydrofolate Reductase (TbDHFR) inhibition. Lastly the derivation of CHARMM force-field parameters that can be used to accurately model the geometry and dynamics of the T. brucei Phosphodiesterase B1 enzyme (TbrPDEB1) bimetallic active site center. The derived parameters were then used in MD simulations to characterise protein-ligand residue interactions that are important in TbrPDEB1 inhibition with the goal of targeting the cyclic Adenosine Monophosphate (cAMP) signalling pathway. In the CGAS we were unable to detect any genetic associations in the Ugandan cohort analysed that passed correction for multiple testing in spite of the study being sufficiently powered. Additionally, our study found no association of the Apo lipoprotein 1 (APOL1) G2 allele association with protection against acute HAT that has been previously reported. Future investigations for example, Genome Wide Association Studies using larger samples sizes (>3000 cases and controls) are required. Macrophage migration inhibitory factor (MIF) is a cytokine that is important in both innate and adaptive immunity that has been shown to play a role in T. brucei pathogenicity using murine models. A total of 27 missense SNVs were modelled using homology modelling to create MIF protein mutants that were investigated using in silico effect prediction tools, molecular dynamics (MD), Principal Component Analysis (PCA), and Dynamic Residue Network (DRN) analysis. Our results demonstrate that mutations P2Q, I5M, P16Q, L23F, T24S, T31I, Y37H, H41P, M48V, P44L, G52C, S54R, I65M, I68T, S75F, N106S, and T113S caused significant conformational changes. Further, DRN analysis showed that residues P2, T31, Y37, G52, I65, I68, S75, N106, and T113S are part of a similar local residue interaction network with functional significance. These results show how polymorphisms such as missense SNVs can affect protein conformation, dynamics, and function. Trypanosomes are auxotrophic for folates and pterins but require them for survival. They scavenge them from their hosts. PTR1 is a multifunctional enzyme that is unique to trypanosomatids that reduces both pterins and folates. In the presence of DHFR inhibitors, PTR1 is over-expressed thus providing an escape from the effects of DHFR inhibition. Both TbPTR1 and TbDHFR are pharmacologically and genetically validated drug targets. In this study 5742 compounds were screened using molecular docking, and 13 promising binding modes were further analysed using MD simulations. The trajectories were analysed using RMSD, Rg, RMSF, PCA, Essential Dynamics Analysis (EDA), Molecular Mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and DRN analysis. The computational screening approach allowed us to identify five of the compounds, named RUBi004, RUBi007, RUBi014, RUBi016 and RUBi018 that exhibited antitrypanosomal growth activities against trypanosomes in culture with IC50 values of 12.5 ± 4.8 μM, 32.4 ± 4.2 μM, 5.9 ± 1.4 μM, 28.2 ± 3.3 μM, and 9.7 ± 2.1 μM, respectively. Further when used in combination with WR99210 a known TbDHFR inhibitor RUBi004, RUBi007, RUBi014 and RUBi018 showed antagonism while RUBi016 showed an additive effect. These results indicate that the four compounds might be competing with TbDHFR while RUBi016 might be more specific for TbPTR1. These compounds provide scaffolds that can be further optimised to improve their potency and specificity. Lastly, using a systematic approach we derived CHARMM force-field parameters to accurately describe the TbrPDEB1 bi-metal catalytic center. For dynamics, we employed mixed bonded and non-bonded approach. We optimised the structure using a two-layer QM/MM ONIOM (B3LYP/6-31(g): UFF). The TbrPDEB1 bi-metallic center bonds, angles, and dihedrals were parameterized by fitting the energy profiles from Potential Energy Surface (PES) scans to the CHARMM potential energy function. The parameters were validated by means of MD simulations and analysed using RMSD, Rg, RMSF, hydrogen bonding, bond/angle/dihedral evaluations, EDA, PCA, and DRN analysis. The force-field parameters were able to accurately reproduce the geometry and dynamics of the TbrPDEB1 bi-metal catalytic center during MD simulations. Molecular docking was used to identify 6 potential hits, that inhibited trypanosome growth in vitro. The derived force-field parameters were used to simulate the 6 protein-ligand complexes with the aim of elucidating crucial protein-ligand residue interactions. Using the most potent ligand RUBi022 that had an IC50 of 14.96 μM we were able to identify key residue interactions that can be of use in in silico prediction of potential TbrPDEB1 inhibitors. Overall we demonstrate how bioinformatics tools can complement current disease eradication strategies. Future work will focus on identifying variants identified in Genome Wide Association Studies and partnering with wet labs to carry out further enzyme-ligand activity relationship studies, structure determination or characterisation of appropriate protein-ligand complexes by crystallography, and site specific mutation studies
- Full Text:
- Date Issued: 2020
Hop as an anti-cancer drug target
- Vaaltyn, Michaelone Chantelle
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164704 , vital:41156 , doi:10.21504/10962/164704
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164704 , vital:41156 , doi:10.21504/10962/164704
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Mechanism of action of non-synonymous single nucleotide variations associated with α-carbonic anhydrases II, IV and VIII
- Authors: Sanyanga, T. Allan
- Date: 2020
- Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/167346 , vital:41470
- Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
- Full Text:
- Date Issued: 2020
- Authors: Sanyanga, T. Allan
- Date: 2020
- Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/167346 , vital:41470
- Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
- Full Text:
- Date Issued: 2020
Multiplexed Mass Spectrometry: Single, On-Bead, Detection Analysis Using MALDI-TOF MS
- Authors: Twala, Busisiwe Victoria
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164693 , vital:41155 , doi:10.21504/10962/164693
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Twala, Busisiwe Victoria
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164693 , vital:41155 , doi:10.21504/10962/164693
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Nutrient impacts on grasses and legumes growing in communal pasture soil in relation to mycorrhizal activity
- Authors: Mkile, Zolani
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164512 , vital:41125 , doi:10.21504/10962/164512
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Mkile, Zolani
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164512 , vital:41125 , doi:10.21504/10962/164512
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
The role of the Hop co-chaperone in the formation of Hsp90 complexes: chaperone link to glycolysis
- Authors: Maharaj, Shantal
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163593 , vital:41051 , doi:10.21504/10962/163593
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Maharaj, Shantal
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163593 , vital:41051 , doi:10.21504/10962/163593
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
A computational analysis to decipher the pathways of stability, uncoating and antigenicity of human enterovirus capsids
- Authors: Ross, Caroline Jane
- Date: 2019
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/114788 , vital:34035 , 10.21504/10962/114788
- Description: Expected release date-April 2021
- Full Text: false
- Date Issued: 2019
- Authors: Ross, Caroline Jane
- Date: 2019
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/114788 , vital:34035 , 10.21504/10962/114788
- Description: Expected release date-April 2021
- Full Text: false
- Date Issued: 2019
A dynamics based analysis of allosteric modulation in heat shock proteins
- Authors: Penkler, David Lawrence
- Date: 2019
- Subjects: Heat shock proteins , Molecular chaperones , Allosteric regulation , Homeostasis , Protein kinases , Transcription factors , Adenosine triphosphatase , Cancer -- Chemotherapy , Molecular dynamics , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115948 , vital:34273
- Description: The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies.
- Full Text:
- Date Issued: 2019
- Authors: Penkler, David Lawrence
- Date: 2019
- Subjects: Heat shock proteins , Molecular chaperones , Allosteric regulation , Homeostasis , Protein kinases , Transcription factors , Adenosine triphosphatase , Cancer -- Chemotherapy , Molecular dynamics , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115948 , vital:34273
- Description: The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies.
- Full Text:
- Date Issued: 2019
An investigation into the bacterial biosynthetic origins of bioactive natural products isolated from South African latrunculid sponges
- Authors: Waterworth, Samantha Che
- Date: 2018
- Subjects: Marine biodiversity , Metagenomics , Sponges Biotechnology , Spirochetes , Natural products Biotechnology
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61826 , vital:28065
- Description: Several pyrroloiminoquinone alkaloids exhibiting cytotoxic, anti-tumour activity have been isolated from sponges within the Latrunculiidae family that are endemic to the South African coastline. Other, structurally similar pyrroloiminoquinone compounds have been isolated from geographically distant and phylogenetically distinct marine sponges, as well as terrestrial myxomycetes which suggested that sponge-associated bacteria may be the true biosynthetic origin of pyrroloiminoquinone compounds. Previous studies have shown that there is conservation of spirochete and betaproteobacterial species in the bacterial communities associated with South African Latrunculiidae sponges and it was proposed that these conserved bacteria represented candidate pyrroloiminoquinone-producers. This study aimed to confirm the conserved dominance of betaproteobacteria and spirochetes within bacterial communities associated with South African latrunculid sponges and employed a shotgun metagenomic approach to assess the functional and biosynthetic potential of associated microbiota in Tsitsikamma favus sponges. Clustering of assembled contigs revealed twenty-three putative bacterial genomes, of which, two were identified as representatives of the conserved betaproteobacteria and spirochete species previously identified in Tsitsikamma sponges. It was shown that the spirochete was most likely an obligate symbiont that benefitted the host sponge through possible defence against pathogenic bacteria and/or nutrient acquisition. The putative genome representing the conserved betaproteobacteria was found to be heavily contaminated and further sequencing is required to accurately resolve the genome for functional characterization. Several biosynthetic gene clusters were identified and demonstrated the bioactive potential of Tsitsikamma favus-associated bacteria. A biosynthetic gene cluster was identified on an unclustered contig that included several genetic features that were indicative of possible pyrroloiminoquinone production.
- Full Text:
- Date Issued: 2018
- Authors: Waterworth, Samantha Che
- Date: 2018
- Subjects: Marine biodiversity , Metagenomics , Sponges Biotechnology , Spirochetes , Natural products Biotechnology
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61826 , vital:28065
- Description: Several pyrroloiminoquinone alkaloids exhibiting cytotoxic, anti-tumour activity have been isolated from sponges within the Latrunculiidae family that are endemic to the South African coastline. Other, structurally similar pyrroloiminoquinone compounds have been isolated from geographically distant and phylogenetically distinct marine sponges, as well as terrestrial myxomycetes which suggested that sponge-associated bacteria may be the true biosynthetic origin of pyrroloiminoquinone compounds. Previous studies have shown that there is conservation of spirochete and betaproteobacterial species in the bacterial communities associated with South African Latrunculiidae sponges and it was proposed that these conserved bacteria represented candidate pyrroloiminoquinone-producers. This study aimed to confirm the conserved dominance of betaproteobacteria and spirochetes within bacterial communities associated with South African latrunculid sponges and employed a shotgun metagenomic approach to assess the functional and biosynthetic potential of associated microbiota in Tsitsikamma favus sponges. Clustering of assembled contigs revealed twenty-three putative bacterial genomes, of which, two were identified as representatives of the conserved betaproteobacteria and spirochete species previously identified in Tsitsikamma sponges. It was shown that the spirochete was most likely an obligate symbiont that benefitted the host sponge through possible defence against pathogenic bacteria and/or nutrient acquisition. The putative genome representing the conserved betaproteobacteria was found to be heavily contaminated and further sequencing is required to accurately resolve the genome for functional characterization. Several biosynthetic gene clusters were identified and demonstrated the bioactive potential of Tsitsikamma favus-associated bacteria. A biosynthetic gene cluster was identified on an unclustered contig that included several genetic features that were indicative of possible pyrroloiminoquinone production.
- Full Text:
- Date Issued: 2018
Analysis of the human HSP70-HSP90 organising protein (HOP) gene - characterisation of the promoter and identification of a novel isoform
- Authors: Mattison, Stacey
- Date: 2018
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62821 , vital:28296
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
- Authors: Mattison, Stacey
- Date: 2018
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62821 , vital:28296
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Date Issued: 2018
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Date Issued: 2018