Development of an enzyme-synergy based bioreactor system for the beneficiation of apple pomace lignocellulosic waste
- Authors: Abboo, Sagaran
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/315 , vital:19947
- Description: Due to the finite supply of non-renewable fossil fuels, agro-industrial wastes are identified as alternate, renewable sources for energy supply. Large amounts of fruit waste are generated in South Africa due to fruit juice and wine processing from apples, grapes and citrus fruit. Apple pomace is the solid residue that is left over after juice, cider and wine processing and constitutes between 25-30% of the total fruit. On a global scale millions of tonnes of apple pomace are produced; between 2006-2007 over 46 million tonnes were produced. In South Africa a total production of 244 469 tonnes were produced during the 2011- 2012 season. Initially, apple pomace was regarded as a waste by-product used for animal feed and compost in soil, however presently it is considered a source of dietary fiber and natural antioxidants like polyphenols. In addition, apple pomace has a high carbohydrate content and can be enzymatically hydrolysed to produce sugar monomers which, in turn, can be fermented by yeasts to produce bioethanol. The polyphenols present in apple pomace can be used for their health properties, and the bioethanol can be used as a replacement for fossil fuel. Apple pomace is lignocellulosic in nature and consists of hemicellulose, cellulose, lignin and pectin. A combination of enzymes such as cellulases, hemicellulases, pectinases and lignases are required to operate in synergy for the degradation of lignocellulosic biomass. This is due to the recalcitrant nature of lignocellulose. This study investigated the degradation of apple pomace using a combination of commercially obtained enzyme cocktails viz. Viscozyme L , Celluclast 1.5L and Novozyme 188. The commercial enzymes Viscozyme L and Celluclast 1.5L were added in a ratio of 1:1 (50%:50%). The final concentrations of the enzymes were 0.019 mg/ml each. Novozyme 188 was added to provide a final concentration of 0.0024 mg/ml. A novel cost effective 20L bioreactor was designed, constructed and implemented for the degradation of apple pomace to produce value added products. The hydrolysis of the apple pomace was performed initially in 1 L flasks (batch fed) and, once optimized, scaled up to a 20 L bioreactor in batch mode. The bioreactors were operated at room temperature (22 ± 2ºC) and in an unbuffered system. The sugars released were detected and quantified using an optimized validated HPLC method established in this study. The sugars released in the bioreactors were mainly glucose, galactose, arabinose, cellobiose and fructose. The polyphenols released in this study were gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloridzin, which have a number of health benefits. The simultaneous analyses of the polyphenols were performed using a newly developed and validated HPLC method established in this study. This method was developed to detect nine polyphenols simultaneously. The two HPLC methods developed and validated in this study for the analysis of sugars and polyphenols demonstrated good accuracy, precision, reproducibility, linearity, robustness and sensitivity. Both analytical methods were validated according to the International Convention on Harmonization (ICH). The HPLC parameters for sugar analysis were: refractive index (RI) as the detection mode, the stationary phase was a ligand-exchange sugar column (Shodex SP0810) and an aqueous mobile phase in isocratic mode was used. The HPLC method for polyphenols employed UV diode array detection (DAD) as the detection mode, a reverse phase column as the stationary phase and a mobile phase of consisting of 0.01 M phosphoric acid in water and 100% methanol using gradient elution mode. The highest concentrations of sugars released in the novel 20 L bioreactor with 20% apple pomace (w/v) substrate loading were as follow: glucose (6.5 mg/ml), followed by galactose (2.1 mg/ml), arabinose (1.4 mg/ml), cellobiose (0.7 mg/ml) and fructose (0.5 mg/ml). The amounts of polyphenols released at 20% (w/v) apple pomace substrate were epicatechin (0.01 mg/ml), catechin (0.002 mg/ml), rutin (0.03 mg/ml), chlorogenic acid (0.002 mg/ml) and gallic acid 0.01 (mg/ml). Two mathematical models were developed in this study for kinetic analysis of lignocellulose (apple pomace) hydrolysis in the novel 20 L bioreactor, using the experimental data generated by the above HPLC analyses. The first model, modelling with regression, defines the hydrolysis of the sugars glucose, galactose, cellobiose and arabinose produced in the novel 20 L bioreactor at 5%, 10%, 15% and 20% (w/v) substrate concentrations. The regression model describes the sugars produced in the 20 L bioreactor by minimizing the error of the sugars released by finding a value for K which minimises the function which computes the sum of squares of errors between the solution curves and the data points. The second, more complex, model developed in this study used a system of differential equations model (ODE). This model solved the system by using a numerical method, such as the Runge-Kutta method, then fitted the solution curves to the data. Both models simulated (and had the ability to predict) the production of sugars in the novel 20 L bioreactor for apple pomace hydrolysis. These two models also revealed the time at which the maximum amount of sugars were released, which revealed the optimum time to run the 20 L bioreactor in order to be more cost effective. The optimum time for maximum glucose (the main sugar used in fermentation for biofuel production) release was determined to be around 60 h. The ODE model, in addition, determined the rate at which the substrate became depleted, as well as the rate at which the enzymes became deactivated for the various substrate loadings in the 20 L bioreactor. A third model was developed to determine the optimal running cost of the bioreactor which incorporated the substrate loading and the amount of glucose (g/L) produced. The novel 20 L bioreactor constructed from cost effective materials demonstrated that agro-industrial waste can be converted to value-added products by lignocellolytic enzymes. The sugars released from apple pomace can be used in biofuel production and the polyphenols as food supplements and nutraceuticals for health benefits. This novel study contributes to agro-industrial waste beneficiation via fuel production. In addition, using agro-industrial waste for the generation of value added products (instead of mere disposal) will help prevent environmental pollution.
- Full Text:
- Date Issued: 2016
- Authors: Abboo, Sagaran
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/315 , vital:19947
- Description: Due to the finite supply of non-renewable fossil fuels, agro-industrial wastes are identified as alternate, renewable sources for energy supply. Large amounts of fruit waste are generated in South Africa due to fruit juice and wine processing from apples, grapes and citrus fruit. Apple pomace is the solid residue that is left over after juice, cider and wine processing and constitutes between 25-30% of the total fruit. On a global scale millions of tonnes of apple pomace are produced; between 2006-2007 over 46 million tonnes were produced. In South Africa a total production of 244 469 tonnes were produced during the 2011- 2012 season. Initially, apple pomace was regarded as a waste by-product used for animal feed and compost in soil, however presently it is considered a source of dietary fiber and natural antioxidants like polyphenols. In addition, apple pomace has a high carbohydrate content and can be enzymatically hydrolysed to produce sugar monomers which, in turn, can be fermented by yeasts to produce bioethanol. The polyphenols present in apple pomace can be used for their health properties, and the bioethanol can be used as a replacement for fossil fuel. Apple pomace is lignocellulosic in nature and consists of hemicellulose, cellulose, lignin and pectin. A combination of enzymes such as cellulases, hemicellulases, pectinases and lignases are required to operate in synergy for the degradation of lignocellulosic biomass. This is due to the recalcitrant nature of lignocellulose. This study investigated the degradation of apple pomace using a combination of commercially obtained enzyme cocktails viz. Viscozyme L , Celluclast 1.5L and Novozyme 188. The commercial enzymes Viscozyme L and Celluclast 1.5L were added in a ratio of 1:1 (50%:50%). The final concentrations of the enzymes were 0.019 mg/ml each. Novozyme 188 was added to provide a final concentration of 0.0024 mg/ml. A novel cost effective 20L bioreactor was designed, constructed and implemented for the degradation of apple pomace to produce value added products. The hydrolysis of the apple pomace was performed initially in 1 L flasks (batch fed) and, once optimized, scaled up to a 20 L bioreactor in batch mode. The bioreactors were operated at room temperature (22 ± 2ºC) and in an unbuffered system. The sugars released were detected and quantified using an optimized validated HPLC method established in this study. The sugars released in the bioreactors were mainly glucose, galactose, arabinose, cellobiose and fructose. The polyphenols released in this study were gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloridzin, which have a number of health benefits. The simultaneous analyses of the polyphenols were performed using a newly developed and validated HPLC method established in this study. This method was developed to detect nine polyphenols simultaneously. The two HPLC methods developed and validated in this study for the analysis of sugars and polyphenols demonstrated good accuracy, precision, reproducibility, linearity, robustness and sensitivity. Both analytical methods were validated according to the International Convention on Harmonization (ICH). The HPLC parameters for sugar analysis were: refractive index (RI) as the detection mode, the stationary phase was a ligand-exchange sugar column (Shodex SP0810) and an aqueous mobile phase in isocratic mode was used. The HPLC method for polyphenols employed UV diode array detection (DAD) as the detection mode, a reverse phase column as the stationary phase and a mobile phase of consisting of 0.01 M phosphoric acid in water and 100% methanol using gradient elution mode. The highest concentrations of sugars released in the novel 20 L bioreactor with 20% apple pomace (w/v) substrate loading were as follow: glucose (6.5 mg/ml), followed by galactose (2.1 mg/ml), arabinose (1.4 mg/ml), cellobiose (0.7 mg/ml) and fructose (0.5 mg/ml). The amounts of polyphenols released at 20% (w/v) apple pomace substrate were epicatechin (0.01 mg/ml), catechin (0.002 mg/ml), rutin (0.03 mg/ml), chlorogenic acid (0.002 mg/ml) and gallic acid 0.01 (mg/ml). Two mathematical models were developed in this study for kinetic analysis of lignocellulose (apple pomace) hydrolysis in the novel 20 L bioreactor, using the experimental data generated by the above HPLC analyses. The first model, modelling with regression, defines the hydrolysis of the sugars glucose, galactose, cellobiose and arabinose produced in the novel 20 L bioreactor at 5%, 10%, 15% and 20% (w/v) substrate concentrations. The regression model describes the sugars produced in the 20 L bioreactor by minimizing the error of the sugars released by finding a value for K which minimises the function which computes the sum of squares of errors between the solution curves and the data points. The second, more complex, model developed in this study used a system of differential equations model (ODE). This model solved the system by using a numerical method, such as the Runge-Kutta method, then fitted the solution curves to the data. Both models simulated (and had the ability to predict) the production of sugars in the novel 20 L bioreactor for apple pomace hydrolysis. These two models also revealed the time at which the maximum amount of sugars were released, which revealed the optimum time to run the 20 L bioreactor in order to be more cost effective. The optimum time for maximum glucose (the main sugar used in fermentation for biofuel production) release was determined to be around 60 h. The ODE model, in addition, determined the rate at which the substrate became depleted, as well as the rate at which the enzymes became deactivated for the various substrate loadings in the 20 L bioreactor. A third model was developed to determine the optimal running cost of the bioreactor which incorporated the substrate loading and the amount of glucose (g/L) produced. The novel 20 L bioreactor constructed from cost effective materials demonstrated that agro-industrial waste can be converted to value-added products by lignocellolytic enzymes. The sugars released from apple pomace can be used in biofuel production and the polyphenols as food supplements and nutraceuticals for health benefits. This novel study contributes to agro-industrial waste beneficiation via fuel production. In addition, using agro-industrial waste for the generation of value added products (instead of mere disposal) will help prevent environmental pollution.
- Full Text:
- Date Issued: 2016
Exploring targeted metagenomics and untargeted metabolomics for characterising aquaponics bacterial ecology and phytochemistry
- Authors: Abraham, Benjamin Melakail
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192453 , vital:45227
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Abraham, Benjamin Melakail
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192453 , vital:45227
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
An investigation into the potential immunogenicity of various extracts of the South African bont tick Amblyomma hebraeum
- Authors: Adamson, Deborah Jane
- Date: 1993
- Subjects: Amblyomma -- South Africa , Ticks -- South Africa , Ticks -- Control -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4127 , http://hdl.handle.net/10962/d1015640
- Description: Rabbits and goats were inoculated with crude, membrane-associated and soluble components extracted from unengorged adult females and nymphs of the bont tick Amblyomma hebraeum. Inoculation provided some protection against nymphal infestation, however it had little effect on adult feeding. Histological examination of adults fed on inoculated hosts showed evidence of gut damage. Skin provocation testing with tick extracts elicited a Type I immediate hypersensitivity which was influenced by antihistamine. A delayed skin reaction was also evident. Whether this was attributable to Type III Arthus reaction or Type IV cell-mediated hypersensitivity was not determined. A comparative histological study of sites of tick extract injection, on inoculated and naive hosts, demonstrated the role of eosinophils in the hosts response to tick feeding. Serological examination revealed elevated anti-A hebraeum lgG titres following inoculation. These titres were found to decrease in the ten weeks after inoculation, despite the hosts being repeatedly infested with A hebraeum. Although the IgG titres of naive control hosts increased after each tick infestation, they failed to reach the titres achieved through inoculation. Western blot analysis of serum from inoculated hosts recognized most of the A. hebraeum proteins against which it was screened.
- Full Text:
- Date Issued: 1993
- Authors: Adamson, Deborah Jane
- Date: 1993
- Subjects: Amblyomma -- South Africa , Ticks -- South Africa , Ticks -- Control -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4127 , http://hdl.handle.net/10962/d1015640
- Description: Rabbits and goats were inoculated with crude, membrane-associated and soluble components extracted from unengorged adult females and nymphs of the bont tick Amblyomma hebraeum. Inoculation provided some protection against nymphal infestation, however it had little effect on adult feeding. Histological examination of adults fed on inoculated hosts showed evidence of gut damage. Skin provocation testing with tick extracts elicited a Type I immediate hypersensitivity which was influenced by antihistamine. A delayed skin reaction was also evident. Whether this was attributable to Type III Arthus reaction or Type IV cell-mediated hypersensitivity was not determined. A comparative histological study of sites of tick extract injection, on inoculated and naive hosts, demonstrated the role of eosinophils in the hosts response to tick feeding. Serological examination revealed elevated anti-A hebraeum lgG titres following inoculation. These titres were found to decrease in the ten weeks after inoculation, despite the hosts being repeatedly infested with A hebraeum. Although the IgG titres of naive control hosts increased after each tick infestation, they failed to reach the titres achieved through inoculation. Western blot analysis of serum from inoculated hosts recognized most of the A. hebraeum proteins against which it was screened.
- Full Text:
- Date Issued: 1993
Bioprospecting for amylases, cellulases and xylanases from ericoid associated fungi, their production and characterisation for the bio-economy
- Authors: Adeoyo, Olusegun Richard
- Date: 2018
- Subjects: Mycorrhizal fungi , Hydrolases , Ericaceae South Africa , Ericaceae Molecular aspects
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64327 , vital:28533
- Description: South Africa is one of the most productive areas for ericaceous plants with about 850 identified species in the Cape Floral Region. The Albany Centre of Endemism where all fungi used in this study were isolated from, falls within this region. Ericaceous plants interact with some fungi via an association called the ericoid mycorrhizal (ERM) association. All fungi used in this study were isolated from roots of six ericaceous plants; Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Fungal enzymes are known to play a significant role in the food, brewing, detergent, pharmaceutical and biofuel industries. The enzyme industry is among the major sectors of the world, and additional novel sources are being explored from time to time. This study focussed on amylases (amyloglucosidase, AMG), cellulases (endoglucanase) and xylanases (endo-1,4-P-xylanase) production from ERM fungal isolates. Out of the fifty-one (51), fungal isolates screened, ChemRU330 (Leohumicola sp.), EdRU083 and EdRU002 were among the fungi that had the highest activities of all the enzymes. They were tested for the ability to produce amylases and cellulases under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28°C in a modified Melin-Norkrans (MMN) liquid medium. Cellulase specific activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and ChemRU330, respectively, was produced at an optimal pH of 5.0. For amylase, ChemRU330 had the highest specific activity of 1.11 U/mg protein while EdRU083 and EdRU02 had a specific activity of 0.80 and 0.92 U/mg protein, respectively, at the same pH with corresponding biomass yield of 113, 125 and 97 mg/50 ml, respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone, while NaFe-EDTA and cobalt inhibited enzyme activity. ChemRU330 was selected to determine the consistency and amount of amylase, cellulase and xylanase formed after several in vitro subculturing events. AMG and endo-1,4-P-xylanase were found to have the most consistent production throughout the study period. The AMG was stable at 45oC (pH 5.0), retaining approximately 65% activity over a period of 24 h. The molecular mass of AMG and endo-1,4-P-xylanase were estimated to be 101 kDa and 72 kDa, respectively. The Km and kcat were 0.38 mg/ml and 70 s-1, respectively, using soluble starch (AMG). For endo-1,4-P-xylanase, the Km and Vmax were 0.93 mg/ml and 8.54 U/ml, respectively, using beechwood xylan (endo-1,4-P-xylanase) as substrate. Additionally, crude extracts of five root endophytes with unique morphological characteristics were screened for antibacterial properties and was followed by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). L. incrustata (ChemRU330) and Chaetomium sp. extracts exhibited varying degrees of inhibition against two Gram-positive and Gram-negative bacteria. The crude extract of L. incrustata was the most effective which was found to inhibit Staphylococcus aureus (MIC: 1 mg/ml), Bacillus subtilis (MIC: 2 mg/ml) and Proteus vulgaris (MIC: 16 mg/ml). The L. incrustata displayed potential for antibacterial production and could be considered as an additional source of new antimicrobial agents in drug and food preservation. Also, the three isolates used for enzyme production were identified to genus and species levels, i.e., Leohumicola incrustata (ChemRU330), Leohumicola sp. (EdRU083) and Oidiodendron sp. (EdRU002) using both ITS and Cox1 DNA regions. The molecular analysis results indicated that these ERM mycorrhizal fungi were similar to those successfully described by some researchers in South Africa and Australia. Therefore, this study opens new opportunities for exploring ERM fungal biomolecules for the bio-economy. The promising physicochemical properties, starch and xylan hydrolysis end- products, and being non-pathogenic make AMG and endo-1,4-P-xylanase potential candidates for future applications as additives in the food industry for the production of glucose, glucose syrups, high-fructose corn syrups, and as well as the production of bioethanol. Finally, the findings of this study revealed that it is possible to produce hydrolytic enzymes from ERM fungi in vitro using chemically defined media. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Adeoyo, Olusegun Richard
- Date: 2018
- Subjects: Mycorrhizal fungi , Hydrolases , Ericaceae South Africa , Ericaceae Molecular aspects
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64327 , vital:28533
- Description: South Africa is one of the most productive areas for ericaceous plants with about 850 identified species in the Cape Floral Region. The Albany Centre of Endemism where all fungi used in this study were isolated from, falls within this region. Ericaceous plants interact with some fungi via an association called the ericoid mycorrhizal (ERM) association. All fungi used in this study were isolated from roots of six ericaceous plants; Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Fungal enzymes are known to play a significant role in the food, brewing, detergent, pharmaceutical and biofuel industries. The enzyme industry is among the major sectors of the world, and additional novel sources are being explored from time to time. This study focussed on amylases (amyloglucosidase, AMG), cellulases (endoglucanase) and xylanases (endo-1,4-P-xylanase) production from ERM fungal isolates. Out of the fifty-one (51), fungal isolates screened, ChemRU330 (Leohumicola sp.), EdRU083 and EdRU002 were among the fungi that had the highest activities of all the enzymes. They were tested for the ability to produce amylases and cellulases under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28°C in a modified Melin-Norkrans (MMN) liquid medium. Cellulase specific activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and ChemRU330, respectively, was produced at an optimal pH of 5.0. For amylase, ChemRU330 had the highest specific activity of 1.11 U/mg protein while EdRU083 and EdRU02 had a specific activity of 0.80 and 0.92 U/mg protein, respectively, at the same pH with corresponding biomass yield of 113, 125 and 97 mg/50 ml, respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone, while NaFe-EDTA and cobalt inhibited enzyme activity. ChemRU330 was selected to determine the consistency and amount of amylase, cellulase and xylanase formed after several in vitro subculturing events. AMG and endo-1,4-P-xylanase were found to have the most consistent production throughout the study period. The AMG was stable at 45oC (pH 5.0), retaining approximately 65% activity over a period of 24 h. The molecular mass of AMG and endo-1,4-P-xylanase were estimated to be 101 kDa and 72 kDa, respectively. The Km and kcat were 0.38 mg/ml and 70 s-1, respectively, using soluble starch (AMG). For endo-1,4-P-xylanase, the Km and Vmax were 0.93 mg/ml and 8.54 U/ml, respectively, using beechwood xylan (endo-1,4-P-xylanase) as substrate. Additionally, crude extracts of five root endophytes with unique morphological characteristics were screened for antibacterial properties and was followed by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). L. incrustata (ChemRU330) and Chaetomium sp. extracts exhibited varying degrees of inhibition against two Gram-positive and Gram-negative bacteria. The crude extract of L. incrustata was the most effective which was found to inhibit Staphylococcus aureus (MIC: 1 mg/ml), Bacillus subtilis (MIC: 2 mg/ml) and Proteus vulgaris (MIC: 16 mg/ml). The L. incrustata displayed potential for antibacterial production and could be considered as an additional source of new antimicrobial agents in drug and food preservation. Also, the three isolates used for enzyme production were identified to genus and species levels, i.e., Leohumicola incrustata (ChemRU330), Leohumicola sp. (EdRU083) and Oidiodendron sp. (EdRU002) using both ITS and Cox1 DNA regions. The molecular analysis results indicated that these ERM mycorrhizal fungi were similar to those successfully described by some researchers in South Africa and Australia. Therefore, this study opens new opportunities for exploring ERM fungal biomolecules for the bio-economy. The promising physicochemical properties, starch and xylan hydrolysis end- products, and being non-pathogenic make AMG and endo-1,4-P-xylanase potential candidates for future applications as additives in the food industry for the production of glucose, glucose syrups, high-fructose corn syrups, and as well as the production of bioethanol. Finally, the findings of this study revealed that it is possible to produce hydrolytic enzymes from ERM fungi in vitro using chemically defined media. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
In silico analysis of plasmodium falciparum Hsp70-x for potential binding sites and hits
- Authors: Amusengeri, Arnold
- Date: 2017
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59136 , vital:27435
- Description: Restricted access-thesis embargoed for 1 year - release date April 2019
- Full Text:
- Date Issued: 2017
- Authors: Amusengeri, Arnold
- Date: 2017
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59136 , vital:27435
- Description: Restricted access-thesis embargoed for 1 year - release date April 2019
- Full Text:
- Date Issued: 2017
Citizen science, treatment and microbial compliance monitoring in rainwater harvesting in Namibia
- Angala, Hallo Angaleni Nameya
- Authors: Angala, Hallo Angaleni Nameya
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62082 , vital:28105
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
- Authors: Angala, Hallo Angaleni Nameya
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62082 , vital:28105
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
Localizing selected endocytosis protein candidates in Plasmodium falciparum using GFP-tagged fusion constructs
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Date Issued: 2016
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Date Issued: 2016
An in-silico study of the type II NADH: Quinone Oxidoreductase (ndh2). A new anti-malaria drug target
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
The role of Stress Inducible Protein 1 (STI1) in the regulation of actin dynamics
- Authors: Beckley, Samantha Joy
- Date: 2015
- Subjects: Heat shock proteins , Molecular chaperones , Actin , Microfilament proteins , Cell migration , Adenosine triphosphatase , Metastasis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193941 , vital:45409
- Description: Stress-inducible protein 1 (STI1) otherwise known as Hop (Hsp70/Hsp90 organising protein) is a highly conserved abundant co-chaperone of the Hsp70 and Hsp90 chaperones. STI1 acts as an adapter protein, where it regulates the transfer of protein substrates from Hsp70 to Hsp90 during the assembly of a number of chaperone-client protein complexes. The role of STI1 associating independently with non-chaperone proteins has become increasingly prominent. Recent data from colocalisation and co-sedimentation analyses in our laboratory suggested a direct interaction between STI1 and the cytoskeletal protein, actin. However, there was a lack of information on the motifs which mediated this interaction, as well as the exact role of STI1 in the regulation of cytoskeletal dynamics. Two putative actin binding motifs, DAYKKK (within the TPR2A domain) and a polyproline region (after the DP1 domain), were identified in mammalian STI1. Our data from in vitro interaction studies including surface plasmon resonance and high speed co-sedimentation assays suggested that both TPR1 and TPR2AB were required for the STI1-actin interaction, and peptides corresponding to either the DAYKKK or the polyproline motif, alone or in combination, could not block the STI1-actin interaction. Full length mSTI1 was shown to have ATPase activity and when combined with actin an increase in ATPase activity was seen. Ex vivo studies using STI1 knockdown shRNA HEK293T cells and non-targeting control shRNA HEK293T cells showed a change of F-actin morphology as well as reduction in levels of actin-binding proteins profilin, cofilin and tubulin in the STI1 knockdown cells. These data extend our understanding of the role of STI1 in regulating actin dynamics and may have implications for cell migration. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
- Authors: Beckley, Samantha Joy
- Date: 2015
- Subjects: Heat shock proteins , Molecular chaperones , Actin , Microfilament proteins , Cell migration , Adenosine triphosphatase , Metastasis
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193941 , vital:45409
- Description: Stress-inducible protein 1 (STI1) otherwise known as Hop (Hsp70/Hsp90 organising protein) is a highly conserved abundant co-chaperone of the Hsp70 and Hsp90 chaperones. STI1 acts as an adapter protein, where it regulates the transfer of protein substrates from Hsp70 to Hsp90 during the assembly of a number of chaperone-client protein complexes. The role of STI1 associating independently with non-chaperone proteins has become increasingly prominent. Recent data from colocalisation and co-sedimentation analyses in our laboratory suggested a direct interaction between STI1 and the cytoskeletal protein, actin. However, there was a lack of information on the motifs which mediated this interaction, as well as the exact role of STI1 in the regulation of cytoskeletal dynamics. Two putative actin binding motifs, DAYKKK (within the TPR2A domain) and a polyproline region (after the DP1 domain), were identified in mammalian STI1. Our data from in vitro interaction studies including surface plasmon resonance and high speed co-sedimentation assays suggested that both TPR1 and TPR2AB were required for the STI1-actin interaction, and peptides corresponding to either the DAYKKK or the polyproline motif, alone or in combination, could not block the STI1-actin interaction. Full length mSTI1 was shown to have ATPase activity and when combined with actin an increase in ATPase activity was seen. Ex vivo studies using STI1 knockdown shRNA HEK293T cells and non-targeting control shRNA HEK293T cells showed a change of F-actin morphology as well as reduction in levels of actin-binding proteins profilin, cofilin and tubulin in the STI1 knockdown cells. These data extend our understanding of the role of STI1 in regulating actin dynamics and may have implications for cell migration. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism
- Authors: Bizabani, Christine
- Date: 2015
- Subjects: Ericaceae , Ericas , Roots (Botany) -- Diseases and pests , Mycorrhizal fungi , Polymerase chain reaction , Fungi -- Classification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4160 , http://hdl.handle.net/10962/d1018575
- Description: South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.
- Full Text:
- Date Issued: 2015
The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism
- Authors: Bizabani, Christine
- Date: 2015
- Subjects: Ericaceae , Ericas , Roots (Botany) -- Diseases and pests , Mycorrhizal fungi , Polymerase chain reaction , Fungi -- Classification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4160 , http://hdl.handle.net/10962/d1018575
- Description: South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.
- Full Text:
- Date Issued: 2015
Diversified computational approaches for the identification of orthosteric drugs, allosteric modulators and unveiling drug resistance mechanisms: application to infectious diseases
- Authors: Boateng, Rita Afriyie
- Date: 2022-04-08
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/234173 , vital:50169
- Description: Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-08
- Authors: Boateng, Rita Afriyie
- Date: 2022-04-08
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/234173 , vital:50169
- Description: Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-08
In silico characterization of plasmodial transketolases as potential malaria drug target
- Authors: Boateng, Rita Afriyie
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63540 , vital:28433
- Description: Expected release date-April 2019
- Full Text:
- Date Issued: 2018
- Authors: Boateng, Rita Afriyie
- Date: 2018
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63540 , vital:28433
- Description: Expected release date-April 2019
- Full Text:
- Date Issued: 2018
The novobiocin-induced turnover of fibronectin via low density lipoprotein receptor-related protein 1 alters matrix morphology with physiological consequences on cell growth and migration
- Authors: Boёl, Natasha Marie-Eraine
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/114778 , vital:34034 , 10.21504/10962/114778
- Description: Fibronectin (FN), an extracellular matrix protein, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion thereby contributing to the metastatic potential of cancer cells. Previous studies have shown the direct binding of Heat Shock Protein 90 kDa (Hsp90) and FN in vitro, and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. Low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitous receptor known to bind both Hsp90 and FN. Using an LRP1 expressing Hs578T breast cancer cell line and an isogenic mouse embryonic fibroblast (MEF) model system of differential LRP1 expression we demonstrate that LRP1 is involved in turnover of FN in response to C-terminal Hsp90 inhibition. The first objective of this study was to identify the mechanism of NOV-induced LRP1-mediated FN turnover. Our data show that NOV-mediated FN turnover via LRP1 did not require the activity of matrix metalloproteinases (MMPs), which play an important role in processing and degradation of the extracellular matrix and FN. In addition, the levels of the main FN receptor responsible for its extracellular assembly, β1-integrin, did not change in response to NOV. LRP1 is known to undergo regulated intramembrane proteolysis (RIP) which generates smaller fragments that may translocate to the nucleus and modulate gene transcription. Using inhibitors of LRP1 cleavage and nuclear fractionation we determined that LRP1 processing was not required for the NOV-induced FN response suggesting that a mechanism unrelated to LRP1 RIP is involved. A possible mechanism may be in altered Hsp90-LRP1 cell signalling as we observed disruption of the FN-Hsp90-LRP1 complex at the cell surface in NOV treated cells. How this affects downstream eHsp90-LRP1 signalling is still to be determined but may be related to a significant increase in phospho-AKT and loss of phospho-ERK upon NOV-treatment; two key signalling proteins involved in FN matrix regulation and which are downstream of LRP1 signalling. The second objective of this study was to determine the physiological consequences associated with FN turnover in response to NOV treatment. Using migration assays we demonstrated that levels of insoluble matrix-associated FN and FN concentration are not solely responsible for migratory capacity of cells on decellularized extracellular matrices, but rather that structural composition and integrity of the matrix plays a bigger role. Using confocal and scanning electron microscopy, we identified NOV treated matrices to be flatter, less mature and contain thicker, rope-like FN fibrils to which cells adhered better but were generally less proliferative. Comparatively, cells adhered less to the more mature and 3-dimensional untreated matrices but exhibited increased spreading and cell growth, which may in part be due to the thinner fibrils and web-like matrix. In summary, this study substantiates the role of LRP1 in NOV-mediated FN turnover, and provides new insights into the possible mechanisms of the Hsp90-LRP1 mediated loss of FN matrix. This is the first study to demonstrate some of the functional consequences related to FN turnover by NOV at the ECM level. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
- Authors: Boёl, Natasha Marie-Eraine
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/114778 , vital:34034 , 10.21504/10962/114778
- Description: Fibronectin (FN), an extracellular matrix protein, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion thereby contributing to the metastatic potential of cancer cells. Previous studies have shown the direct binding of Heat Shock Protein 90 kDa (Hsp90) and FN in vitro, and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. Low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitous receptor known to bind both Hsp90 and FN. Using an LRP1 expressing Hs578T breast cancer cell line and an isogenic mouse embryonic fibroblast (MEF) model system of differential LRP1 expression we demonstrate that LRP1 is involved in turnover of FN in response to C-terminal Hsp90 inhibition. The first objective of this study was to identify the mechanism of NOV-induced LRP1-mediated FN turnover. Our data show that NOV-mediated FN turnover via LRP1 did not require the activity of matrix metalloproteinases (MMPs), which play an important role in processing and degradation of the extracellular matrix and FN. In addition, the levels of the main FN receptor responsible for its extracellular assembly, β1-integrin, did not change in response to NOV. LRP1 is known to undergo regulated intramembrane proteolysis (RIP) which generates smaller fragments that may translocate to the nucleus and modulate gene transcription. Using inhibitors of LRP1 cleavage and nuclear fractionation we determined that LRP1 processing was not required for the NOV-induced FN response suggesting that a mechanism unrelated to LRP1 RIP is involved. A possible mechanism may be in altered Hsp90-LRP1 cell signalling as we observed disruption of the FN-Hsp90-LRP1 complex at the cell surface in NOV treated cells. How this affects downstream eHsp90-LRP1 signalling is still to be determined but may be related to a significant increase in phospho-AKT and loss of phospho-ERK upon NOV-treatment; two key signalling proteins involved in FN matrix regulation and which are downstream of LRP1 signalling. The second objective of this study was to determine the physiological consequences associated with FN turnover in response to NOV treatment. Using migration assays we demonstrated that levels of insoluble matrix-associated FN and FN concentration are not solely responsible for migratory capacity of cells on decellularized extracellular matrices, but rather that structural composition and integrity of the matrix plays a bigger role. Using confocal and scanning electron microscopy, we identified NOV treated matrices to be flatter, less mature and contain thicker, rope-like FN fibrils to which cells adhered better but were generally less proliferative. Comparatively, cells adhered less to the more mature and 3-dimensional untreated matrices but exhibited increased spreading and cell growth, which may in part be due to the thinner fibrils and web-like matrix. In summary, this study substantiates the role of LRP1 in NOV-mediated FN turnover, and provides new insights into the possible mechanisms of the Hsp90-LRP1 mediated loss of FN matrix. This is the first study to demonstrate some of the functional consequences related to FN turnover by NOV at the ECM level. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
Bioinformatics tool development with a focus on structural bioinformatics and the analysis of genetic variation in humans
- Authors: Brown, David K
- Date: 2018
- Subjects: Bioinformatics , Human genetics -- Variation , High performance computing , Workflow management systems , Molecular dynamics , Next generation sequencing , Human Mutation Analysis (HUMA)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/60708 , vital:27820
- Description: This thesis is divided into three parts, united under the general theme of bioinformatics tool development and variation analysis. Part 1 describes the design and development of the Job Management System (JMS), a workflow management system for high performance computing (HPC). HPC has become an integral part of bioinformatics. Computational methods for molecular dynamics and next generation sequencing (NGS) analysis, which require complex calculations on large datasets, are not yet feasible on desktop computers. As such, powerful computer clusters have been employed to perform these calculations. However, making use of these HPC clusters requires familiarity with command line interfaces. This excludes a large number of researchers from taking advantage of these resources. JMS was developed as a tool to make it easier for researchers without a computer science background to make use of HPC. Additionally, JMS can be used to host computational tools and pipelines and generates both web-based interfaces and RESTful APIs for those tools. The web-based interfaces can be used to quickly and easily submit jobs to the underlying cluster. The RESTful web API, on the other hand, allows JMS to provided backend functionality for external tools and web servers that want to run jobs on the cluster. Numerous tools and workflows have already been added to JMS, several of which have been incorporated into external web servers. One such web server is the Human Mutation Analysis (HUMA) web server and database. HUMA, the topic of part 2 of this thesis, is a platform for the analysis of genetic variation in humans. HUMA aggregates data from various existing databases into a single, connected and related database. The advantages of this are realized in the powerful querying abilities that it provides. HUMA includes protein, gene, disease, and variation data and can be searched from the angle of any one of these categories. For example, searching for a protein will return the protein data (e.g. protein sequences, structures, domains and families, and other meta-data). However, the related nature of the database means that genes, diseases, variation, and literature related to the protein will also be returned, giving users a powerful and holistic view of all data associated with the protein. HUMA also provides links to the original sources of the data, allowing users to follow the links to find additional details. HUMA aims to be a platform for the analysis of genetic variation. As such, it also provides tools to visualize and analyse the data (several of which run on the underlying cluster, via JMS). These tools include alignment and 3D structure visualization, homology modeling, variant analysis, and the ability to upload custom variation datasets and map them to proteins, genes and diseases. HUMA also provides collaboration features, allowing users to share and discuss datasets and job results. Finally, part 3 of this thesis focused on the development of a suite of tools, MD-TASK, to analyse genetic variation at the protein structure level via network analysis of molecular dynamics simulations. The use of MD-TASK in combination with the tools developed in the previous parts of this thesis is showcased via the analysis of variation in the renin-angiotensinogen complex, a vital part of the renin-angiotensin system.
- Full Text:
- Date Issued: 2018
- Authors: Brown, David K
- Date: 2018
- Subjects: Bioinformatics , Human genetics -- Variation , High performance computing , Workflow management systems , Molecular dynamics , Next generation sequencing , Human Mutation Analysis (HUMA)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/60708 , vital:27820
- Description: This thesis is divided into three parts, united under the general theme of bioinformatics tool development and variation analysis. Part 1 describes the design and development of the Job Management System (JMS), a workflow management system for high performance computing (HPC). HPC has become an integral part of bioinformatics. Computational methods for molecular dynamics and next generation sequencing (NGS) analysis, which require complex calculations on large datasets, are not yet feasible on desktop computers. As such, powerful computer clusters have been employed to perform these calculations. However, making use of these HPC clusters requires familiarity with command line interfaces. This excludes a large number of researchers from taking advantage of these resources. JMS was developed as a tool to make it easier for researchers without a computer science background to make use of HPC. Additionally, JMS can be used to host computational tools and pipelines and generates both web-based interfaces and RESTful APIs for those tools. The web-based interfaces can be used to quickly and easily submit jobs to the underlying cluster. The RESTful web API, on the other hand, allows JMS to provided backend functionality for external tools and web servers that want to run jobs on the cluster. Numerous tools and workflows have already been added to JMS, several of which have been incorporated into external web servers. One such web server is the Human Mutation Analysis (HUMA) web server and database. HUMA, the topic of part 2 of this thesis, is a platform for the analysis of genetic variation in humans. HUMA aggregates data from various existing databases into a single, connected and related database. The advantages of this are realized in the powerful querying abilities that it provides. HUMA includes protein, gene, disease, and variation data and can be searched from the angle of any one of these categories. For example, searching for a protein will return the protein data (e.g. protein sequences, structures, domains and families, and other meta-data). However, the related nature of the database means that genes, diseases, variation, and literature related to the protein will also be returned, giving users a powerful and holistic view of all data associated with the protein. HUMA also provides links to the original sources of the data, allowing users to follow the links to find additional details. HUMA aims to be a platform for the analysis of genetic variation. As such, it also provides tools to visualize and analyse the data (several of which run on the underlying cluster, via JMS). These tools include alignment and 3D structure visualization, homology modeling, variant analysis, and the ability to upload custom variation datasets and map them to proteins, genes and diseases. HUMA also provides collaboration features, allowing users to share and discuss datasets and job results. Finally, part 3 of this thesis focused on the development of a suite of tools, MD-TASK, to analyse genetic variation at the protein structure level via network analysis of molecular dynamics simulations. The use of MD-TASK in combination with the tools developed in the previous parts of this thesis is showcased via the analysis of variation in the renin-angiotensinogen complex, a vital part of the renin-angiotensin system.
- Full Text:
- Date Issued: 2018
Molecular simulations of potential agents and targets of Alzheimer’s disease
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
- Full Text:
- Date Issued: 2020
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
- Full Text:
- Date Issued: 2020
Towards a Mobile Bioethanol Unit for point of source conversion of sugar sources to bioethanol: design and feasibility study for South Africa
- Authors: Cech, Alexandra Louise
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59141 , vital:27439
- Description: Restricted access-thesis embargoed for 5 years
- Full Text:
- Date Issued: 2015
- Authors: Cech, Alexandra Louise
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59141 , vital:27439
- Description: Restricted access-thesis embargoed for 5 years
- Full Text:
- Date Issued: 2015
The Role of HSP70/HSP90 Organizing Protein (Hop) in the Heat Shock Factor 1 (HSF1)-mediated Stress Response
- Authors: Chakraborty, Abantika
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163204 , vital:41018 , doi:10.21504/10962/163204
- Description: Molecular chaperones regulate cellular proteostasis. They control protein conformation and prevent misfolding and aggregation under both normal and stressful environments, ultimately resulting in cell survival. The project aimed to understand the role of the HSP70 – HSP90 Organizing Protein (Hop/STIP1) in the survival of stressed cells and the function of the stress-responsive transcription factor, Heat Shock Factor 1 (HSF1). HSF1 protein levels were significantly reduced in Hop-depleted HEK293T cells compared to controls by ELISA, western blot, and mass spectrometry. HSF1 transcriptional activity at the HSP70 promoter, and binding of a biotinylated HSE oligonucleotide under basal conditions were significantly reduced, consistent with the reduced levels of HSF1. In response to heat shock, HSF1 levels in Hop-depleted cells increased to that of controls, but there was still significantly lowerHSF1 transcriptional activity and HSE binding. Hop-depleted HEK293T cells were more sensitive than controls to the HSF1 inhibitor KRIBB11 and showed reduced short-term and long-term proliferation. Unlike the HSP90 inhibitor 17-DMAG, which had no effect, the HSP70 inhibitor JG98, further decreased the levels of HSF1 in Hop-depleted cells, suggesting a role for HSP70 in the Hop-mediated effects. There was punctate nuclear staining for HSF1 in Hop-depleted cells under both basal and heat shock conditions, as well as reduced nuclear localization and increased cytoplasmic accumulation of HSF1 in response to heat shock. Hop and HSF1 colocalized in cells, and HSF1 could be isolated in complex with Hop and HSP70. Loss of Hop reduced HSF1 in HSP70complexes but did not affect HSF1 abundance in HSP90 complexes. Hop-depleted cells showed reduced short-term and long-term survival compared to controls, an effect that was potentiated by the JG98 HSP70 inhibitor. Taken together, these data suggest that Hop regulation of HSF1activity is via a mechanism involving reductions in HSP70 interaction, as well as reduced nuclear localization, and DNA binding, and is consistent with reduced cellular fitness under basal and stress conditions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Chakraborty, Abantika
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163204 , vital:41018 , doi:10.21504/10962/163204
- Description: Molecular chaperones regulate cellular proteostasis. They control protein conformation and prevent misfolding and aggregation under both normal and stressful environments, ultimately resulting in cell survival. The project aimed to understand the role of the HSP70 – HSP90 Organizing Protein (Hop/STIP1) in the survival of stressed cells and the function of the stress-responsive transcription factor, Heat Shock Factor 1 (HSF1). HSF1 protein levels were significantly reduced in Hop-depleted HEK293T cells compared to controls by ELISA, western blot, and mass spectrometry. HSF1 transcriptional activity at the HSP70 promoter, and binding of a biotinylated HSE oligonucleotide under basal conditions were significantly reduced, consistent with the reduced levels of HSF1. In response to heat shock, HSF1 levels in Hop-depleted cells increased to that of controls, but there was still significantly lowerHSF1 transcriptional activity and HSE binding. Hop-depleted HEK293T cells were more sensitive than controls to the HSF1 inhibitor KRIBB11 and showed reduced short-term and long-term proliferation. Unlike the HSP90 inhibitor 17-DMAG, which had no effect, the HSP70 inhibitor JG98, further decreased the levels of HSF1 in Hop-depleted cells, suggesting a role for HSP70 in the Hop-mediated effects. There was punctate nuclear staining for HSF1 in Hop-depleted cells under both basal and heat shock conditions, as well as reduced nuclear localization and increased cytoplasmic accumulation of HSF1 in response to heat shock. Hop and HSF1 colocalized in cells, and HSF1 could be isolated in complex with Hop and HSP70. Loss of Hop reduced HSF1 in HSP70complexes but did not affect HSF1 abundance in HSP90 complexes. Hop-depleted cells showed reduced short-term and long-term survival compared to controls, an effect that was potentiated by the JG98 HSP70 inhibitor. Taken together, these data suggest that Hop regulation of HSF1activity is via a mechanism involving reductions in HSP70 interaction, as well as reduced nuclear localization, and DNA binding, and is consistent with reduced cellular fitness under basal and stress conditions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
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
Identification of novel compounds against Plasmodium falciparum Cytochrome bc1 Complex inhibiting the trans-membrane electron transfer pathway: an In Silico study
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Isolation, expression and purification of the hydantoin hydrolysing enzymes of agrobacterium tumefaciens
- Authors: Clark, Sally-Ann
- Date: 2003
- Subjects: Agrobacterium tumefaciens , Amino acids Hydantoin Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4140 , http://hdl.handle.net/10962/d1016233
- Description: The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.
- Full Text:
- Date Issued: 2003
- Authors: Clark, Sally-Ann
- Date: 2003
- Subjects: Agrobacterium tumefaciens , Amino acids Hydantoin Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4140 , http://hdl.handle.net/10962/d1016233
- Description: The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.
- Full Text:
- Date Issued: 2003