Human FN1 is regulated by the heat-shock response
- Authors: Dhanani, Karim Colin Hassan
- Date: 2015
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193487 , vital:45336
- Description: Heat shock protein 90 (Hsp90) and heat shock factors (HSFs) are known to be involved in the epigenetic regulation of several fundamental oncogenic genes. Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein which plays key roles in cell adhesion and migration. Hsp90 binds directly to FN and Hsp90 inhibition has been shown to regulate FN protein levels and matrix formation. Where inhibition of Hsp90 with a C-terminal inhibitor (novobiocin) induced the loss of FN matrix, treatment with an N-terminal inhibitor (geldanamycin) increased FN matrix levels. GA treatment induced a strong dose and time dependent increase in FN1 promoter activity and increased total FN mRNA respectively. By contrast, NOV showed no increase in the promoter activity and no change in the expression of FN mRNA. As GA is known to induce the stress response, we investigated the relationship between the cell stress machinery and the transcriptional regulation of FN. Three putative heat shock elements (HSEs) were identified in the FN1 promoter. The loss of two of the three identified putative HSEs resulted in a loss in the basal transcriptional activity of the FN1 promoter in our reporter model. This was in addition to the loss of the induction of transcriptional activity with GA treatment observed with the full-length promoter. Binding of HSF1 to one of the putative HSEs, which was identified as potentially functional from the truncation analysis, was confirmed using ChIP. The occupancy of this HSE by HSF1 was shown to increase with GA treatment. These data support the hypothesis that FN1 is a functional HSF1 target gene. The 5' promoter regions of seven additional ECM protein encoding genes were analysed and mRNA levels were detected by quantitative RT-PCR upon treatment with GA. Collagen 4 _2 and laminin _3 mRNA were found to increase in the presence of GA, whereas collagen 4 _3 and osteopontin showed no change. Similarly to FN1, these data indicate that a subset of ECM genes may be under the regulation of the HSF1 mediated heat-shock response. This may have implications for our understanding of ECM dynamics in cancer, where the clinical application of Hsp90 inhibitors is intended. Additionally, our data provide a poten- tial underpinning for the role of the HSF1 mediated heat-shock response in several fibrotic and metabolic stress related pathologies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
- Authors: Dhanani, Karim Colin Hassan
- Date: 2015
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193487 , vital:45336
- Description: Heat shock protein 90 (Hsp90) and heat shock factors (HSFs) are known to be involved in the epigenetic regulation of several fundamental oncogenic genes. Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein which plays key roles in cell adhesion and migration. Hsp90 binds directly to FN and Hsp90 inhibition has been shown to regulate FN protein levels and matrix formation. Where inhibition of Hsp90 with a C-terminal inhibitor (novobiocin) induced the loss of FN matrix, treatment with an N-terminal inhibitor (geldanamycin) increased FN matrix levels. GA treatment induced a strong dose and time dependent increase in FN1 promoter activity and increased total FN mRNA respectively. By contrast, NOV showed no increase in the promoter activity and no change in the expression of FN mRNA. As GA is known to induce the stress response, we investigated the relationship between the cell stress machinery and the transcriptional regulation of FN. Three putative heat shock elements (HSEs) were identified in the FN1 promoter. The loss of two of the three identified putative HSEs resulted in a loss in the basal transcriptional activity of the FN1 promoter in our reporter model. This was in addition to the loss of the induction of transcriptional activity with GA treatment observed with the full-length promoter. Binding of HSF1 to one of the putative HSEs, which was identified as potentially functional from the truncation analysis, was confirmed using ChIP. The occupancy of this HSE by HSF1 was shown to increase with GA treatment. These data support the hypothesis that FN1 is a functional HSF1 target gene. The 5' promoter regions of seven additional ECM protein encoding genes were analysed and mRNA levels were detected by quantitative RT-PCR upon treatment with GA. Collagen 4 _2 and laminin _3 mRNA were found to increase in the presence of GA, whereas collagen 4 _3 and osteopontin showed no change. Similarly to FN1, these data indicate that a subset of ECM genes may be under the regulation of the HSF1 mediated heat-shock response. This may have implications for our understanding of ECM dynamics in cancer, where the clinical application of Hsp90 inhibitors is intended. Additionally, our data provide a poten- tial underpinning for the role of the HSF1 mediated heat-shock response in several fibrotic and metabolic stress related pathologies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
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
Investigating the expression of three small open reading frames encoded on Helicoverpa armigera stunt virus RNA 1
- Authors: De Bruyn, Mart-Mari
- Date: 2017
- Subjects: Helicoverpa armigera , RNA viruses , Insects Viruses , Proteins
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59168 , vital:27448
- Description: The Helicoverpa armigera stunt virus (HaSV), belonging to the Family Alphatetraviridae (Genus: Omegatetravirus), is a non-enveloped insect virus encapsidating a bi-partite, positive-sense single-stranded RNA genome. RNA1 encodes the replicase, as well as three small open reading frames (ORFs) arranged in tandem, and overlapping with the 3’ end of the replicase ORF. These ORFs, designated p11, p15 and p8, encode putative proteins of unknown function. The p11 and p15 ORFs are conserved in the genome of the related Omegatetravirus, Dendrolimus punctatus tetravirus. In HaSV, the stop codon of p11 is followed immediately by the start of p15, whereas the stop of p15 and start of p8 are separated by a glycine intercodon. Furthermore, only p11 is known to have a recognizable Kozak sequence. The aim of this study was to determine the expression and function of these three small proteins in the HaSV infectious lifecycle. The authenticity of the viral cDNA sequence, encoding the three small ORFs, was validated by sequencing multiple cDNA clones of the relevant region in viral RNA (vRNA), purified from infectious HaSV particles. The sequence of all three ORFs was conserved in seven cDNA clones, while point mutations were observed in each of two remaining cDNA clones, suggesting that the ORFs were conserved in infectious virus. Polyclonal antisera were raised against a p11 peptide, and a recombinant p15-p8 fusion protein (p23) expressed and purified from Escherichia coli. The affinity of the anti-p23 antiserum was confirmed by western blot analysis, while that of the anti-p11 antiserum was confirmed using immunofluorescence microscopy, as attempted expression of recombinant p11 in E. coli appeared to be toxic. The antisera were used to detect expression of the small proteins in HaSV-infected H. armigera larvae by western blot analysis. A band migrating at approximately 34 kDa was detected by both antisera in infected larvae, absent in uninfected larvae, suggesting the expression of a p11-p15-p8 polyprotein. Protein bands of 11 kDa and 8 kDa were also detected by the anti-p11 and anti-p23 antisera, respectively. Bioinformatic analysis revealed that the polyprotein would be produced by a novel type of stop codon read-through, however the mechanism required for individual expression could not be definitively determined. The mechanism by which these ORFs are translated was further investigated by expressing p11-p15, tagged with FLAG and enhanced green flourescent protein (EGFP) at its amino- and carboxyl-termini respectively (FLAG-p11-p15-EGFP), in Spodoptera frugiperda (Sf9) cells detected by flourescence microscopy. Punctate structures were observed throughout the cytoplasm that were also detected with antiFLAG, anti-p11 and anti-p23 antisera, complementing results obtained in previous studies. Since p15 does not exhibit a strong recognizable Kozak like p11, the dependency of p15 expression on that of p11 was investigated by mutating this construct such that p15 occurred in a +1 frame to p11. Both EGFP and anti-p23 fluorescence was detected with the same cytoplasmic distribution as the unmutated construct, whereas nothing was detected by anti-FLAG and anti-p11. Preliminary results therefore suggested p15 may also be expressed as a discrete protein, independent of p11. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2017
- Authors: De Bruyn, Mart-Mari
- Date: 2017
- Subjects: Helicoverpa armigera , RNA viruses , Insects Viruses , Proteins
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59168 , vital:27448
- Description: The Helicoverpa armigera stunt virus (HaSV), belonging to the Family Alphatetraviridae (Genus: Omegatetravirus), is a non-enveloped insect virus encapsidating a bi-partite, positive-sense single-stranded RNA genome. RNA1 encodes the replicase, as well as three small open reading frames (ORFs) arranged in tandem, and overlapping with the 3’ end of the replicase ORF. These ORFs, designated p11, p15 and p8, encode putative proteins of unknown function. The p11 and p15 ORFs are conserved in the genome of the related Omegatetravirus, Dendrolimus punctatus tetravirus. In HaSV, the stop codon of p11 is followed immediately by the start of p15, whereas the stop of p15 and start of p8 are separated by a glycine intercodon. Furthermore, only p11 is known to have a recognizable Kozak sequence. The aim of this study was to determine the expression and function of these three small proteins in the HaSV infectious lifecycle. The authenticity of the viral cDNA sequence, encoding the three small ORFs, was validated by sequencing multiple cDNA clones of the relevant region in viral RNA (vRNA), purified from infectious HaSV particles. The sequence of all three ORFs was conserved in seven cDNA clones, while point mutations were observed in each of two remaining cDNA clones, suggesting that the ORFs were conserved in infectious virus. Polyclonal antisera were raised against a p11 peptide, and a recombinant p15-p8 fusion protein (p23) expressed and purified from Escherichia coli. The affinity of the anti-p23 antiserum was confirmed by western blot analysis, while that of the anti-p11 antiserum was confirmed using immunofluorescence microscopy, as attempted expression of recombinant p11 in E. coli appeared to be toxic. The antisera were used to detect expression of the small proteins in HaSV-infected H. armigera larvae by western blot analysis. A band migrating at approximately 34 kDa was detected by both antisera in infected larvae, absent in uninfected larvae, suggesting the expression of a p11-p15-p8 polyprotein. Protein bands of 11 kDa and 8 kDa were also detected by the anti-p11 and anti-p23 antisera, respectively. Bioinformatic analysis revealed that the polyprotein would be produced by a novel type of stop codon read-through, however the mechanism required for individual expression could not be definitively determined. The mechanism by which these ORFs are translated was further investigated by expressing p11-p15, tagged with FLAG and enhanced green flourescent protein (EGFP) at its amino- and carboxyl-termini respectively (FLAG-p11-p15-EGFP), in Spodoptera frugiperda (Sf9) cells detected by flourescence microscopy. Punctate structures were observed throughout the cytoplasm that were also detected with antiFLAG, anti-p11 and anti-p23 antisera, complementing results obtained in previous studies. Since p15 does not exhibit a strong recognizable Kozak like p11, the dependency of p15 expression on that of p11 was investigated by mutating this construct such that p15 occurred in a +1 frame to p11. Both EGFP and anti-p23 fluorescence was detected with the same cytoplasmic distribution as the unmutated construct, whereas nothing was detected by anti-FLAG and anti-p11. Preliminary results therefore suggested p15 may also be expressed as a discrete protein, independent of p11. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2017
An investigation into the bacterial communities associated with pyrroloiminoquinone-producing South African latrunculid sponges
- Authors: Hilliar, Storm Hannah
- Date: 2018
- Subjects: Sponges South Africa Algoa Bay , Betaproteobacteria , Spirochaeta , Symbiosis , Bacterial communities
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62112 , vital:28128
- Description: Marine sponges belonging to the family Latrunculiidae are known for their production of cytotoxic pyrroloiminoquinone alkaloids and the South African coast provides a unique environment for the exploitation of these potent bioactive compounds. The isolation of structurally similar pyrroloiminoquinone compounds from unrelated, non poriferan sources has led to the suggestion that South African latrunculid pyrroloiminoquinones may be secondary metabolites produced by sponge associated microbial symbionts. Previous studies investigating the bacterial communities of South African latrunculid sponges have shown the conservation of distinct microbial populations with unusual bacterial taxa dominated by a novel betaproteobacterial and spirochete species. This study describes the further investigation into these associated bacterial communities, their conservation and sponge microbiome comparisons across spatial, temporal and environmental scales. The bacterial communities associated with seven latrunculid species representing three genera (Tsitsikamma, Cyclacanthia and Latrunculia) were characterized as well as a Mycale and Tethya rubra species. Latrunculid sponge microbiomes were significantly different from those associated with sympatric outlier sponge species and the surrounding environment. The bacterial communities associated with latrunculid sponges appear host specific with the conservation of two dominant bacterial symbionts which mirror the phylogeny of their host species. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Hilliar, Storm Hannah
- Date: 2018
- Subjects: Sponges South Africa Algoa Bay , Betaproteobacteria , Spirochaeta , Symbiosis , Bacterial communities
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62112 , vital:28128
- Description: Marine sponges belonging to the family Latrunculiidae are known for their production of cytotoxic pyrroloiminoquinone alkaloids and the South African coast provides a unique environment for the exploitation of these potent bioactive compounds. The isolation of structurally similar pyrroloiminoquinone compounds from unrelated, non poriferan sources has led to the suggestion that South African latrunculid pyrroloiminoquinones may be secondary metabolites produced by sponge associated microbial symbionts. Previous studies investigating the bacterial communities of South African latrunculid sponges have shown the conservation of distinct microbial populations with unusual bacterial taxa dominated by a novel betaproteobacterial and spirochete species. This study describes the further investigation into these associated bacterial communities, their conservation and sponge microbiome comparisons across spatial, temporal and environmental scales. The bacterial communities associated with seven latrunculid species representing three genera (Tsitsikamma, Cyclacanthia and Latrunculia) were characterized as well as a Mycale and Tethya rubra species. Latrunculid sponge microbiomes were significantly different from those associated with sympatric outlier sponge species and the surrounding environment. The bacterial communities associated with latrunculid sponges appear host specific with the conservation of two dominant bacterial symbionts which mirror the phylogeny of their host species. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Production, purification, and characterisation of proteases from an ericoid mycorrhizal fungus, Oidiodendron maius
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Vachellia erioloba (camel thorn) and microbial interactions
- Authors: Van Aswegen, Sunet
- Date: 2018
- Subjects: Vesicular-arbuscular mycorrhizas , Cadmium , Rhizobacteria , Plant growth-promoting rhizobacteria , Acacia erioloba
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63716 , vital:28475
- Description: Vachellia erioloba (camel thorn) is one of South Africa’s economically important tree species and therefore requires further investigation to improve its health and growth. Beneficial soil microbes have positive effects on plants through various mechanisms such as nitrogen fixation, phosphate solubilisation, indole acetic acid and siderophore production and biofilm formation. These traits enhance plant growth and protect the host plant against parasitic organisms that are present in soil. The arbuscular mycorrhizal (AM) fungi are well known for their beneficial symbiotic effects on host plants. The objective of this study was to determine the role of AM fungi and associated beneficial rhizobacteria in improving the growth of V. erioloba seedlings. Soil and root samples were collected from a farm in the Northern Cape, South Africa. Fifty-seven bacterial cultures were isolated from the soil and tested for plant growth promoting characteristics. Fourteen isolates showing at least four beneficial traits were molecularly identified using the GenBank database. The AM fungal and bacterial populations in the soil samples were assessed using Illumina sequencing. Sequences were identified using the MaarJAM and GenBank databases, respectively. Three separate pot trials were conducted to determine; 1) the effects of cadmium (Cd) on seedling growth; 2) the individual effects of three selected bacterial isolates and AM fungi alone and combined on seedling growth, and 3) the combined effects of the selected bacteria on AM fungal inoculated and uninoculated seedlings. Of the fourteen isolates the Enterobacter genera was the dominant species identified, with Acinetobacter, Pantoea and Bacillus each having one isolate. All were described as plant growth promoting rhizobacteria. One isolate from each genus, excluding Pantoea, was used in the pot trials. Three genera were identified in the AM fungal population that was assessed, namely Ambispora, Paraglomus and Glomus with Ambispora being the dominant genus. The bacterial population assessed showed a high diversity of bacteria from the Actinobacteria phylum being the dominant group. The results of the heavy metal pot trial showed that the symbiotic relationship between the seedlings and AM fungi increased the seedlings’ health and growth during heavy metal stress. The combination of bacteria and AM fungi increased growth parameters in all the inoculated seedlings, but not when compared to uninoculated seedlings indicating a possible competition for nutrients. The results were influenced by the presence of a nematode, which was suspected to have been seed borne. Further investigations on these interactions are required. Inoculation of AM fungi and selected PGPR is recommended for V. erioloba seedling production. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Van Aswegen, Sunet
- Date: 2018
- Subjects: Vesicular-arbuscular mycorrhizas , Cadmium , Rhizobacteria , Plant growth-promoting rhizobacteria , Acacia erioloba
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63716 , vital:28475
- Description: Vachellia erioloba (camel thorn) is one of South Africa’s economically important tree species and therefore requires further investigation to improve its health and growth. Beneficial soil microbes have positive effects on plants through various mechanisms such as nitrogen fixation, phosphate solubilisation, indole acetic acid and siderophore production and biofilm formation. These traits enhance plant growth and protect the host plant against parasitic organisms that are present in soil. The arbuscular mycorrhizal (AM) fungi are well known for their beneficial symbiotic effects on host plants. The objective of this study was to determine the role of AM fungi and associated beneficial rhizobacteria in improving the growth of V. erioloba seedlings. Soil and root samples were collected from a farm in the Northern Cape, South Africa. Fifty-seven bacterial cultures were isolated from the soil and tested for plant growth promoting characteristics. Fourteen isolates showing at least four beneficial traits were molecularly identified using the GenBank database. The AM fungal and bacterial populations in the soil samples were assessed using Illumina sequencing. Sequences were identified using the MaarJAM and GenBank databases, respectively. Three separate pot trials were conducted to determine; 1) the effects of cadmium (Cd) on seedling growth; 2) the individual effects of three selected bacterial isolates and AM fungi alone and combined on seedling growth, and 3) the combined effects of the selected bacteria on AM fungal inoculated and uninoculated seedlings. Of the fourteen isolates the Enterobacter genera was the dominant species identified, with Acinetobacter, Pantoea and Bacillus each having one isolate. All were described as plant growth promoting rhizobacteria. One isolate from each genus, excluding Pantoea, was used in the pot trials. Three genera were identified in the AM fungal population that was assessed, namely Ambispora, Paraglomus and Glomus with Ambispora being the dominant genus. The bacterial population assessed showed a high diversity of bacteria from the Actinobacteria phylum being the dominant group. The results of the heavy metal pot trial showed that the symbiotic relationship between the seedlings and AM fungi increased the seedlings’ health and growth during heavy metal stress. The combination of bacteria and AM fungi increased growth parameters in all the inoculated seedlings, but not when compared to uninoculated seedlings indicating a possible competition for nutrients. The results were influenced by the presence of a nematode, which was suspected to have been seed borne. Further investigations on these interactions are required. Inoculation of AM fungi and selected PGPR is recommended for V. erioloba seedling production. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta
- Authors: Van der Merwe, Marcel
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62963 , vital:28347
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Van der Merwe, Marcel
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62963 , vital:28347
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- 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
Exploring targeted metagenomics and untargeted metabolomics for characterising aquaponics bacterial ecology and phytochemistry
- Authors: Abraham, Benjamin Melakail
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192453 , vital:45227
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Abraham, Benjamin Melakail
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192453 , vital:45227
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Exploring the use of in vitro colorimetric and bioluminescence assays to distinguish between Arf GTPase isoforms and detect Arf GTPase activity
- Authors: Woolf, Alexander Robert
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192582 , vital:45240
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Woolf, Alexander Robert
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192582 , vital:45240
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Hop-mediated alteration of cellular metabolism in KSHV infection
- Authors: Kirigin, Elisa
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192477 , vital:45229
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Kirigin, Elisa
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192477 , vital:45229
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
In silico identification of natural inhibitory compounds against the Mycobacterium tuberculosis Enzyme Pyrazinamidase using high-throughput virtual screening techniques
- Authors: Kenyon, Thomas
- Date: 2021-10-29
- Subjects: Mycobacterium tuberculosis , Pyrazinamide , Molecular dynamics , High throughput screening (Drug development) , Mutagenesis , South African Natural Compounds database (SANCDB)
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192074 , vital:45193
- Description: Tuberculosis (TB) is most commonly a pulmonary infection caused by the bacterium Mycobacterium tuberculosis. With the exception of the COVID-19 pandemic, TB was the most common cause of death due to an infectious disease for a number of years up until 2020. In 2019, 10 million people fell ill with TB worldwide and 1.4 million people died (WHO, 2020a). Additionally, multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat. A global total of 206 030 people with multidrug- or rifampicin-resistant TB (MDR/RR-TB) were reported in 2019, a 10% increase from 186 883 in 2018. South Africa is ranked among the 48 high TB burden countries, with an estimated 360 000 people falling ill in 2019, resulting in 58 000 deaths, the majority of which being among people living with HIV. Unlike HIV, however, TB is a curable disease when managed correctly with long durations of antitubercular chemotherapy. Pyrazinamide (PZA) is an important first-line tuberculosis drug unique for its activity against latent TB. PZA is a prodrug, being converted into its active form, pyrazinoic acid (POA) by the Mtb gene pncA, coding for the pyrazinamidase enzyme (PZase). TB resistance to first-line drugs such as PZA is commonly associated with mutations in the pncA/PZase enzyme. This study aimed to identify potential novel inhibitors that bind to the active site of PZase. By making use of molecular docking studies and molecular dynamics (MD) simulations, high throughput virtual screening was performed on 623 compounds from the South African Natural Compounds database (SANCDB; https://sancdb.rubi.ru.ac.za). Ligands that selectively bound to the PZase active site were identified using docking studies, followed by MD simulations to assess ligand-PZase complex stability, Finally, hit compounds identified from the first round of MD simulations were screened again against PZase structures with high confidence point mutations known to infer PZA resistance in order to identify any novel compounds which had inhibitory potential against both WT and mutant forms of the PZase enzyme. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Kenyon, Thomas
- Date: 2021-10-29
- Subjects: Mycobacterium tuberculosis , Pyrazinamide , Molecular dynamics , High throughput screening (Drug development) , Mutagenesis , South African Natural Compounds database (SANCDB)
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192074 , vital:45193
- Description: Tuberculosis (TB) is most commonly a pulmonary infection caused by the bacterium Mycobacterium tuberculosis. With the exception of the COVID-19 pandemic, TB was the most common cause of death due to an infectious disease for a number of years up until 2020. In 2019, 10 million people fell ill with TB worldwide and 1.4 million people died (WHO, 2020a). Additionally, multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat. A global total of 206 030 people with multidrug- or rifampicin-resistant TB (MDR/RR-TB) were reported in 2019, a 10% increase from 186 883 in 2018. South Africa is ranked among the 48 high TB burden countries, with an estimated 360 000 people falling ill in 2019, resulting in 58 000 deaths, the majority of which being among people living with HIV. Unlike HIV, however, TB is a curable disease when managed correctly with long durations of antitubercular chemotherapy. Pyrazinamide (PZA) is an important first-line tuberculosis drug unique for its activity against latent TB. PZA is a prodrug, being converted into its active form, pyrazinoic acid (POA) by the Mtb gene pncA, coding for the pyrazinamidase enzyme (PZase). TB resistance to first-line drugs such as PZA is commonly associated with mutations in the pncA/PZase enzyme. This study aimed to identify potential novel inhibitors that bind to the active site of PZase. By making use of molecular docking studies and molecular dynamics (MD) simulations, high throughput virtual screening was performed on 623 compounds from the South African Natural Compounds database (SANCDB; https://sancdb.rubi.ru.ac.za). Ligands that selectively bound to the PZase active site were identified using docking studies, followed by MD simulations to assess ligand-PZase complex stability, Finally, hit compounds identified from the first round of MD simulations were screened again against PZase structures with high confidence point mutations known to infer PZA resistance in order to identify any novel compounds which had inhibitory potential against both WT and mutant forms of the PZase enzyme. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
The de novo biosynthesis of biotin is required for the optimal growth of Salmonella enterica serovar Typhimurium in the intracellular environment
- Authors: McLaughlin, Claire
- Date: 2021-10-29
- Subjects: Salmonella , Biotin , Biosynthesis , Salmonella typhimurium , Antibacterial agents , Anti-infective agents , Pathogenic bacteria , Salmonella food poisoning
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192097 , vital:45195
- Description: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne pathogen infecting humans and animals, contributing to significant morbidity and mortality worldwide each year. The increase in antibiotic-resistant S. Typhimurium infections in recent years has highlighted the need for new antibacterial drugs and drug targets. S. Typhimurium can acquire biotin through de novo biosynthesis or via transport from its extracellular environment. The importance of the vitamin for bacterial survival, coupled with the absence of the biotin biosynthetic pathway in humans, makes the biotin biosynthetic enzymes attractive targets for drug discovery. The study's primary aim was to determine the relative importance of the biotin biosynthesis and transport pathways for the in vitro and ex vivo growth and survival of S. Typhimurium, with the goal of validating the pathways as valid targets for antimicrobial drug development. In order to achieve this aim, we generated S. Typhimurium mutant strains harbouring deletions in either the biotin biosynthetic gene, bioB, or putative high-affinity biotin transporter, yigM (ΔbioB and ΔyigM, respectively), as well as a double mutant in which the two mutations were combined (ΔbioB ΔyigM). Since the simultaneous disruption of biotin biosynthesis and transport in the double mutant may form a synthetic lethal combination, preventing further analysis of the strain, we also constructed a conditional mutant in which the promoter of the yigM gene was replaced by the arabinose-regulatable, PBAD promoter in the ΔbioB background (ΔbioB PBAD::yigM). Since the expression of the YigM in this strain is arabinose-regulatable, its role as a biotin transporter can be evaluated by altering the arabinose concentration in the growth media. Once the mutant strains were isolated and verified genetically, their growth and that of their genetically complemented counterparts were analysed in liquid and/or solid M9 minimal medium in the absence of biotin. Consistent with previous observations, the ΔbioB auxotrophic mutant's growth was severely compromised in minimal media in the absence of biotin. The growth of the strain could, however, be restored by supplementation with exogenous biotin or expression of the wild type bioB gene from an episomal plasmid. The ability of biotin to reverse the growth defect of the ΔbioB mutant strain was, however, dependent on the presence of a functional YigM, since biotin supplementation did not affect the growth of the ΔbioB ΔyigM double mutant strain. The introduction of a second copy of the yigM gene in the ΔbioB ΔyigM background, however, restored the growth of the strain in the presence, but not absence, of biotin. The dependence of the double mutant on both YigM and biotin for growth supports the idea that the protein functions as the sole or primary biotin transporter in S. Typhimurium, as it has recently been shown for E. coli (Ringsletter, 2010; Finkenwirth et al, 2013). The essentiality of YigM for biotin transport was subsequently verified by two independent means. Firstly, the growth of the ΔbioB PBAD::yigM promoter-replacement mutant was strictly dependent on the inclusion of arabinose in biotin-supplemented M9 minimal media supplemented, indicating that the expression of YigM from the PBAD promoter is essential for biotin transport. Secondly, following treatment with a known small-molecule inhibitor of the biotin biosynthesis, MAC-13772, exogenous biotin was capable of restoring the growth defect of the YigM+ wild type S. Typhimurium strain, but not the YigM− ΔyigM mutant. Taken together, these findings confirm that YigM serves as the biotin transporter for S. Typhimurium and that the corresponding ΔyigM mutant is, as a result, defective for biotin transport. Having confirmed the genotypes and phenotypes of the ΔbioB, ΔyigM, and ΔbioB ΔyigM mutants, we next analysed the importance of the biotin biosynthesis and transport pathways for the growth and survival of S. Typhimurium within the intracellular environment. To this end, we determined the proliferation of each of the mutant strains following infection of HeLa epithelial and RAW264.7 macrophage-like cell lines. Our results revealed that the de novo biosynthesis of biotin is required for the optimal growth of S. Typhimurium following infection of both epithelial and macrophage-like cell lines. Disruption of biotin transport, by contrast, had no significant effect on the intracellular proliferation of S. Typhimurium when a functional pathway for the biosynthesis of biotin was present. The simultaneous disruption of biotin biosynthesis and transport, however, resulted in significant attenuation of S. Typhimurium in epithelial cells, while bacterial survival in macrophages decreased to below the limit of detection. Overall, our results suggest the S. Typhimurium relies primarily on biotin produced by the de novo biosynthesis pathway to support its growth in the intracellular environment. While YigM-mediated biotin transport is essential for sustaining the viability of intracellular S. Typhimurium in the absence of de novo biosynthesis, it appears to play a relatively minor role in the acquisition of biotin during growth in the nutrient-limited Salmonella containing vacuole. Our findings suggest that inhibiting biotin biosynthesis may be a viable strategy for combating systemic infections caused by Salmonella, as has been recently proposed for other medically important bacterial pathogens (Carfrae et al., 2020). , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: McLaughlin, Claire
- Date: 2021-10-29
- Subjects: Salmonella , Biotin , Biosynthesis , Salmonella typhimurium , Antibacterial agents , Anti-infective agents , Pathogenic bacteria , Salmonella food poisoning
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192097 , vital:45195
- Description: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a foodborne pathogen infecting humans and animals, contributing to significant morbidity and mortality worldwide each year. The increase in antibiotic-resistant S. Typhimurium infections in recent years has highlighted the need for new antibacterial drugs and drug targets. S. Typhimurium can acquire biotin through de novo biosynthesis or via transport from its extracellular environment. The importance of the vitamin for bacterial survival, coupled with the absence of the biotin biosynthetic pathway in humans, makes the biotin biosynthetic enzymes attractive targets for drug discovery. The study's primary aim was to determine the relative importance of the biotin biosynthesis and transport pathways for the in vitro and ex vivo growth and survival of S. Typhimurium, with the goal of validating the pathways as valid targets for antimicrobial drug development. In order to achieve this aim, we generated S. Typhimurium mutant strains harbouring deletions in either the biotin biosynthetic gene, bioB, or putative high-affinity biotin transporter, yigM (ΔbioB and ΔyigM, respectively), as well as a double mutant in which the two mutations were combined (ΔbioB ΔyigM). Since the simultaneous disruption of biotin biosynthesis and transport in the double mutant may form a synthetic lethal combination, preventing further analysis of the strain, we also constructed a conditional mutant in which the promoter of the yigM gene was replaced by the arabinose-regulatable, PBAD promoter in the ΔbioB background (ΔbioB PBAD::yigM). Since the expression of the YigM in this strain is arabinose-regulatable, its role as a biotin transporter can be evaluated by altering the arabinose concentration in the growth media. Once the mutant strains were isolated and verified genetically, their growth and that of their genetically complemented counterparts were analysed in liquid and/or solid M9 minimal medium in the absence of biotin. Consistent with previous observations, the ΔbioB auxotrophic mutant's growth was severely compromised in minimal media in the absence of biotin. The growth of the strain could, however, be restored by supplementation with exogenous biotin or expression of the wild type bioB gene from an episomal plasmid. The ability of biotin to reverse the growth defect of the ΔbioB mutant strain was, however, dependent on the presence of a functional YigM, since biotin supplementation did not affect the growth of the ΔbioB ΔyigM double mutant strain. The introduction of a second copy of the yigM gene in the ΔbioB ΔyigM background, however, restored the growth of the strain in the presence, but not absence, of biotin. The dependence of the double mutant on both YigM and biotin for growth supports the idea that the protein functions as the sole or primary biotin transporter in S. Typhimurium, as it has recently been shown for E. coli (Ringsletter, 2010; Finkenwirth et al, 2013). The essentiality of YigM for biotin transport was subsequently verified by two independent means. Firstly, the growth of the ΔbioB PBAD::yigM promoter-replacement mutant was strictly dependent on the inclusion of arabinose in biotin-supplemented M9 minimal media supplemented, indicating that the expression of YigM from the PBAD promoter is essential for biotin transport. Secondly, following treatment with a known small-molecule inhibitor of the biotin biosynthesis, MAC-13772, exogenous biotin was capable of restoring the growth defect of the YigM+ wild type S. Typhimurium strain, but not the YigM− ΔyigM mutant. Taken together, these findings confirm that YigM serves as the biotin transporter for S. Typhimurium and that the corresponding ΔyigM mutant is, as a result, defective for biotin transport. Having confirmed the genotypes and phenotypes of the ΔbioB, ΔyigM, and ΔbioB ΔyigM mutants, we next analysed the importance of the biotin biosynthesis and transport pathways for the growth and survival of S. Typhimurium within the intracellular environment. To this end, we determined the proliferation of each of the mutant strains following infection of HeLa epithelial and RAW264.7 macrophage-like cell lines. Our results revealed that the de novo biosynthesis of biotin is required for the optimal growth of S. Typhimurium following infection of both epithelial and macrophage-like cell lines. Disruption of biotin transport, by contrast, had no significant effect on the intracellular proliferation of S. Typhimurium when a functional pathway for the biosynthesis of biotin was present. The simultaneous disruption of biotin biosynthesis and transport, however, resulted in significant attenuation of S. Typhimurium in epithelial cells, while bacterial survival in macrophages decreased to below the limit of detection. Overall, our results suggest the S. Typhimurium relies primarily on biotin produced by the de novo biosynthesis pathway to support its growth in the intracellular environment. While YigM-mediated biotin transport is essential for sustaining the viability of intracellular S. Typhimurium in the absence of de novo biosynthesis, it appears to play a relatively minor role in the acquisition of biotin during growth in the nutrient-limited Salmonella containing vacuole. Our findings suggest that inhibiting biotin biosynthesis may be a viable strategy for combating systemic infections caused by Salmonella, as has been recently proposed for other medically important bacterial pathogens (Carfrae et al., 2020). , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Cloning, expression, partial characterisation and application of a recombinant GH10 xylanase, XT6, from Geobacillus stearothermophilus T6 as an additive to chicken feeds
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Effects of hydraulic fracking fluid on soil microbial composition and diversity
- Authors: Sianyuka, Nicolette
- Date: 2022-04-06
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/232366 , vital:49985
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Sianyuka, Nicolette
- Date: 2022-04-06
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/232366 , vital:49985
- Description: Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Molecular characterization of microbial communities in the Sundays and Swartkops estuaries impacted by anthropogenic activities
- Kgomokhumo, Tlhoafalang Evah
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Assessment of cytotoxic artemisinin and its derivatives as DNA damaging inducing agents in triple-negative breast cancer cells
- Authors: Mkhwanazi, Ntando
- Date: 2022-10-14
- Subjects: Breast Cancer , Artemisinin , DNA damage , Antineoplastic agents , Breast Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362960 , vital:65378
- Description: In developing countries, including South Africa, breast cancer is the primary cause of cancer-related deaths among women. TNBC (triple-negative breast cancer) is an aggressive breast cancer subtype that is more prevalent in women of African descent. This subtype lacks the key receptors, namely the estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 (HER2-) that are the basis of successful targeted therapies for other subtypes of the disease. To date, there are no effective, standardized targeted therapies for TNBC. Artemisinin is an anti-malarial drug and numerous derivatives of the compound have been developed to improve the potency and solubility of the parent compound. Artemisinin and its derivatives have gained attention as potential anti-cancer agents; however, such studies have not yet progressed to clinical trials and the precise mechanism of action of these compounds is yet to be fully explained. In this study, artemisinin, and its known derivative artesunate, as well as a novel derivative, WHN11, were investigated as DNA damage-inducing agents in TNBC. WHN11 was found to be the most potent of the three compounds, displaying an IC50 of 3.20 μM against HCC70 cells, artemisinin displayed an IC50 of 214.70 μM and artesunate displayed an IC50 of 25.48 μM. The compounds were less toxic to the MCF12A non-cancerous cells, with IC50 values 298.30, 87.53, and 8.35 μM for artemisinin, artesunate, and WHN11, respectively, and displayed selectivity indices of 1.39, 3.44 and 2.61 μM for artemisinin, artesunate, and WHN11, respectively. In silico and in vitro studies revealed that the artemisinin compounds bind to DNA through the minor groove. While all three compounds were able to bind to DNA, a comet assay revealed that only artemisinin and artesunate, and not WHN11, were able to cause DNA damage compared to the vehicle control, DMSO. Finally, a topoisomerase I (TOPO I) enzyme assay demonstrated that while the compounds appeared to display a degree of inhibition of TOPO I, as evidenced by a downward shift in the plasmid band on the agarose gel, they were not able to fully inhibit the enzyme to return the plasmid to the supercoiled conformation. In addition, combination studies revealed that artemisinin, artesunate, and WHN11 acted synergistically in combination with camptothecin, but displayed either an additive (artemisinin) or antagonistic (artesunate and WHN11) relationship when used in combination with etoposide. In conclusion, artemisinin, its known derivative artesunate, and novel and highly toxic derivative WHN11, all bind to DNA via the minor groove, however only artemisinin and artesunate, and not WHN11, cause DNA damage, indicating a potentially different mechanism of action of the three artemisinins. All three compounds act synergistically with camptothecin, which suggests interference with topoisomerase activity, partially supported by slight inhibition of TOPO I activity, and could indicate either direct inhibition of the enzyme or interference with enzyme function by competitive binding to the DNA. Further studies could help explore alternate DNA damage assays, to validate these findings, and the effect of the compounds on TOPO II activity could also be assessed. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mkhwanazi, Ntando
- Date: 2022-10-14
- Subjects: Breast Cancer , Artemisinin , DNA damage , Antineoplastic agents , Breast Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362960 , vital:65378
- Description: In developing countries, including South Africa, breast cancer is the primary cause of cancer-related deaths among women. TNBC (triple-negative breast cancer) is an aggressive breast cancer subtype that is more prevalent in women of African descent. This subtype lacks the key receptors, namely the estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 (HER2-) that are the basis of successful targeted therapies for other subtypes of the disease. To date, there are no effective, standardized targeted therapies for TNBC. Artemisinin is an anti-malarial drug and numerous derivatives of the compound have been developed to improve the potency and solubility of the parent compound. Artemisinin and its derivatives have gained attention as potential anti-cancer agents; however, such studies have not yet progressed to clinical trials and the precise mechanism of action of these compounds is yet to be fully explained. In this study, artemisinin, and its known derivative artesunate, as well as a novel derivative, WHN11, were investigated as DNA damage-inducing agents in TNBC. WHN11 was found to be the most potent of the three compounds, displaying an IC50 of 3.20 μM against HCC70 cells, artemisinin displayed an IC50 of 214.70 μM and artesunate displayed an IC50 of 25.48 μM. The compounds were less toxic to the MCF12A non-cancerous cells, with IC50 values 298.30, 87.53, and 8.35 μM for artemisinin, artesunate, and WHN11, respectively, and displayed selectivity indices of 1.39, 3.44 and 2.61 μM for artemisinin, artesunate, and WHN11, respectively. In silico and in vitro studies revealed that the artemisinin compounds bind to DNA through the minor groove. While all three compounds were able to bind to DNA, a comet assay revealed that only artemisinin and artesunate, and not WHN11, were able to cause DNA damage compared to the vehicle control, DMSO. Finally, a topoisomerase I (TOPO I) enzyme assay demonstrated that while the compounds appeared to display a degree of inhibition of TOPO I, as evidenced by a downward shift in the plasmid band on the agarose gel, they were not able to fully inhibit the enzyme to return the plasmid to the supercoiled conformation. In addition, combination studies revealed that artemisinin, artesunate, and WHN11 acted synergistically in combination with camptothecin, but displayed either an additive (artemisinin) or antagonistic (artesunate and WHN11) relationship when used in combination with etoposide. In conclusion, artemisinin, its known derivative artesunate, and novel and highly toxic derivative WHN11, all bind to DNA via the minor groove, however only artemisinin and artesunate, and not WHN11, cause DNA damage, indicating a potentially different mechanism of action of the three artemisinins. All three compounds act synergistically with camptothecin, which suggests interference with topoisomerase activity, partially supported by slight inhibition of TOPO I activity, and could indicate either direct inhibition of the enzyme or interference with enzyme function by competitive binding to the DNA. Further studies could help explore alternate DNA damage assays, to validate these findings, and the effect of the compounds on TOPO II activity could also be assessed. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Bioactivity evaluation of manno-oligosaccharides produced from spent coffee grounds using a Bacillus sp. derived endo-1,4-β-mannanase
- Authors: Magengelele, Mihle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365233 , vital:65719
- Description: Thesis embargoed. Possible release date set for early 2024. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Magengelele, Mihle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365233 , vital:65719
- Description: Thesis embargoed. Possible release date set for early 2024. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Evaluating the role of Stress Induced Phosphoprotein 1 isoforms in Kaposi's Sarcoma-Associated Herpesvirus biology
- Authors: Ruck, Duncan Kyle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365280 , vital:65723
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Ruck, Duncan Kyle
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365280 , vital:65723
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14