A case-control approach to assess variability in distribution of distance between transcription factor binding site and transcription start site
- Authors: Moos, Abdul Ragmaan
- Date: 2017
- Subjects: Transcription factors , Proteomics , Chromatin , Chromatin immunoprecipitation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5315 , vital:20808
- Description: Using the in-silico approach, with ENCODE ChIP-seq data for various transcription factors and different cell types; we systematically compared the distance between the transcription factor binding site (TFBS) and the transcription start (TSS). Our aim was to determine if the same transcription factor binds at a different position relative to the TSS in a normal and an abnormal cell type. We compare distribution of distance of binding sites from the TSS; to make description less verbose we call this “distance” where there is no possibility of confusion. We used a case-control methodology where the distance between the TFBS and the TSS in the normal, non-cancerous or untreated cell type is the control. The distance between the TFBS and the TSS in the cancerous or treated cell type is the case. We use the distance between the TFBS and the TSS in the control as the standard. We compared the distance between the TFBS and the TSS in the case and the control. If the distance between the TFBS and the TSS in the control was greater than the distance between the TFBS and the TSS in the case, we can infer the following. The transcription factor in the case binds closer to the TSS compared to the control. If the distance between the TFBS and the TSS in the control is smaller than the distance between the TFBS and the TSS in the case, we can infer the following. The TF in the case binds further away from the TSS compared to the control. Our method is a screening method whereby we compare ChIP-seq data to determine if there is a difference in the distribution distance between the TFBS and the TSS for normal and abnormal cell types. We used the R package ChIP-Enrich to compare the distribution of distance between ChIP-seq peak and the nearest TSS. ChIP-Enrich produces a histogram with the number of ChIP-seq peaks at a certain distance from the TSS. The results indicate for some transcription factors like GM12878-cMyc and K562-cMyc there is a difference between the distribution of distance between the TFBS and the nearest TSS. cMyc has more binding sites within a distance of 1kb from the TSS in GM12878 when compared to K562. GM12878-CTCF and K562-CTCF have slight differences when comparing their distribution of distance from the TSS. This means CTCF binds almost the same distance from the TSS in both GM12878 and K562. A549-gr treated with dexamethasone is interesting because with increase dose of dexamethasone the distribution of distance from the TSS changes as well.
- Full Text:
- Date Issued: 2017
- Authors: Moos, Abdul Ragmaan
- Date: 2017
- Subjects: Transcription factors , Proteomics , Chromatin , Chromatin immunoprecipitation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5315 , vital:20808
- Description: Using the in-silico approach, with ENCODE ChIP-seq data for various transcription factors and different cell types; we systematically compared the distance between the transcription factor binding site (TFBS) and the transcription start (TSS). Our aim was to determine if the same transcription factor binds at a different position relative to the TSS in a normal and an abnormal cell type. We compare distribution of distance of binding sites from the TSS; to make description less verbose we call this “distance” where there is no possibility of confusion. We used a case-control methodology where the distance between the TFBS and the TSS in the normal, non-cancerous or untreated cell type is the control. The distance between the TFBS and the TSS in the cancerous or treated cell type is the case. We use the distance between the TFBS and the TSS in the control as the standard. We compared the distance between the TFBS and the TSS in the case and the control. If the distance between the TFBS and the TSS in the control was greater than the distance between the TFBS and the TSS in the case, we can infer the following. The transcription factor in the case binds closer to the TSS compared to the control. If the distance between the TFBS and the TSS in the control is smaller than the distance between the TFBS and the TSS in the case, we can infer the following. The TF in the case binds further away from the TSS compared to the control. Our method is a screening method whereby we compare ChIP-seq data to determine if there is a difference in the distribution distance between the TFBS and the TSS for normal and abnormal cell types. We used the R package ChIP-Enrich to compare the distribution of distance between ChIP-seq peak and the nearest TSS. ChIP-Enrich produces a histogram with the number of ChIP-seq peaks at a certain distance from the TSS. The results indicate for some transcription factors like GM12878-cMyc and K562-cMyc there is a difference between the distribution of distance between the TFBS and the nearest TSS. cMyc has more binding sites within a distance of 1kb from the TSS in GM12878 when compared to K562. GM12878-CTCF and K562-CTCF have slight differences when comparing their distribution of distance from the TSS. This means CTCF binds almost the same distance from the TSS in both GM12878 and K562. A549-gr treated with dexamethasone is interesting because with increase dose of dexamethasone the distribution of distance from the TSS changes as well.
- Full Text:
- Date Issued: 2017
A computational analysis to decipher the pathways of stability, uncoating and antigenicity of human enterovirus capsids
- Authors: Ross, Caroline Jane
- Date: 2019
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/114788 , vital:34035 , 10.21504/10962/114788
- Description: Expected release date-April 2021
- Full Text: false
- Date Issued: 2019
- Authors: Ross, Caroline Jane
- Date: 2019
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/114788 , vital:34035 , 10.21504/10962/114788
- Description: Expected release date-April 2021
- Full Text: false
- Date Issued: 2019
A dynamics based analysis of allosteric modulation in heat shock proteins
- Authors: Penkler, David Lawrence
- Date: 2019
- Subjects: Heat shock proteins , Molecular chaperones , Allosteric regulation , Homeostasis , Protein kinases , Transcription factors , Adenosine triphosphatase , Cancer -- Chemotherapy , Molecular dynamics , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115948 , vital:34273
- Description: The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies.
- Full Text:
- Date Issued: 2019
- Authors: Penkler, David Lawrence
- Date: 2019
- Subjects: Heat shock proteins , Molecular chaperones , Allosteric regulation , Homeostasis , Protein kinases , Transcription factors , Adenosine triphosphatase , Cancer -- Chemotherapy , Molecular dynamics , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115948 , vital:34273
- Description: The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies.
- Full Text:
- Date Issued: 2019
A novel, improved throughput bioassay for determining the delative speed of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/139902 , vital:37810
- Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials, there has been little progress towards a proven vaccine. Pending a long-term solution, endemic countries rely heavily on the development of innovative drugs that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
- Full Text:
- Date Issued: 2020
- Authors: Laming, Dustin
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/139902 , vital:37810
- Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials, there has been little progress towards a proven vaccine. Pending a long-term solution, endemic countries rely heavily on the development of innovative drugs that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
- Full Text:
- Date Issued: 2020
Algal biotechnology and the beneficiation of saline effluent wastes
- Authors: Rose, P D (Peter Dale)
- Date: 1992
- Subjects: Algae -- Biotechnology , Algae culture , Tanneries -- Waste disposal
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4135 , http://hdl.handle.net/10962/d1015967
- Description: Saline deterioration in the South African public water system has been documented and disposal of brine wastes has been identified as part of the problem. The broad aim of this research programme was to undertake an initial technical study to evaluate the feasibility of integrating algal biotechnology into a disposal function for these wastes. A demonstration of utility in the form of products and waste treatment could produce a beneficiation of saline effluents and provide incentives necessary to deal with the disposal issue. The study attempted to demonstrate a synthesis between the two main thrusts in algal biotechnology that have produced large-scale practical applications - stable, predictable algal production in saline media and the cost effective High Rate Oxidation Ponding (HROP) process for incorporating algal production into a waste treatment function. Tannery organic saline effluents and the biotechnology of Dunaliella salina culture producing β- carotene were chosen as paradigms for the study. 1. The alga was shown to grow in certain tannery effluents producing enhanced biomass yields compared to defined inorganic medium cultivation. The potential for amino acid or protein supplementation of defmed culture media was noted. 2. A reduction in organic load simultaneous with the growth of D.salina was recorded in laboratory-scale simulations of the HROP process. Rates similar to the fresh water HROP equivalent were demonstrated. 3. These results suggested the uptake and storage of organic nitrogen by D.salina. The consequent inhibition of β-carotene accumulation by the organism presented a potentially insurmountable obstacle to the feasibility of β-carotene production in this medium. Uptake and release of organic compounds, previously demonstrated in phytoplankton and other micro-algae, was confirmed in this study for D.salina. The evidence acquired indicated the internalization of both glycine and bovine serum albumin. An ultrastructural study demonstrated mechanisms by which this process might occur. 4. The release of substantial quantities of glycerol was shown. A mechanism whereby D. salina may use this to regulate ammonia availability via control of its associated bacterial population was observed. Glycerol release was identified as presenting an application in treating refractory organic wastes, such as secondary sewage sludges, by elevating C:N ratios. This could demonstrate a significant utility for brine waste impoundments. 5. A multistage production process was proposed to deal with the problem of β-carotene inhibition by separation of the growth and metabolite accumulation functions into separate unit operations. It was shown in this study that the stress of nitrogen deficiency combined with high salinity provides for effectiveβ-carotene accumulation under the conditions of low illumination that pertain in dense cultures. Subjected to these conditions effluent-grown cells show delayed but unimpaired {j-carotene accumulation. 6. A role for the plant hormone abscisic acid in mediating the stress response was demonstrated in D.salina. Fluorescence induction studies suggested the presence of a signalling process forming part of a sensitivity control mechanism. Stress induction of β-carotene accumulation could occur through four clearly defined stages. Potential was identified for using this response as a physiological probe for monitoring and regulating the stress induction process. 7. The multistage processing concept requires effective algal cell separation technology. The use of cross-flow ultrafiltration and diafiltration with a polyethersulfone tubular membrane system was demonstrated as an effective process for the recovery and washing of D. salina. Cell concentrates were produced in a viable form. 8. Process designs incorporating the findings of the research programme are presented demonstrating how effluent and organic waste treatment functions may be combined with the production of D.salina and its products. Application of the multi-stage processing concept to β-carotene production in a defined medium process was identified as offering a potential four-fold yield enhancement. This could have a significant impact on a high cost, marginal algal biotechnology process. Aspects of novelty have been claimed in provisional patents applications. A provisional demonstration of the feasibility of D.salina production in tannery effluent indicates that algal biotechnology may provide a utility for, and hence the beneficiation of saline effluent wastes.
- Full Text:
- Date Issued: 1992
- Authors: Rose, P D (Peter Dale)
- Date: 1992
- Subjects: Algae -- Biotechnology , Algae culture , Tanneries -- Waste disposal
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4135 , http://hdl.handle.net/10962/d1015967
- Description: Saline deterioration in the South African public water system has been documented and disposal of brine wastes has been identified as part of the problem. The broad aim of this research programme was to undertake an initial technical study to evaluate the feasibility of integrating algal biotechnology into a disposal function for these wastes. A demonstration of utility in the form of products and waste treatment could produce a beneficiation of saline effluents and provide incentives necessary to deal with the disposal issue. The study attempted to demonstrate a synthesis between the two main thrusts in algal biotechnology that have produced large-scale practical applications - stable, predictable algal production in saline media and the cost effective High Rate Oxidation Ponding (HROP) process for incorporating algal production into a waste treatment function. Tannery organic saline effluents and the biotechnology of Dunaliella salina culture producing β- carotene were chosen as paradigms for the study. 1. The alga was shown to grow in certain tannery effluents producing enhanced biomass yields compared to defined inorganic medium cultivation. The potential for amino acid or protein supplementation of defmed culture media was noted. 2. A reduction in organic load simultaneous with the growth of D.salina was recorded in laboratory-scale simulations of the HROP process. Rates similar to the fresh water HROP equivalent were demonstrated. 3. These results suggested the uptake and storage of organic nitrogen by D.salina. The consequent inhibition of β-carotene accumulation by the organism presented a potentially insurmountable obstacle to the feasibility of β-carotene production in this medium. Uptake and release of organic compounds, previously demonstrated in phytoplankton and other micro-algae, was confirmed in this study for D.salina. The evidence acquired indicated the internalization of both glycine and bovine serum albumin. An ultrastructural study demonstrated mechanisms by which this process might occur. 4. The release of substantial quantities of glycerol was shown. A mechanism whereby D. salina may use this to regulate ammonia availability via control of its associated bacterial population was observed. Glycerol release was identified as presenting an application in treating refractory organic wastes, such as secondary sewage sludges, by elevating C:N ratios. This could demonstrate a significant utility for brine waste impoundments. 5. A multistage production process was proposed to deal with the problem of β-carotene inhibition by separation of the growth and metabolite accumulation functions into separate unit operations. It was shown in this study that the stress of nitrogen deficiency combined with high salinity provides for effectiveβ-carotene accumulation under the conditions of low illumination that pertain in dense cultures. Subjected to these conditions effluent-grown cells show delayed but unimpaired {j-carotene accumulation. 6. A role for the plant hormone abscisic acid in mediating the stress response was demonstrated in D.salina. Fluorescence induction studies suggested the presence of a signalling process forming part of a sensitivity control mechanism. Stress induction of β-carotene accumulation could occur through four clearly defined stages. Potential was identified for using this response as a physiological probe for monitoring and regulating the stress induction process. 7. The multistage processing concept requires effective algal cell separation technology. The use of cross-flow ultrafiltration and diafiltration with a polyethersulfone tubular membrane system was demonstrated as an effective process for the recovery and washing of D. salina. Cell concentrates were produced in a viable form. 8. Process designs incorporating the findings of the research programme are presented demonstrating how effluent and organic waste treatment functions may be combined with the production of D.salina and its products. Application of the multi-stage processing concept to β-carotene production in a defined medium process was identified as offering a potential four-fold yield enhancement. This could have a significant impact on a high cost, marginal algal biotechnology process. Aspects of novelty have been claimed in provisional patents applications. A provisional demonstration of the feasibility of D.salina production in tannery effluent indicates that algal biotechnology may provide a utility for, and hence the beneficiation of saline effluent wastes.
- Full Text:
- Date Issued: 1992
An in-silico investigation of Morita-Baylis-Hillman accessible heterocyclic analogues for applications as novel HIV-1 C protease inhibitors
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
- Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
- Full Text:
- Date Issued: 2015
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , HIV infections , Drug resistance , Cheminformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4152 , http://hdl.handle.net/10962/d1017913
- Description: Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.
- Full Text:
- Date Issued: 2015
An investigation into the bacterial biosynthetic origins of bioactive natural products isolated from South African latrunculid sponges
- Authors: Waterworth, Samantha Che
- Date: 2018
- Subjects: Marine biodiversity , Metagenomics , Sponges Biotechnology , Spirochetes , Natural products Biotechnology
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61826 , vital:28065
- Description: Several pyrroloiminoquinone alkaloids exhibiting cytotoxic, anti-tumour activity have been isolated from sponges within the Latrunculiidae family that are endemic to the South African coastline. Other, structurally similar pyrroloiminoquinone compounds have been isolated from geographically distant and phylogenetically distinct marine sponges, as well as terrestrial myxomycetes which suggested that sponge-associated bacteria may be the true biosynthetic origin of pyrroloiminoquinone compounds. Previous studies have shown that there is conservation of spirochete and betaproteobacterial species in the bacterial communities associated with South African Latrunculiidae sponges and it was proposed that these conserved bacteria represented candidate pyrroloiminoquinone-producers. This study aimed to confirm the conserved dominance of betaproteobacteria and spirochetes within bacterial communities associated with South African latrunculid sponges and employed a shotgun metagenomic approach to assess the functional and biosynthetic potential of associated microbiota in Tsitsikamma favus sponges. Clustering of assembled contigs revealed twenty-three putative bacterial genomes, of which, two were identified as representatives of the conserved betaproteobacteria and spirochete species previously identified in Tsitsikamma sponges. It was shown that the spirochete was most likely an obligate symbiont that benefitted the host sponge through possible defence against pathogenic bacteria and/or nutrient acquisition. The putative genome representing the conserved betaproteobacteria was found to be heavily contaminated and further sequencing is required to accurately resolve the genome for functional characterization. Several biosynthetic gene clusters were identified and demonstrated the bioactive potential of Tsitsikamma favus-associated bacteria. A biosynthetic gene cluster was identified on an unclustered contig that included several genetic features that were indicative of possible pyrroloiminoquinone production.
- Full Text:
- Date Issued: 2018
- Authors: Waterworth, Samantha Che
- Date: 2018
- Subjects: Marine biodiversity , Metagenomics , Sponges Biotechnology , Spirochetes , Natural products Biotechnology
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61826 , vital:28065
- Description: Several pyrroloiminoquinone alkaloids exhibiting cytotoxic, anti-tumour activity have been isolated from sponges within the Latrunculiidae family that are endemic to the South African coastline. Other, structurally similar pyrroloiminoquinone compounds have been isolated from geographically distant and phylogenetically distinct marine sponges, as well as terrestrial myxomycetes which suggested that sponge-associated bacteria may be the true biosynthetic origin of pyrroloiminoquinone compounds. Previous studies have shown that there is conservation of spirochete and betaproteobacterial species in the bacterial communities associated with South African Latrunculiidae sponges and it was proposed that these conserved bacteria represented candidate pyrroloiminoquinone-producers. This study aimed to confirm the conserved dominance of betaproteobacteria and spirochetes within bacterial communities associated with South African latrunculid sponges and employed a shotgun metagenomic approach to assess the functional and biosynthetic potential of associated microbiota in Tsitsikamma favus sponges. Clustering of assembled contigs revealed twenty-three putative bacterial genomes, of which, two were identified as representatives of the conserved betaproteobacteria and spirochete species previously identified in Tsitsikamma sponges. It was shown that the spirochete was most likely an obligate symbiont that benefitted the host sponge through possible defence against pathogenic bacteria and/or nutrient acquisition. The putative genome representing the conserved betaproteobacteria was found to be heavily contaminated and further sequencing is required to accurately resolve the genome for functional characterization. Several biosynthetic gene clusters were identified and demonstrated the bioactive potential of Tsitsikamma favus-associated bacteria. A biosynthetic gene cluster was identified on an unclustered contig that included several genetic features that were indicative of possible pyrroloiminoquinone production.
- Full Text:
- Date Issued: 2018
An investigation into the potential immunogenicity of various extracts of the South African bont tick Amblyomma hebraeum
- Authors: Adamson, Deborah Jane
- Date: 1993
- Subjects: Amblyomma -- South Africa , Ticks -- South Africa , Ticks -- Control -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4127 , http://hdl.handle.net/10962/d1015640
- Description: Rabbits and goats were inoculated with crude, membrane-associated and soluble components extracted from unengorged adult females and nymphs of the bont tick Amblyomma hebraeum. Inoculation provided some protection against nymphal infestation, however it had little effect on adult feeding. Histological examination of adults fed on inoculated hosts showed evidence of gut damage. Skin provocation testing with tick extracts elicited a Type I immediate hypersensitivity which was influenced by antihistamine. A delayed skin reaction was also evident. Whether this was attributable to Type III Arthus reaction or Type IV cell-mediated hypersensitivity was not determined. A comparative histological study of sites of tick extract injection, on inoculated and naive hosts, demonstrated the role of eosinophils in the hosts response to tick feeding. Serological examination revealed elevated anti-A hebraeum lgG titres following inoculation. These titres were found to decrease in the ten weeks after inoculation, despite the hosts being repeatedly infested with A hebraeum. Although the IgG titres of naive control hosts increased after each tick infestation, they failed to reach the titres achieved through inoculation. Western blot analysis of serum from inoculated hosts recognized most of the A. hebraeum proteins against which it was screened.
- Full Text:
- Date Issued: 1993
- Authors: Adamson, Deborah Jane
- Date: 1993
- Subjects: Amblyomma -- South Africa , Ticks -- South Africa , Ticks -- Control -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4127 , http://hdl.handle.net/10962/d1015640
- Description: Rabbits and goats were inoculated with crude, membrane-associated and soluble components extracted from unengorged adult females and nymphs of the bont tick Amblyomma hebraeum. Inoculation provided some protection against nymphal infestation, however it had little effect on adult feeding. Histological examination of adults fed on inoculated hosts showed evidence of gut damage. Skin provocation testing with tick extracts elicited a Type I immediate hypersensitivity which was influenced by antihistamine. A delayed skin reaction was also evident. Whether this was attributable to Type III Arthus reaction or Type IV cell-mediated hypersensitivity was not determined. A comparative histological study of sites of tick extract injection, on inoculated and naive hosts, demonstrated the role of eosinophils in the hosts response to tick feeding. Serological examination revealed elevated anti-A hebraeum lgG titres following inoculation. These titres were found to decrease in the ten weeks after inoculation, despite the hosts being repeatedly infested with A hebraeum. Although the IgG titres of naive control hosts increased after each tick infestation, they failed to reach the titres achieved through inoculation. Western blot analysis of serum from inoculated hosts recognized most of the A. hebraeum proteins against which it was screened.
- Full Text:
- Date Issued: 1993
An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
An investigation into the use of anaerobic digestion for the treatment of tannery wastewaters
- Authors: Jackson-Moss, Clive Alan
- Date: 1991
- Subjects: Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4059 , http://hdl.handle.net/10962/d1004120 , Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Description: The anaerobic digestion of tannery wastewaters was investigated with a view to using this form of treatment in the tanning industry. As these wastewaters are extremely complex and contain high concentrations of both inorganic and organic compounds, the effect of these individual compounds on the anaerobic digestion process was investigated in detail, in order to ascertain the fate of these compounds during the digestion process. The experiments comprising the initial toxicity study were carried out as adaptation experiments using a synthetic wastewater. It was found that the heavy metals such as chrome, aluminium and iron precipitated and accumulated in the sludge bed of the digesters . The soluble ions such as sodium and chloride were not retained and passed through the digesters. Approximately 20 % of the calcium ions were removed through precipitation, with the remainder being present in the digester effluent . Under the anaerobic conditions, ammonification of the organic nitrogen occurred, and influent sulphates were reduced to sulphides . These sulphides were present as either H2S, HS or insoluble sulphides. As these compounds under investigation on caused no inhibition of the anaerobic digestion process at the concentrations found in tannery wastewaters, the anaerobic treatment of these wastewaters appeared to be possible, provided the bacteria were given sufficient time to adapt to the potentially toxic compounds. However, despite the findings of the synthetic study, the successful anaerobic digestion of the tannery effluents could not be achieved. Although the use of acid was found to be essential in order to control the digester pH in the optimum range, the metabolism of the methanogenic bacteria was inhibited by the presence or absence of unknown compounds. Neither the addition of essential trace nutrients, nor the prevention of the competition between the methanogens and the sulphate-reducing bacteria were able to reverse this inhibition. As tannery effluents contain very low concentrations of phosphorous, it is possible that the methanogens were inhibited by a lack of phosphorous, which is essential during methanogenesis. In contrast to the results obtained from the effluent experiments, the anaerobic digestion of tannery sludge was found to be possible. Of the organic solids present in the sludge, 60 % were degraded and converted into biogas, which had a methane content greater than 70 %. The degradation of the organic solids ensured that COD and PV reductions of greater than 90 % were achieved, and the fate of the compounds in the digesters were in agreement with the findings of the v synthetic study. Efforts to improve the efficiency of the digestion process through the addition of trace nutrients and the use of a two-stage process were only successful in bringing about a minor improvement in digester performance. The overall results of this investigation show, therefore, that although the anaerobic treatment of the tannery effluent was not achieved, the successful anaerobic digestion of tannery sludge is possible at low loading rates. As many difficulties still need to be solved, a great deal of further research is necessary if anaerobic digestion is to be used on an industrial scale for the treatment and disposal of tannery wastewaters.
- Full Text:
- Date Issued: 1991
- Authors: Jackson-Moss, Clive Alan
- Date: 1991
- Subjects: Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4059 , http://hdl.handle.net/10962/d1004120 , Tanneries -- Waste disposal , Sewage sludge -- South Africa -- Management , Sewage -- Purification -- Biological treatment -- South Africa , Water quality management -- South Africa , Water -- Purification -- Biological treatment -- South Africa
- Description: The anaerobic digestion of tannery wastewaters was investigated with a view to using this form of treatment in the tanning industry. As these wastewaters are extremely complex and contain high concentrations of both inorganic and organic compounds, the effect of these individual compounds on the anaerobic digestion process was investigated in detail, in order to ascertain the fate of these compounds during the digestion process. The experiments comprising the initial toxicity study were carried out as adaptation experiments using a synthetic wastewater. It was found that the heavy metals such as chrome, aluminium and iron precipitated and accumulated in the sludge bed of the digesters . The soluble ions such as sodium and chloride were not retained and passed through the digesters. Approximately 20 % of the calcium ions were removed through precipitation, with the remainder being present in the digester effluent . Under the anaerobic conditions, ammonification of the organic nitrogen occurred, and influent sulphates were reduced to sulphides . These sulphides were present as either H2S, HS or insoluble sulphides. As these compounds under investigation on caused no inhibition of the anaerobic digestion process at the concentrations found in tannery wastewaters, the anaerobic treatment of these wastewaters appeared to be possible, provided the bacteria were given sufficient time to adapt to the potentially toxic compounds. However, despite the findings of the synthetic study, the successful anaerobic digestion of the tannery effluents could not be achieved. Although the use of acid was found to be essential in order to control the digester pH in the optimum range, the metabolism of the methanogenic bacteria was inhibited by the presence or absence of unknown compounds. Neither the addition of essential trace nutrients, nor the prevention of the competition between the methanogens and the sulphate-reducing bacteria were able to reverse this inhibition. As tannery effluents contain very low concentrations of phosphorous, it is possible that the methanogens were inhibited by a lack of phosphorous, which is essential during methanogenesis. In contrast to the results obtained from the effluent experiments, the anaerobic digestion of tannery sludge was found to be possible. Of the organic solids present in the sludge, 60 % were degraded and converted into biogas, which had a methane content greater than 70 %. The degradation of the organic solids ensured that COD and PV reductions of greater than 90 % were achieved, and the fate of the compounds in the digesters were in agreement with the findings of the v synthetic study. Efforts to improve the efficiency of the digestion process through the addition of trace nutrients and the use of a two-stage process were only successful in bringing about a minor improvement in digester performance. The overall results of this investigation show, therefore, that although the anaerobic treatment of the tannery effluent was not achieved, the successful anaerobic digestion of tannery sludge is possible at low loading rates. As many difficulties still need to be solved, a great deal of further research is necessary if anaerobic digestion is to be used on an industrial scale for the treatment and disposal of tannery wastewaters.
- Full Text:
- Date Issued: 1991
Analysis of predictive power of binding affinity of PBM-derived sequences
- Authors: Matereke, Lavious Tapiwa
- Date: 2015
- Subjects: Transcription factors , Protein binding , DNA-binding proteins , Chromatin , Protein microarrays
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4161 , http://hdl.handle.net/10962/d1018666
- Description: A transcription factor (TF) is a protein that binds to specific DNA sequences as part of the initiation stage of transcription. Various methods of finding these transcription factor binding sites (TFBS) have been developed. In vivo technologies analyze DNA binding regions known to have bound to a TF in a living cell. Most widely used in vivo methods at the moment are chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and DNase I hypersensitive sites sequencing. In vitro methods derive TFBS based on experiments with TFs and DNA usually in artificial settings or computationally. An example is the Protein Binding Microarray which uses artificially constructed DNA sequences to determine the short sequences that are most likely to bind to a TF. The major drawback of this approach is that binding of TFs in vivo is also dependent on other factors such as chromatin accessibility and the presence of cofactors. Therefore TFBS derived from the PBM technique might not resemble the true DNA binding sequences. In this work, we use PBM data from the UniPROBE motif database, ChIP-seq data and DNase I hypersensitive sites data. Using the Spearman’s rank correlation and area under receiver operating characteristic curve, we compare the enrichment scores which the PBM approach assigns to its identified sequences and the frequency of these sequences in likely binding regions and the human genome as a whole. We also use central motif enrichment analysis (CentriMo) to compare the enrichment of UniPROBE motifs with in vivo derived motifs (from the JASPAR CORE database) in their respective TF ChIP-seq peak region. CentriMo is applied to 14 TF ChIP-seq peak regions from different cell lines. We aim to establish if there is a relationship between the occurrences of UniPROBE 8-mer patterns in likely binding regions and their enrichment score and how well the in vitro derived motifs match in vivo binding specificity. We did not come out with a particular trend showing failure of the PBM approach to predict in vivo binding specificity. Our results show Ets1, Hnf4a and Tcf3 show prediction failure by the PBM technique in terms of our Spearman’s rank correlation for ChIP-seq data and central motif enrichment analysis. However, the PBM technique also matched the in vivo binding specificities of FoxA2, Pou2f2 and Mafk. Failure of the PBM approach was found to be a result of variability in the TF’s binding specificity, the presence of cofactors, narrow binding specificity and the presence ubiquitous binding patterns.
- Full Text:
- Date Issued: 2015
- Authors: Matereke, Lavious Tapiwa
- Date: 2015
- Subjects: Transcription factors , Protein binding , DNA-binding proteins , Chromatin , Protein microarrays
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4161 , http://hdl.handle.net/10962/d1018666
- Description: A transcription factor (TF) is a protein that binds to specific DNA sequences as part of the initiation stage of transcription. Various methods of finding these transcription factor binding sites (TFBS) have been developed. In vivo technologies analyze DNA binding regions known to have bound to a TF in a living cell. Most widely used in vivo methods at the moment are chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and DNase I hypersensitive sites sequencing. In vitro methods derive TFBS based on experiments with TFs and DNA usually in artificial settings or computationally. An example is the Protein Binding Microarray which uses artificially constructed DNA sequences to determine the short sequences that are most likely to bind to a TF. The major drawback of this approach is that binding of TFs in vivo is also dependent on other factors such as chromatin accessibility and the presence of cofactors. Therefore TFBS derived from the PBM technique might not resemble the true DNA binding sequences. In this work, we use PBM data from the UniPROBE motif database, ChIP-seq data and DNase I hypersensitive sites data. Using the Spearman’s rank correlation and area under receiver operating characteristic curve, we compare the enrichment scores which the PBM approach assigns to its identified sequences and the frequency of these sequences in likely binding regions and the human genome as a whole. We also use central motif enrichment analysis (CentriMo) to compare the enrichment of UniPROBE motifs with in vivo derived motifs (from the JASPAR CORE database) in their respective TF ChIP-seq peak region. CentriMo is applied to 14 TF ChIP-seq peak regions from different cell lines. We aim to establish if there is a relationship between the occurrences of UniPROBE 8-mer patterns in likely binding regions and their enrichment score and how well the in vitro derived motifs match in vivo binding specificity. We did not come out with a particular trend showing failure of the PBM approach to predict in vivo binding specificity. Our results show Ets1, Hnf4a and Tcf3 show prediction failure by the PBM technique in terms of our Spearman’s rank correlation for ChIP-seq data and central motif enrichment analysis. However, the PBM technique also matched the in vivo binding specificities of FoxA2, Pou2f2 and Mafk. Failure of the PBM approach was found to be a result of variability in the TF’s binding specificity, the presence of cofactors, narrow binding specificity and the presence ubiquitous binding patterns.
- Full Text:
- Date Issued: 2015
Analysis of the human HSP70-HSP90 organising protein (HOP) gene - characterisation of the promoter and identification of a novel isoform
- Authors: Mattison, Stacey
- Date: 2018
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62821 , vital:28296
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
- Authors: Mattison, Stacey
- Date: 2018
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62821 , vital:28296
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
Application of machine learning, molecular modelling and structural data mining against antiretroviral drug resistance in HIV-1
- Sheik Amamuddy, Olivier Serge André
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Date Issued: 2020
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Date Issued: 2020
Arbuscular mycorrhizal fungi as a bio-indicator of soil health under agricultural management practices in South Africa
- Authors: Sekgota, Wendy Maphefo
- Date: 2019
- Subjects: Soils -- Quality -- South Africa , Soil fertility -- South Africa , Fungi in agriculture -- South Africa , Mycorrhizal fungi , Vesicular-arbuscular mycorrhizas , Fungi -- Spores , Soils -- Agricultural chemical content
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72161 , vital:30011
- Description: This study investigated the activity of arbuscular mycorrhizal (AM) fungi as a potential biological indicator of soil health under conventional and conservation agricultural management in South Africa. An experimental trial consisting of three replicates plots under conventional and reduced tillage subdivided into twelve treatments of six crops and two fertilizer inputs was assessed over four growing seasons for various AM fungal parameters such as spore density, most probable number (MPN) of propagules percentage root colonisation and easily extractable glomalin (EEG). Cropping combinations were maize monoculture; maize soybean rotation; maize cowpea rotation; maize cowpea intercropping; maize oats intercropping and maize vetch intercropping. Resident AM fungal spore numbers and EEG protein levels were very low and no root colonization was recorded in the first two growing seasons. These findings prompted the need for the inoculation of the study site in the third growing season with a commercial AM fungal product (MycorootTM). Spore numbers, EEG concentrations and percentage root colonisation increased 8 weeks after inoculation but were significantly reduced in the fourth growing season that was not inoculated. MPN infectivity increased with inoculation particularly under conventional tillage and maize monoculture. Resident spore taxa were morphologically identified into three genera Gigaspora, Scutellospora, and Glomus. For the first two growing seasons, the maize roots were heavily colonized by a pathogenic fungus after mycorrhizal inoculation no evidence of pathogenic fungi was observed. In the fourth growing season which did not receive inoculation, root colonization started to decline. Reduced tillage, high fertilizer input combined with maize cowpea rotation (MC) and maize hairy vetch intercropping (Mv) had a significant effect (P = 0.01) on AM fungal spore numbers. Cropping systems and high fertilizer input had a significant effect on EEG concentrations in the second growing season. Overall, fertilizer application and crop type had implications for mycorrhizal activity. The soil health status in this study site was deemed low as measured by the impaired mycorrhizal activity due to agricultural management practices. Field inoculation combined with classical and molecular tools could provide a more realistic assessment of the effect of agricultural management practices on AM fungi as potential bioindicators of soil health. Therefore, AM fungi could be used as bioindicators of soil health under agricultural management practices in South African soil conditions.
- Full Text:
- Date Issued: 2019
- Authors: Sekgota, Wendy Maphefo
- Date: 2019
- Subjects: Soils -- Quality -- South Africa , Soil fertility -- South Africa , Fungi in agriculture -- South Africa , Mycorrhizal fungi , Vesicular-arbuscular mycorrhizas , Fungi -- Spores , Soils -- Agricultural chemical content
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72161 , vital:30011
- Description: This study investigated the activity of arbuscular mycorrhizal (AM) fungi as a potential biological indicator of soil health under conventional and conservation agricultural management in South Africa. An experimental trial consisting of three replicates plots under conventional and reduced tillage subdivided into twelve treatments of six crops and two fertilizer inputs was assessed over four growing seasons for various AM fungal parameters such as spore density, most probable number (MPN) of propagules percentage root colonisation and easily extractable glomalin (EEG). Cropping combinations were maize monoculture; maize soybean rotation; maize cowpea rotation; maize cowpea intercropping; maize oats intercropping and maize vetch intercropping. Resident AM fungal spore numbers and EEG protein levels were very low and no root colonization was recorded in the first two growing seasons. These findings prompted the need for the inoculation of the study site in the third growing season with a commercial AM fungal product (MycorootTM). Spore numbers, EEG concentrations and percentage root colonisation increased 8 weeks after inoculation but were significantly reduced in the fourth growing season that was not inoculated. MPN infectivity increased with inoculation particularly under conventional tillage and maize monoculture. Resident spore taxa were morphologically identified into three genera Gigaspora, Scutellospora, and Glomus. For the first two growing seasons, the maize roots were heavily colonized by a pathogenic fungus after mycorrhizal inoculation no evidence of pathogenic fungi was observed. In the fourth growing season which did not receive inoculation, root colonization started to decline. Reduced tillage, high fertilizer input combined with maize cowpea rotation (MC) and maize hairy vetch intercropping (Mv) had a significant effect (P = 0.01) on AM fungal spore numbers. Cropping systems and high fertilizer input had a significant effect on EEG concentrations in the second growing season. Overall, fertilizer application and crop type had implications for mycorrhizal activity. The soil health status in this study site was deemed low as measured by the impaired mycorrhizal activity due to agricultural management practices. Field inoculation combined with classical and molecular tools could provide a more realistic assessment of the effect of agricultural management practices on AM fungi as potential bioindicators of soil health. Therefore, AM fungi could be used as bioindicators of soil health under agricultural management practices in South African soil conditions.
- Full Text:
- Date Issued: 2019
Bacterial degradation of fossil fuel waste in aqueous and solid media
- Authors: Edeki, Oghenekume Gerald
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54565 , vital:26588
- Description: The generation of environmental pollutants worldwide is mainly due to over reliance on fossil fuels as a source of energy. As a result of the negative impacts of these pollutants on the health of humans, animals, plants and microorganisms, global attention has been directed towards ways of containing this problem. Biodegradation of fossil fuel is one of the most effective methods used to remediate contaminated systems. However with regard to coal waste, much of what is known is based on the ability of fungal species to biosolubilize this material under enrichment conditions in a laboratory setting. For effective biodegradation as a remediation technique, there is an immediate need to source, isolate, enrich and incorporate other microorganisms such as bacteria into bioremediation technologies. The goal of this dissertation was to isolate bacteria from fossil fuel contaminated environments and to demonstrate competence for petroleum hydrocarbon degradation which was achieved using a combination of analytical methods such as spectrophotometry, FT-IR, SEM and GC-MS. Screening for biodegradation of coal and petroleum hydrocarbon waste resulted in the isolation of 75 bacterial strains of which 15 showed good potential for use in developing remedial biotechnologies. Spectrophotometric analysis of bacteria both in coal and petroleum hydrocarbons (all in aqueous media) revealed a high proliferation of bacteria in these media suggesting that these microbes can effectively utilize the various substrates as a source of carbon. The isolated bacteria effectively degraded and converted waste coal to humic and fulvic acids; products required to enrich coal mine dumps to support re-vegetation. Scanning electron microscopy showed the attachment of bacteria to waste coal surfaces and the disintegration of coal structures while FT-IR analysis of extracted humic-like substances from biodegraded waste coal revealed these to have the same functional groups as commercial humic acid. Specific consortia which were established using the isolated bacterial strains, showed greater potential to biodegrade coal than did individual isolates. This was evident in experiments carried out on coal and hydrocarbons where the efficient colonization and utilization of these substrates by each bacterial consortium was observed due to the effect of added nutrients such as algae. The biodegradation of liquid petroleum hydrocarbons (diesel and BTEX) was also achieved using the 15 bacterial isolates. GC-MS analysis of extracted residual PHC from aqueous and solid media revealed rapid breakdown of these contaminants by bacteria. Different bacterial consortia established from the individual isolates were shown to be more efficient than single isolates indicating that formulated consortia are the biocatalysts of choice for fossil fuel biodegradation. This study represents one of the most detailed screenings for bacteria from fossil fuel contaminated sites and the isolation of strains with potential to biodegrade coal and petroleum hydrocarbon wastes. Several consortia have been developed and these show potential for further development as biocatalysts for use in bioremediation technology development. An evaluation of efficiency of each established bacterial consortium for biodegradation in a commercial and/or industrial setting at pilot scale is now needed.
- Full Text:
- Date Issued: 2015
- Authors: Edeki, Oghenekume Gerald
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54565 , vital:26588
- Description: The generation of environmental pollutants worldwide is mainly due to over reliance on fossil fuels as a source of energy. As a result of the negative impacts of these pollutants on the health of humans, animals, plants and microorganisms, global attention has been directed towards ways of containing this problem. Biodegradation of fossil fuel is one of the most effective methods used to remediate contaminated systems. However with regard to coal waste, much of what is known is based on the ability of fungal species to biosolubilize this material under enrichment conditions in a laboratory setting. For effective biodegradation as a remediation technique, there is an immediate need to source, isolate, enrich and incorporate other microorganisms such as bacteria into bioremediation technologies. The goal of this dissertation was to isolate bacteria from fossil fuel contaminated environments and to demonstrate competence for petroleum hydrocarbon degradation which was achieved using a combination of analytical methods such as spectrophotometry, FT-IR, SEM and GC-MS. Screening for biodegradation of coal and petroleum hydrocarbon waste resulted in the isolation of 75 bacterial strains of which 15 showed good potential for use in developing remedial biotechnologies. Spectrophotometric analysis of bacteria both in coal and petroleum hydrocarbons (all in aqueous media) revealed a high proliferation of bacteria in these media suggesting that these microbes can effectively utilize the various substrates as a source of carbon. The isolated bacteria effectively degraded and converted waste coal to humic and fulvic acids; products required to enrich coal mine dumps to support re-vegetation. Scanning electron microscopy showed the attachment of bacteria to waste coal surfaces and the disintegration of coal structures while FT-IR analysis of extracted humic-like substances from biodegraded waste coal revealed these to have the same functional groups as commercial humic acid. Specific consortia which were established using the isolated bacterial strains, showed greater potential to biodegrade coal than did individual isolates. This was evident in experiments carried out on coal and hydrocarbons where the efficient colonization and utilization of these substrates by each bacterial consortium was observed due to the effect of added nutrients such as algae. The biodegradation of liquid petroleum hydrocarbons (diesel and BTEX) was also achieved using the 15 bacterial isolates. GC-MS analysis of extracted residual PHC from aqueous and solid media revealed rapid breakdown of these contaminants by bacteria. Different bacterial consortia established from the individual isolates were shown to be more efficient than single isolates indicating that formulated consortia are the biocatalysts of choice for fossil fuel biodegradation. This study represents one of the most detailed screenings for bacteria from fossil fuel contaminated sites and the isolation of strains with potential to biodegrade coal and petroleum hydrocarbon wastes. Several consortia have been developed and these show potential for further development as biocatalysts for use in bioremediation technology development. An evaluation of efficiency of each established bacterial consortium for biodegradation in a commercial and/or industrial setting at pilot scale is now needed.
- Full Text:
- Date Issued: 2015
Bacteriophage growth on stationary phase achromabacter strains
- Authors: Robb, Susan Mary
- Date: 1980
- Subjects: Bacteriophages , Strains and stresses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4125 , http://hdl.handle.net/10962/d1014131
- Description: Achromobacter w.t. and strain 14 both support phage α3a growth in stationary phase, but unlike the w.t. strain, exponential phase cultures of strain 14 block phage development. A standard method was developed for determining phage growth in stationary phase cultures. Lyophilised cells were used to eliminate variations due to the unstable phenotype of Achromobacter strain 14 cells. Phage α3a growth in stationary phase was characterized by a long and variable latent period of 6 to 9 h and an increased burst size of 709 p.f.u. per cell as compared with 153 p.f.u. per cell in exponential wild type cells. During the latent period the infected cells were very sensitive to changes in growth conditions and in particular, dilution. Pre-conditioning of the bacterial cells by allowing them to stand for 24 h after shaking for 3 days was an important aspect of the stationary phase phage growth system. Cells which had been allowed to stand retained the ability to be infected and to support phage growth for at least 16 days. Shaking cultures gradually lost the ability to support phage growth but the phage could persist in the host cell for 10 days until removal from shaking when the lytic cycle could proceed after allowing the cultures to stand. In comparison the latent period and burst size in Achromobacter w.t. stationary phase cells were reduced to less than 2 h and less than 200 respectively. Stationary phase cultures differed physiologically and morphologically depending on the aeration conditions. In comparison with non-aerated standing cultures, vigorously aerated cultures showed a decrease in viability, RNA synthesis, membrane transport, intracellular ATP levels, UV resistance and heat resistance but had markedly higher protein synthesis levels. Aerated cells were small non-motile rods which did not support phage growth. They developed into large motile rods under conditions of limited aeration and were able to propagate phage. It was proposed that changes in the host control mechanisms for macromolecular synthesis may be instrumental in either blocking or permitting phage development. A spontaneous mutant of Achromobacter strain 14 (14x) which liberated phage and was resistant to superinfection was isolated. The phage-host relationship was unstable and similar to the phage carrier state. The liberated phage were able to grow in exponential strain 14 cells. It was proposed that strain 14 was a defective lysogen and that an immunity phase shift model may account for the differential phage growth in exponential and stationary phase cells. Host transcriptional control appears to be implicated in control of phage development in exponential and stationary phase cells. Achromobacter Lp only supported phage in exponential phase but a rifampicin resistant mutant of this strain was able to propagate phage in stationary phase. In vitro RNA synthesis assays showed that the rifampicin resistance was caused by an alteration in the RNA polymerase. Preliminary experiments to determine intracellular phage macromolecular synthesis were carried out using exponential Achromobacter w.t. cells which had been irradiated with UV prior to infection. In irradiated cells, infection with phage resulted in stimulation of DNA synthesis but no stimulation of protein synthesis. Phage production was drastically reduced in cells which had been treated with very low UV doses. It was proposed that α3a development may rely heavily on host cell functions which are destroyed by UV. Achromobacter mutants with defective leucine transport systems were isolated. Mutants which lost the leucine uptake system completely were totally resistant to phage infection and were unable to adsorb phage α3a. This is the first report to implicate an amino-acid transport system in phage adsorption.
- Full Text:
- Date Issued: 1980
- Authors: Robb, Susan Mary
- Date: 1980
- Subjects: Bacteriophages , Strains and stresses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4125 , http://hdl.handle.net/10962/d1014131
- Description: Achromobacter w.t. and strain 14 both support phage α3a growth in stationary phase, but unlike the w.t. strain, exponential phase cultures of strain 14 block phage development. A standard method was developed for determining phage growth in stationary phase cultures. Lyophilised cells were used to eliminate variations due to the unstable phenotype of Achromobacter strain 14 cells. Phage α3a growth in stationary phase was characterized by a long and variable latent period of 6 to 9 h and an increased burst size of 709 p.f.u. per cell as compared with 153 p.f.u. per cell in exponential wild type cells. During the latent period the infected cells were very sensitive to changes in growth conditions and in particular, dilution. Pre-conditioning of the bacterial cells by allowing them to stand for 24 h after shaking for 3 days was an important aspect of the stationary phase phage growth system. Cells which had been allowed to stand retained the ability to be infected and to support phage growth for at least 16 days. Shaking cultures gradually lost the ability to support phage growth but the phage could persist in the host cell for 10 days until removal from shaking when the lytic cycle could proceed after allowing the cultures to stand. In comparison the latent period and burst size in Achromobacter w.t. stationary phase cells were reduced to less than 2 h and less than 200 respectively. Stationary phase cultures differed physiologically and morphologically depending on the aeration conditions. In comparison with non-aerated standing cultures, vigorously aerated cultures showed a decrease in viability, RNA synthesis, membrane transport, intracellular ATP levels, UV resistance and heat resistance but had markedly higher protein synthesis levels. Aerated cells were small non-motile rods which did not support phage growth. They developed into large motile rods under conditions of limited aeration and were able to propagate phage. It was proposed that changes in the host control mechanisms for macromolecular synthesis may be instrumental in either blocking or permitting phage development. A spontaneous mutant of Achromobacter strain 14 (14x) which liberated phage and was resistant to superinfection was isolated. The phage-host relationship was unstable and similar to the phage carrier state. The liberated phage were able to grow in exponential strain 14 cells. It was proposed that strain 14 was a defective lysogen and that an immunity phase shift model may account for the differential phage growth in exponential and stationary phase cells. Host transcriptional control appears to be implicated in control of phage development in exponential and stationary phase cells. Achromobacter Lp only supported phage in exponential phase but a rifampicin resistant mutant of this strain was able to propagate phage in stationary phase. In vitro RNA synthesis assays showed that the rifampicin resistance was caused by an alteration in the RNA polymerase. Preliminary experiments to determine intracellular phage macromolecular synthesis were carried out using exponential Achromobacter w.t. cells which had been irradiated with UV prior to infection. In irradiated cells, infection with phage resulted in stimulation of DNA synthesis but no stimulation of protein synthesis. Phage production was drastically reduced in cells which had been treated with very low UV doses. It was proposed that α3a development may rely heavily on host cell functions which are destroyed by UV. Achromobacter mutants with defective leucine transport systems were isolated. Mutants which lost the leucine uptake system completely were totally resistant to phage infection and were unable to adsorb phage α3a. This is the first report to implicate an amino-acid transport system in phage adsorption.
- Full Text:
- Date Issued: 1980
Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Date Issued: 2018
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Date Issued: 2018
Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
Biochemical characterization of the β-mannanase activity of Bacillus paralicheniformis SVD1
- Authors: Clarke, Matthew David
- Date: 2019
- Subjects: Mycobacterium avium paratuberculosis , Enzymes -- Biotechnology , Lignocellulose -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67570 , vital:29112
- Description: Products produced via the enzymatic hydrolysis of lignocellulosic biomass, the most abundant renewable terrestrial source of carbon, can potentially replace a lot of the fuels and chemicals currently produced using non-renewable hydrocarbons. Mannan is a polysaccharide component of lignocellulose that is abundant in softwoods and legume seeds. Enzymatic hydrolysis of mannan by β-mannanases has various industrial applications, including use in biofuel and prebiotic mannooligosaccharide (MOS) production for the improvement of human and animal health. The industrial use of β-mannanases depends on their biochemical characteristics, such as their activity, stability and substrate specificity. Knowledge of their synergistic interactions with other enzymes is also useful for effective hydrolysis. Bacillus paralicheniformis SVD1 was used as a source for β-mannanases. The two mannanases of B. paralicheniformis SVD1 have not been biochemically characterized apart from minor characterization of crude β-mannanase activity. The protein sequences of the two β-mannanases, of glycosyl hydrolase family 5 and 26, have a 95% - 96% identity to the β-mannanases of B. licheniformis DSM13T (=ATCC14580T). These small protein sequence differences could lead to quite different biochemical characteristics. These mannanases were characterized as these enzymes may have industrially useful characteristics. To induce mannanase production, B. paralicheniformis SVD1 was cultured in broth containing the mannan substrate locust bean gum. Various growth curve parameters were measured over 72 h. Mannanase activity was the highest after 48 h of growth - this was the time at which mannanase activity was concentrated, using 3 kDa centrifugal filtration devices, for biochemical characterization of the crude activity. Zymography revealed that the crude concentrated mannanase fraction consisted of at least two mannanases with relative molecular weights (MWs) of 29.6 kDa and 33 kDa. This was smaller than expected – based on their theoretical molecular masses. Protease activity, which was detected in the broth, was probably the reason. There were two pH optima, pH 5.0 and pH 7.0, which also indicated the presence of two mannanases. The concentrated mannanase displayed characteristics that were expected of a B. paralicheniformis β-mannanase. The temperature optimum was 50°C and the activity loss was less than 7% at 50°C after 24 h. Substrate specificity assays revealed that there was predominantly mannanase activity present. Thin layer chromatography (TLC) analysis of mannan and MOS hydrolysis showed that mainly M2 and M3 MOS were produced; only MOS with a degree of polymerization of 4 or higher were hydrolyzed. Hydrolysis was minimal on mannoligosaccharides with galactose substituents. Activity and MOS production was the highest on soluble, low branched mannan substrates. The highest activity observed was on konjac glucomannan. Purification of the mannanase activity was then attempted using various methods. Ammonium sulfate precipitation, acetone precipitation, as well as centrifugal filtration device concentration was assessed for concentration of the mannanase activity.Concentration was not very successful due to low activity yields (≤ 20%). Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) was used for purification. AEC gave good activity yield and fold purification, but SDS-PAGE analysis revealed the presence of many different proteins so further purification was necessary. SDS-PAGE analysis showed that there were only a few protein contaminants in the SEC fraction. However, the yield was too low to allow for biochemical characterization. The optimized purification procedure, which partially purified the mannanase activity, used 85% ammonium sulfate precipitation, followed by AEC. The fold purification was high (88.9) and the specific activity was 29.5 U.mg-1. A zymogram of the partially purified mannanase showed a mannanase active band with a MW of 40 - 41 kDa. A serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was added during the purification steps. This indicated that the mannanase/s in the crude concentrate, without PMSF added, was hydrolyzed by serine protease activity. Native PAGE zymograms suggested that at least two different isoforms of mannanases were present. Additional purification would be required to determine the true characteristics of the mannanase/s. The biochemical characteristics of the crude and partially purified mannanases were similar. The pH optima of the partially purified mannanases were different; the pH optima were 6.0 and 9.0. The substrate specificities were similar, except that the partially purified mannanases displayed no cellulase and β-D-galactosidase activity, but showed a small amount of α-L-arabinase activity. The partially purified mannanase and a Cyamopsis tetragonolobus GH27 α-galactosidase synergistically hydrolyzed locust bean gum. The M50G50 combination displayed the highest extent of hydrolysis; after 24 h there was a 1.39 fold increase in reducing sugar release and the degree of synergy (DS) was 4.64. TLC analysis indicated that synergy increased the release of small MOS. These MOS could be useful as prebiotics. The synergy between the partially purified mannanase and the commercial cellulase mixture Cellic® CTec2 (Novozymes) on spent coffee grounds (SCG) was also determined. SCG is an abundant industrial waste product that has high mannan content. The SCG was pretreated using NaOH, and the monosaccharide, soluble phenolics and insoluble contents were determined. Glucose and mannose were the dominant monosaccharides in the SCG; the pretreated SCG contained 20.4% (w/w) glucose and 18.5% (w/w) mannose, respectively. The NaOH pretreatment improved mannanase hydrolysis of SCG. It resulted in the opening up and swelling of the SCG particles and removed some of the insoluble solids. The partially purified B. paralicheniformis SVD1 mannanase displayed no detectable activity on SCG, but showed synergy with CTec2, in terms of DS, on untreated and NaOH pretreated SCG. This is the first report of mannanasecellulase synergy on SCG; other studies found that increased hydrolysis was due to additive effects. The results obtained in this study are only an initial assessment of the biochemical properties of B. paralicheniformis SVD1 mannanase activity and its synergy with other enzymes. These results can be used to inform future studies.
- Full Text:
- Date Issued: 2019
- Authors: Clarke, Matthew David
- Date: 2019
- Subjects: Mycobacterium avium paratuberculosis , Enzymes -- Biotechnology , Lignocellulose -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67570 , vital:29112
- Description: Products produced via the enzymatic hydrolysis of lignocellulosic biomass, the most abundant renewable terrestrial source of carbon, can potentially replace a lot of the fuels and chemicals currently produced using non-renewable hydrocarbons. Mannan is a polysaccharide component of lignocellulose that is abundant in softwoods and legume seeds. Enzymatic hydrolysis of mannan by β-mannanases has various industrial applications, including use in biofuel and prebiotic mannooligosaccharide (MOS) production for the improvement of human and animal health. The industrial use of β-mannanases depends on their biochemical characteristics, such as their activity, stability and substrate specificity. Knowledge of their synergistic interactions with other enzymes is also useful for effective hydrolysis. Bacillus paralicheniformis SVD1 was used as a source for β-mannanases. The two mannanases of B. paralicheniformis SVD1 have not been biochemically characterized apart from minor characterization of crude β-mannanase activity. The protein sequences of the two β-mannanases, of glycosyl hydrolase family 5 and 26, have a 95% - 96% identity to the β-mannanases of B. licheniformis DSM13T (=ATCC14580T). These small protein sequence differences could lead to quite different biochemical characteristics. These mannanases were characterized as these enzymes may have industrially useful characteristics. To induce mannanase production, B. paralicheniformis SVD1 was cultured in broth containing the mannan substrate locust bean gum. Various growth curve parameters were measured over 72 h. Mannanase activity was the highest after 48 h of growth - this was the time at which mannanase activity was concentrated, using 3 kDa centrifugal filtration devices, for biochemical characterization of the crude activity. Zymography revealed that the crude concentrated mannanase fraction consisted of at least two mannanases with relative molecular weights (MWs) of 29.6 kDa and 33 kDa. This was smaller than expected – based on their theoretical molecular masses. Protease activity, which was detected in the broth, was probably the reason. There were two pH optima, pH 5.0 and pH 7.0, which also indicated the presence of two mannanases. The concentrated mannanase displayed characteristics that were expected of a B. paralicheniformis β-mannanase. The temperature optimum was 50°C and the activity loss was less than 7% at 50°C after 24 h. Substrate specificity assays revealed that there was predominantly mannanase activity present. Thin layer chromatography (TLC) analysis of mannan and MOS hydrolysis showed that mainly M2 and M3 MOS were produced; only MOS with a degree of polymerization of 4 or higher were hydrolyzed. Hydrolysis was minimal on mannoligosaccharides with galactose substituents. Activity and MOS production was the highest on soluble, low branched mannan substrates. The highest activity observed was on konjac glucomannan. Purification of the mannanase activity was then attempted using various methods. Ammonium sulfate precipitation, acetone precipitation, as well as centrifugal filtration device concentration was assessed for concentration of the mannanase activity.Concentration was not very successful due to low activity yields (≤ 20%). Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) was used for purification. AEC gave good activity yield and fold purification, but SDS-PAGE analysis revealed the presence of many different proteins so further purification was necessary. SDS-PAGE analysis showed that there were only a few protein contaminants in the SEC fraction. However, the yield was too low to allow for biochemical characterization. The optimized purification procedure, which partially purified the mannanase activity, used 85% ammonium sulfate precipitation, followed by AEC. The fold purification was high (88.9) and the specific activity was 29.5 U.mg-1. A zymogram of the partially purified mannanase showed a mannanase active band with a MW of 40 - 41 kDa. A serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was added during the purification steps. This indicated that the mannanase/s in the crude concentrate, without PMSF added, was hydrolyzed by serine protease activity. Native PAGE zymograms suggested that at least two different isoforms of mannanases were present. Additional purification would be required to determine the true characteristics of the mannanase/s. The biochemical characteristics of the crude and partially purified mannanases were similar. The pH optima of the partially purified mannanases were different; the pH optima were 6.0 and 9.0. The substrate specificities were similar, except that the partially purified mannanases displayed no cellulase and β-D-galactosidase activity, but showed a small amount of α-L-arabinase activity. The partially purified mannanase and a Cyamopsis tetragonolobus GH27 α-galactosidase synergistically hydrolyzed locust bean gum. The M50G50 combination displayed the highest extent of hydrolysis; after 24 h there was a 1.39 fold increase in reducing sugar release and the degree of synergy (DS) was 4.64. TLC analysis indicated that synergy increased the release of small MOS. These MOS could be useful as prebiotics. The synergy between the partially purified mannanase and the commercial cellulase mixture Cellic® CTec2 (Novozymes) on spent coffee grounds (SCG) was also determined. SCG is an abundant industrial waste product that has high mannan content. The SCG was pretreated using NaOH, and the monosaccharide, soluble phenolics and insoluble contents were determined. Glucose and mannose were the dominant monosaccharides in the SCG; the pretreated SCG contained 20.4% (w/w) glucose and 18.5% (w/w) mannose, respectively. The NaOH pretreatment improved mannanase hydrolysis of SCG. It resulted in the opening up and swelling of the SCG particles and removed some of the insoluble solids. The partially purified B. paralicheniformis SVD1 mannanase displayed no detectable activity on SCG, but showed synergy with CTec2, in terms of DS, on untreated and NaOH pretreated SCG. This is the first report of mannanasecellulase synergy on SCG; other studies found that increased hydrolysis was due to additive effects. The results obtained in this study are only an initial assessment of the biochemical properties of B. paralicheniformis SVD1 mannanase activity and its synergy with other enzymes. These results can be used to inform future studies.
- Full Text:
- Date Issued: 2019
Bioinformatics tool development with a focus on structural bioinformatics and the analysis of genetic variation in humans
- Authors: Brown, David K
- Date: 2018
- Subjects: Bioinformatics , Human genetics -- Variation , High performance computing , Workflow management systems , Molecular dynamics , Next generation sequencing , Human Mutation Analysis (HUMA)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/60708 , vital:27820
- Description: This thesis is divided into three parts, united under the general theme of bioinformatics tool development and variation analysis. Part 1 describes the design and development of the Job Management System (JMS), a workflow management system for high performance computing (HPC). HPC has become an integral part of bioinformatics. Computational methods for molecular dynamics and next generation sequencing (NGS) analysis, which require complex calculations on large datasets, are not yet feasible on desktop computers. As such, powerful computer clusters have been employed to perform these calculations. However, making use of these HPC clusters requires familiarity with command line interfaces. This excludes a large number of researchers from taking advantage of these resources. JMS was developed as a tool to make it easier for researchers without a computer science background to make use of HPC. Additionally, JMS can be used to host computational tools and pipelines and generates both web-based interfaces and RESTful APIs for those tools. The web-based interfaces can be used to quickly and easily submit jobs to the underlying cluster. The RESTful web API, on the other hand, allows JMS to provided backend functionality for external tools and web servers that want to run jobs on the cluster. Numerous tools and workflows have already been added to JMS, several of which have been incorporated into external web servers. One such web server is the Human Mutation Analysis (HUMA) web server and database. HUMA, the topic of part 2 of this thesis, is a platform for the analysis of genetic variation in humans. HUMA aggregates data from various existing databases into a single, connected and related database. The advantages of this are realized in the powerful querying abilities that it provides. HUMA includes protein, gene, disease, and variation data and can be searched from the angle of any one of these categories. For example, searching for a protein will return the protein data (e.g. protein sequences, structures, domains and families, and other meta-data). However, the related nature of the database means that genes, diseases, variation, and literature related to the protein will also be returned, giving users a powerful and holistic view of all data associated with the protein. HUMA also provides links to the original sources of the data, allowing users to follow the links to find additional details. HUMA aims to be a platform for the analysis of genetic variation. As such, it also provides tools to visualize and analyse the data (several of which run on the underlying cluster, via JMS). These tools include alignment and 3D structure visualization, homology modeling, variant analysis, and the ability to upload custom variation datasets and map them to proteins, genes and diseases. HUMA also provides collaboration features, allowing users to share and discuss datasets and job results. Finally, part 3 of this thesis focused on the development of a suite of tools, MD-TASK, to analyse genetic variation at the protein structure level via network analysis of molecular dynamics simulations. The use of MD-TASK in combination with the tools developed in the previous parts of this thesis is showcased via the analysis of variation in the renin-angiotensinogen complex, a vital part of the renin-angiotensin system.
- Full Text:
- Date Issued: 2018
- Authors: Brown, David K
- Date: 2018
- Subjects: Bioinformatics , Human genetics -- Variation , High performance computing , Workflow management systems , Molecular dynamics , Next generation sequencing , Human Mutation Analysis (HUMA)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/60708 , vital:27820
- Description: This thesis is divided into three parts, united under the general theme of bioinformatics tool development and variation analysis. Part 1 describes the design and development of the Job Management System (JMS), a workflow management system for high performance computing (HPC). HPC has become an integral part of bioinformatics. Computational methods for molecular dynamics and next generation sequencing (NGS) analysis, which require complex calculations on large datasets, are not yet feasible on desktop computers. As such, powerful computer clusters have been employed to perform these calculations. However, making use of these HPC clusters requires familiarity with command line interfaces. This excludes a large number of researchers from taking advantage of these resources. JMS was developed as a tool to make it easier for researchers without a computer science background to make use of HPC. Additionally, JMS can be used to host computational tools and pipelines and generates both web-based interfaces and RESTful APIs for those tools. The web-based interfaces can be used to quickly and easily submit jobs to the underlying cluster. The RESTful web API, on the other hand, allows JMS to provided backend functionality for external tools and web servers that want to run jobs on the cluster. Numerous tools and workflows have already been added to JMS, several of which have been incorporated into external web servers. One such web server is the Human Mutation Analysis (HUMA) web server and database. HUMA, the topic of part 2 of this thesis, is a platform for the analysis of genetic variation in humans. HUMA aggregates data from various existing databases into a single, connected and related database. The advantages of this are realized in the powerful querying abilities that it provides. HUMA includes protein, gene, disease, and variation data and can be searched from the angle of any one of these categories. For example, searching for a protein will return the protein data (e.g. protein sequences, structures, domains and families, and other meta-data). However, the related nature of the database means that genes, diseases, variation, and literature related to the protein will also be returned, giving users a powerful and holistic view of all data associated with the protein. HUMA also provides links to the original sources of the data, allowing users to follow the links to find additional details. HUMA aims to be a platform for the analysis of genetic variation. As such, it also provides tools to visualize and analyse the data (several of which run on the underlying cluster, via JMS). These tools include alignment and 3D structure visualization, homology modeling, variant analysis, and the ability to upload custom variation datasets and map them to proteins, genes and diseases. HUMA also provides collaboration features, allowing users to share and discuss datasets and job results. Finally, part 3 of this thesis focused on the development of a suite of tools, MD-TASK, to analyse genetic variation at the protein structure level via network analysis of molecular dynamics simulations. The use of MD-TASK in combination with the tools developed in the previous parts of this thesis is showcased via the analysis of variation in the renin-angiotensinogen complex, a vital part of the renin-angiotensin system.
- Full Text:
- Date Issued: 2018