Evaluation of functionalized silver and silica nanoparticles for the removal of deoxyribonucleic acid conveying antibiotics resistance genes from water
- Authors: Ezeuko, Adaora Stella
- Date: 2022
- Subjects: DNA , Silica , Water
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27765 , vital:69414
- Description: Antibiotic resistance genes ARGs are recognized as a serious public health emergency linked to extensive use of antibiotics by humans and animals as a prophylactic agent that treats and prevents infections. The occurrence of high concentrations being identified in wastewater treatment plants, rivers, etc is due to untreated effluents being discharged from households, hospitals, agriculture, and pharmaceutical industries. The application of adequate treatment techniques and material for the removal of bacteria DNA conveying ARGs from the effluents before their release to the environment cannot be overemphasized. Adsorption techniques seem to be effective due to their easy design, operation, and ability to regenerate adsorbents for use without producing toxic by-products. This concept was employed for the removal of bacteria DNA conveying ARGs from simulated aqueous solution, effluents from hospital, river and WWTPs using silver and silica metallic nanoparticles. This thesis investigated the effectiveness of metallic nanoparticles containing silver AgNPs and mesoporous silica nanoparticles MSNPs as well as magnetite Fe3O4 functionalized with 4 4hydroxyphenyl 2 262-terpyridine onto their surface, for the removal of bacteria DNA conveying antibiotic resistance genes from water samples from hospitals, river, and wastewater treatment plants WWTPs. Silver nanoparticles AgNPs of different molar concentrations 0.1M, 0.5M and 1.0 M and mesoporous silica nanoparticles MSNPs adsorbents were successfully synthesized in their original states and surface functionalization achieved by incorporating magnetite Fe3O4 and 4 4 hydroxyphenyl 2 2 6 2 terpyridine on the silver AgNPs Fe3O4 and silica MSNPs TPPY surfaces respectively. Their effectiveness as adsorbent for the removal of bacteria DNA conveying ARGs from aqueous solutions and real water/wastewater samples were investigated. The DNA uptake by the as-synthesized AgNPs and MSNPs were compared to the functionalized AgNPs Fe3O4 and MSNPsTPPY by determining the adsorbents with the highest removal efficiencies. All as synthesized and functionalized adsorbents were characterized by SEM, EDX, FTIR, XRD, UV spectroscopy and PZC before the removal process. The extraction of genomic DNA from antibiotic-resistant Enterococcus faecium and Vibrio parahaemolyticus was successfully achieved via the boiling method. Antibiotic susceptibility test was conducted using the disk diffusion method before the commencement of genomic DNA extraction. Molecular characterization via gel electrophoresis confirmed the presence of resistance genes at different base pairs. Adsorption batch experiment were investigated, and the best optimum parameters were evaluated through the influence of pH, contact time, initial DNA concentration, adsorbent dose, and competitive ions for each sorption process. The rate determining step were determined by fitting kinetic models such as Natarajan and Khalaf first order, pseudo first order, pseudo second order, Elovich model to experimental data. Also, the adsorption mechanisms determining adsorption equilibrium were investigated by fitting Freundlich, Langmuir and Sips model into the experimental data. The application of AgNPsFe3O4 nanocomposite and MSNPsTPPY for the removal of bacteria DNA demonstrated much enhancement for DNA uptake than the as-synthesized AgNPs and MSNPs materials. The incorporation of magnetite and 4 4hydroxyphenyl 2 2 6 2-terpyridine onto AgNPs and MSNPs significantly enhanced the binding affinity towards the removal the bacteria DNA via strong electrostatic attraction between the active sites on the adsorbent and the negative DNA molecules. Finally, high adsorption capacities were recorded with AgNPsFe3O4 nanocomposite and MSNPsTPPY compared to AgNPs and MSNPs with chaotropic salts. The kinetic adsorption models were mostly best fitted by the pseudo-second order and Elovich models while the adsorption equilibrium was best described by Langmuir and Sips isotherm models. MSNPs with different chaotropic salts, AgNPsFe3O4 nanocomposite and MSNPsTPPY also proved its effectiveness in DNA removal not only in the simulated aqueous solution but in three different real life water samples obtained from Cofimvaba hospital, Ndevana river and Uitenhage WWTPs. High adsorption efficiencies above 90 percent were achieved during the removal of DNA in all the three real water samples. Therefore, application of these adsorbents for the removal of bacteria DNA conveying ARGs may be a promising option that would tackle the consequences of consuming ARGs infected water globally. , Thesis (MSc) -- Faculty of Science and Agriculture, 2022
- Full Text:
- Date Issued: 2022
- Authors: Ezeuko, Adaora Stella
- Date: 2022
- Subjects: DNA , Silica , Water
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27765 , vital:69414
- Description: Antibiotic resistance genes ARGs are recognized as a serious public health emergency linked to extensive use of antibiotics by humans and animals as a prophylactic agent that treats and prevents infections. The occurrence of high concentrations being identified in wastewater treatment plants, rivers, etc is due to untreated effluents being discharged from households, hospitals, agriculture, and pharmaceutical industries. The application of adequate treatment techniques and material for the removal of bacteria DNA conveying ARGs from the effluents before their release to the environment cannot be overemphasized. Adsorption techniques seem to be effective due to their easy design, operation, and ability to regenerate adsorbents for use without producing toxic by-products. This concept was employed for the removal of bacteria DNA conveying ARGs from simulated aqueous solution, effluents from hospital, river and WWTPs using silver and silica metallic nanoparticles. This thesis investigated the effectiveness of metallic nanoparticles containing silver AgNPs and mesoporous silica nanoparticles MSNPs as well as magnetite Fe3O4 functionalized with 4 4hydroxyphenyl 2 262-terpyridine onto their surface, for the removal of bacteria DNA conveying antibiotic resistance genes from water samples from hospitals, river, and wastewater treatment plants WWTPs. Silver nanoparticles AgNPs of different molar concentrations 0.1M, 0.5M and 1.0 M and mesoporous silica nanoparticles MSNPs adsorbents were successfully synthesized in their original states and surface functionalization achieved by incorporating magnetite Fe3O4 and 4 4 hydroxyphenyl 2 2 6 2 terpyridine on the silver AgNPs Fe3O4 and silica MSNPs TPPY surfaces respectively. Their effectiveness as adsorbent for the removal of bacteria DNA conveying ARGs from aqueous solutions and real water/wastewater samples were investigated. The DNA uptake by the as-synthesized AgNPs and MSNPs were compared to the functionalized AgNPs Fe3O4 and MSNPsTPPY by determining the adsorbents with the highest removal efficiencies. All as synthesized and functionalized adsorbents were characterized by SEM, EDX, FTIR, XRD, UV spectroscopy and PZC before the removal process. The extraction of genomic DNA from antibiotic-resistant Enterococcus faecium and Vibrio parahaemolyticus was successfully achieved via the boiling method. Antibiotic susceptibility test was conducted using the disk diffusion method before the commencement of genomic DNA extraction. Molecular characterization via gel electrophoresis confirmed the presence of resistance genes at different base pairs. Adsorption batch experiment were investigated, and the best optimum parameters were evaluated through the influence of pH, contact time, initial DNA concentration, adsorbent dose, and competitive ions for each sorption process. The rate determining step were determined by fitting kinetic models such as Natarajan and Khalaf first order, pseudo first order, pseudo second order, Elovich model to experimental data. Also, the adsorption mechanisms determining adsorption equilibrium were investigated by fitting Freundlich, Langmuir and Sips model into the experimental data. The application of AgNPsFe3O4 nanocomposite and MSNPsTPPY for the removal of bacteria DNA demonstrated much enhancement for DNA uptake than the as-synthesized AgNPs and MSNPs materials. The incorporation of magnetite and 4 4hydroxyphenyl 2 2 6 2-terpyridine onto AgNPs and MSNPs significantly enhanced the binding affinity towards the removal the bacteria DNA via strong electrostatic attraction between the active sites on the adsorbent and the negative DNA molecules. Finally, high adsorption capacities were recorded with AgNPsFe3O4 nanocomposite and MSNPsTPPY compared to AgNPs and MSNPs with chaotropic salts. The kinetic adsorption models were mostly best fitted by the pseudo-second order and Elovich models while the adsorption equilibrium was best described by Langmuir and Sips isotherm models. MSNPs with different chaotropic salts, AgNPsFe3O4 nanocomposite and MSNPsTPPY also proved its effectiveness in DNA removal not only in the simulated aqueous solution but in three different real life water samples obtained from Cofimvaba hospital, Ndevana river and Uitenhage WWTPs. High adsorption efficiencies above 90 percent were achieved during the removal of DNA in all the three real water samples. Therefore, application of these adsorbents for the removal of bacteria DNA conveying ARGs may be a promising option that would tackle the consequences of consuming ARGs infected water globally. , Thesis (MSc) -- Faculty of Science and Agriculture, 2022
- Full Text:
- Date Issued: 2022
Comparison of protein binding microarray derived and ChIP-seq derived transcription factor binding DNA motifs
- Hlatshwayo, Nkosikhona Rejoyce
- Authors: Hlatshwayo, Nkosikhona Rejoyce
- Date: 2015
- Subjects: Protein binding , DNA , DNA microarrays , Transcription factors , DNA-protein interactions , Gene regulatory networks
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4146 , http://hdl.handle.net/10962/d1017907
- Description: Transcription factors (TFs) are biologically important proteins that interact with transcription machinery and bind DNA regulatory sequences to regulate gene expression by modulating the synthesis of the messenger RNA. The regulatory sequences comprise of short conserved regions of a specific length called motifs . TFs have very diverse roles in different cells and play a very significant role in development. TFs have been associated with carcinogenesis in various tissue types, as well as developmental and hormone response disorders. They may be responsible for the regulation of oncogenes and can be oncogenic. Consequently, understanding TF binding and knowing the motifs to which they bind is worthy of attention and research focus. Various projects have made the study of TF binding their main focus; nevertheless, much about TF binding remains confounding. Chromatin immunoprecipitation in conjunction with deep sequencing (ChIP-seq) techniques are a popular method used to investigate DNA-TF interactions in vivo. This procedure is followed by motif discovery and motif enrichment analysis using relevant tools. Protein Binding Microarrays (PBMs) are an in vitro method for investigating DNA-TF interactions. We use a motif enrichment analysis tools (CentriMo and AME) and an empirical quality assessment tool (Area under the ROC curve) to investigate which method yields motifs that are a true representation of in vivo binding. Motif enrichment analysis: On average, ChIP-seq derived motifs from the JASPAR Core database outperformed PBM derived ones from the UniPROBE mouse database. However, the performance of motifs derived using these two methods is not much different from each other when using CentriMo and AME. The E-values from Motif enrichment analysis were not too different from each other or 0. CentriMo showed that in 35 cases JASPAR Core ChIP-seq derived motifs outperformed UniPROBE mouse PBM derived motifs, while it was only in 11 cases that PBM derived motifs outperformed ChIP-seq derived motifs. AME showed that in 18 cases JASPAR Core ChIP-seq derived motifs did better, while only it was only in 3 cases that UniPROBE motifs outperformed ChIP-seq derived motifs. We could not distinguish the performance in 25 cases. Empirical quality assessment: Area under the ROC curve values computations followed by a two-sided t-test showed that there is no significant difference in the average performances of the motifs from the two databases (with 95% confidence, mean of differences=0.0088125 p-value= 0.4874, DF=47) .
- Full Text:
- Date Issued: 2015
- Authors: Hlatshwayo, Nkosikhona Rejoyce
- Date: 2015
- Subjects: Protein binding , DNA , DNA microarrays , Transcription factors , DNA-protein interactions , Gene regulatory networks
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4146 , http://hdl.handle.net/10962/d1017907
- Description: Transcription factors (TFs) are biologically important proteins that interact with transcription machinery and bind DNA regulatory sequences to regulate gene expression by modulating the synthesis of the messenger RNA. The regulatory sequences comprise of short conserved regions of a specific length called motifs . TFs have very diverse roles in different cells and play a very significant role in development. TFs have been associated with carcinogenesis in various tissue types, as well as developmental and hormone response disorders. They may be responsible for the regulation of oncogenes and can be oncogenic. Consequently, understanding TF binding and knowing the motifs to which they bind is worthy of attention and research focus. Various projects have made the study of TF binding their main focus; nevertheless, much about TF binding remains confounding. Chromatin immunoprecipitation in conjunction with deep sequencing (ChIP-seq) techniques are a popular method used to investigate DNA-TF interactions in vivo. This procedure is followed by motif discovery and motif enrichment analysis using relevant tools. Protein Binding Microarrays (PBMs) are an in vitro method for investigating DNA-TF interactions. We use a motif enrichment analysis tools (CentriMo and AME) and an empirical quality assessment tool (Area under the ROC curve) to investigate which method yields motifs that are a true representation of in vivo binding. Motif enrichment analysis: On average, ChIP-seq derived motifs from the JASPAR Core database outperformed PBM derived ones from the UniPROBE mouse database. However, the performance of motifs derived using these two methods is not much different from each other when using CentriMo and AME. The E-values from Motif enrichment analysis were not too different from each other or 0. CentriMo showed that in 35 cases JASPAR Core ChIP-seq derived motifs outperformed UniPROBE mouse PBM derived motifs, while it was only in 11 cases that PBM derived motifs outperformed ChIP-seq derived motifs. AME showed that in 18 cases JASPAR Core ChIP-seq derived motifs did better, while only it was only in 3 cases that UniPROBE motifs outperformed ChIP-seq derived motifs. We could not distinguish the performance in 25 cases. Empirical quality assessment: Area under the ROC curve values computations followed by a two-sided t-test showed that there is no significant difference in the average performances of the motifs from the two databases (with 95% confidence, mean of differences=0.0088125 p-value= 0.4874, DF=47) .
- Full Text:
- Date Issued: 2015
Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
Marine biotechnology : evaluation and development of methods for the discovery of natural products from fungi
- Authors: Pather, Simisha
- Date: 2005 , 2013-06-18
- Subjects: Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3839 , http://hdl.handle.net/10962/d1007652 , Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Description: One of the major impediments in the development of marine natural products is the provision of biologically active natural products in sufficient quantity for complete pharmacological evaluation, clinical trials and eventual commercial production. Marine microorganisms show great promise in providing a renewable source of biologically active natural products. The main aim of this study was to develop and evaluate methods for the isolation, identification and cultivation of marine fungi from the South African marine environment for the production of biologically active secondary metabolites. Twenty-four species of fungi were isolated from marine algae collected from the intertidal zone near Port Alfred, South Africa. The fungi were cultivated in small-scale under static and agitated conditions and their crude intra- and extracellular organic extracts were screened by ¹H NMR and a series of bioassays. Using this as a basis, one isolate was selected for further study. By analyses of the lTS1 region of the ribosomal DNA, the fungal isolate was identified as a marine-derived isolate of Eurotium rubrum (Aspergillus ruber). Although E. rubrum has been isolated from the marine environment, no investigations have been undertaken to determine the adaptation of these isolates to the marine environment. In order to optimise productivity, creativity and incubation time, the fungus was cultivated in small-scale using a variety of carbon (glucose, fructose, lactose, sucrose, marmitol and maltose) and nitrogen sources (ammonium tartrate, urea, peptone and yeast extract). An HPLC-DAD method was developed to assess the metabolic creativity and productivity under different fermentation conditions. Distinctive variations in the range and yield of metabolites produced as well as morphology and growth time were observed. The crude extracts from all fermentations were combined and six known compounds were isolated by reversed-phase chromatography and their structures elucidated by spectroscopic techniques. The known compounds were fIavoglaucin, aspergin, isodihydroauroglaucin, isotetrahydroauroglaucin, neoechinuline A and physcion. Neoechinuline A, isodihydroauroglaucin and isotetrahydroauroglaucin showed activity against oesophageal and cervical cancer cell lines.
- Full Text:
- Date Issued: 2005
- Authors: Pather, Simisha
- Date: 2005 , 2013-06-18
- Subjects: Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3839 , http://hdl.handle.net/10962/d1007652 , Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Description: One of the major impediments in the development of marine natural products is the provision of biologically active natural products in sufficient quantity for complete pharmacological evaluation, clinical trials and eventual commercial production. Marine microorganisms show great promise in providing a renewable source of biologically active natural products. The main aim of this study was to develop and evaluate methods for the isolation, identification and cultivation of marine fungi from the South African marine environment for the production of biologically active secondary metabolites. Twenty-four species of fungi were isolated from marine algae collected from the intertidal zone near Port Alfred, South Africa. The fungi were cultivated in small-scale under static and agitated conditions and their crude intra- and extracellular organic extracts were screened by ¹H NMR and a series of bioassays. Using this as a basis, one isolate was selected for further study. By analyses of the lTS1 region of the ribosomal DNA, the fungal isolate was identified as a marine-derived isolate of Eurotium rubrum (Aspergillus ruber). Although E. rubrum has been isolated from the marine environment, no investigations have been undertaken to determine the adaptation of these isolates to the marine environment. In order to optimise productivity, creativity and incubation time, the fungus was cultivated in small-scale using a variety of carbon (glucose, fructose, lactose, sucrose, marmitol and maltose) and nitrogen sources (ammonium tartrate, urea, peptone and yeast extract). An HPLC-DAD method was developed to assess the metabolic creativity and productivity under different fermentation conditions. Distinctive variations in the range and yield of metabolites produced as well as morphology and growth time were observed. The crude extracts from all fermentations were combined and six known compounds were isolated by reversed-phase chromatography and their structures elucidated by spectroscopic techniques. The known compounds were fIavoglaucin, aspergin, isodihydroauroglaucin, isotetrahydroauroglaucin, neoechinuline A and physcion. Neoechinuline A, isodihydroauroglaucin and isotetrahydroauroglaucin showed activity against oesophageal and cervical cancer cell lines.
- Full Text:
- Date Issued: 2005
Phylogeography and comparative ecophysiology of Chrysanthemoides Turn. Ex Medik. (Tribe Calenduleae)
- Authors: Howis, Seranne
- Date: 2005
- Subjects: Chrysanthemoides , Phylogeny , Ecophysiology , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4256 , http://hdl.handle.net/10962/d1008189
- Description: Chrysanthem Oides is a common Southern African shrub that grows in a variety of habitats. From coastal shrubland and fynbos to mountainous areas as far north as Kenya. The genus has two species and 8 subspecies. The diagnoses and delimitation of which have been based almost exclusively on morphological characteristics. This project aims to investigate, with the use of phylogenetic species concepts. The validity of these subspecies. Unlike biological species concepts that rely on reproductive isolation as a defining character of a species. Phylogenetic species concepts (PSC) are concerned with delimiting evolutionary significant units (ESUs). ESUs are evolutionarily isolated lineages, and under the PSC a species is an aggregation of organisms consistently diagnosable by a fixed character or combination of characters. This project therefore searched for genetic and physiological characters by which to delimit ESUs within the Cill), samhemoides genus. DNA sequencing was used to investigate the genetic characters, while gas exchange studies were used to investigate the ecophysiological characters. DNA sequence analysis indicated that the ESUs can be diagnosed by genetic means and that one species may be of hybrid origin. Field studies of three disparate genetically identifiable ESUs from three disparate climates found that there are noticeable differences in ecophysiological responses of these ESUs in the field. Plants from each ESU were transferred to a greenhouse and grown under identical conditions for several months and compared to determine if these traits are inherent, or elastic in relation to environmental conditions. Under simulated high rainfall conditions. There does not appear to be a significant difference in the photosynthetic traits.
- Full Text:
- Date Issued: 2005
Phylogeography and comparative ecophysiology of Chrysanthemoides Turn. Ex Medik. (Tribe Calenduleae)
- Authors: Howis, Seranne
- Date: 2005
- Subjects: Chrysanthemoides , Phylogeny , Ecophysiology , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4256 , http://hdl.handle.net/10962/d1008189
- Description: Chrysanthem Oides is a common Southern African shrub that grows in a variety of habitats. From coastal shrubland and fynbos to mountainous areas as far north as Kenya. The genus has two species and 8 subspecies. The diagnoses and delimitation of which have been based almost exclusively on morphological characteristics. This project aims to investigate, with the use of phylogenetic species concepts. The validity of these subspecies. Unlike biological species concepts that rely on reproductive isolation as a defining character of a species. Phylogenetic species concepts (PSC) are concerned with delimiting evolutionary significant units (ESUs). ESUs are evolutionarily isolated lineages, and under the PSC a species is an aggregation of organisms consistently diagnosable by a fixed character or combination of characters. This project therefore searched for genetic and physiological characters by which to delimit ESUs within the Cill), samhemoides genus. DNA sequencing was used to investigate the genetic characters, while gas exchange studies were used to investigate the ecophysiological characters. DNA sequence analysis indicated that the ESUs can be diagnosed by genetic means and that one species may be of hybrid origin. Field studies of three disparate genetically identifiable ESUs from three disparate climates found that there are noticeable differences in ecophysiological responses of these ESUs in the field. Plants from each ESU were transferred to a greenhouse and grown under identical conditions for several months and compared to determine if these traits are inherent, or elastic in relation to environmental conditions. Under simulated high rainfall conditions. There does not appear to be a significant difference in the photosynthetic traits.
- Full Text:
- Date Issued: 2005
Assembly of full-length cDNA, and heterologous expression, of Nudaurelia B virus RNA
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
Physico-chemical and substructural studies on Nudaurelia capensis β virus
- Authors: Struthers, J Keith
- Date: 1974
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4075 , http://hdl.handle.net/10962/d1007327 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: From Introduction: The pine emperor moth, Nudaurelia cytherea capensis Stoll is an insect which, during the larval stage, causes extensive defoliation of the pine tree, Pinus radiata in the Cape province. These insects are susceptible to a virus disease, which on occasions causes large scale mortality. Five nonoccluded viruses have been shown to infect the pine emperor moth, and of these, one found in the greatest concentration, Nudaurelia capensis β virus (NβV) has been characterised to the greatest extent. This virus has been shown to contain RNA, to be isometric with a diameter of 36 mm, and to have a molecular weight of 16 million. The virus occurs in all stages of the insect's development, and by fluorescent antibody staining has been shown to develop in the cytoplasm of the host's cells. There have in recent years been a number of reports describing nonoccluded RNA viruses which appear to be similar to NβV. These are the viruses isolated from the moths Gonometa podocarpi and Antheraea eucalypti, and the one from the citrus red mite, Panonychus citri. These viruses have not been as extensively characterised as NβV, so the extent of the similarity between them and NβV is not known. However it would appear as if their discovery collectively heralds the emergence of a distinct new grouping within the nonoccluded RNA viruses of insects. This work reports the isolation and further characterisation of N. capensis β virus, its protein and nucleic acid.
- Full Text:
- Date Issued: 1974
- Authors: Struthers, J Keith
- Date: 1974
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4075 , http://hdl.handle.net/10962/d1007327 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: From Introduction: The pine emperor moth, Nudaurelia cytherea capensis Stoll is an insect which, during the larval stage, causes extensive defoliation of the pine tree, Pinus radiata in the Cape province. These insects are susceptible to a virus disease, which on occasions causes large scale mortality. Five nonoccluded viruses have been shown to infect the pine emperor moth, and of these, one found in the greatest concentration, Nudaurelia capensis β virus (NβV) has been characterised to the greatest extent. This virus has been shown to contain RNA, to be isometric with a diameter of 36 mm, and to have a molecular weight of 16 million. The virus occurs in all stages of the insect's development, and by fluorescent antibody staining has been shown to develop in the cytoplasm of the host's cells. There have in recent years been a number of reports describing nonoccluded RNA viruses which appear to be similar to NβV. These are the viruses isolated from the moths Gonometa podocarpi and Antheraea eucalypti, and the one from the citrus red mite, Panonychus citri. These viruses have not been as extensively characterised as NβV, so the extent of the similarity between them and NβV is not known. However it would appear as if their discovery collectively heralds the emergence of a distinct new grouping within the nonoccluded RNA viruses of insects. This work reports the isolation and further characterisation of N. capensis β virus, its protein and nucleic acid.
- Full Text:
- Date Issued: 1974
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