Investigating the relationship between Heat Shock Proteins and HIV Transactivator of Transcription
- Authors: Flax, Lili Marie
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163307 , vital:41027
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
- Full Text:
- Date Issued: 2020
- Authors: Flax, Lili Marie
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163307 , vital:41027
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
- Full Text:
- Date Issued: 2020
Investigating the roles of HOP isoforms in KSHV biology
- Matandirotya, Lorraine Tariro
- Authors: Matandirotya, Lorraine Tariro
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365257 , vital:65721
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Matandirotya, Lorraine Tariro
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365257 , vital:65721
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Investigating the use of Arbuscular Mycorrhizas and Plant Growth Promoting Bacteria to improve the drought tolerance of maize (Zea mays L.)
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Date Issued: 2016
- Authors: Moore, Nicolle Maureen
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54587 , vital:26591
- Description: Maize (Zea mays L.) is a direct staple food crop in Africa and remains an essential component of global food security, with maize crops accounting for over 60% of the total harvested area of annual food crops. Stress caused by drought and high soil salinity limits crop growth and productivity more than any other single environmental factor, with grain yield reductions up to 76% depending on the severity of the drought and the plant growth stage. Arbuscular mycorrhizal (AM) fungi and Plant Growth Promotion Rhizobacteria (PGPR) have previously been shown to improve tolerance of plants to drought stress through a number of chemical and physiological processes. The aim of this investigation was to determine whether mycorrhizal fungi and rhizobacteria adapted to drought and saline conditions and possessing plant growth promoting (PGP) traits were able to stimulate plant growth responses when applied to Zea mays seeds growing under greenhouse conditions Bacterial isolates selected were tolerant to concentrations of NaCl up to 600 mM and maintained 50% growth at low water potentials (-1.44 MPa). They were positive for Indole Acetic Acid (IAA) production, phosphate solubilisation and secretion of siderophores. Bacterial isolates showing plant growth promoting potential were identified using 16S rDNA gene sequencing as Achromobacter xylosoxidans strains A8 and C54 and Klebsiella oxytoca strain M1. Mixed inoculum was prepared from indigenous communities of mycorrhizas in soils sampled from the Cerebos Salt Pan and the Kalahari Desert. Mycorrhizal diversity was investigated using 454-Pyrosequencing which revealed that the community composition was dominated by species in the Ambispora, Glomus and Paraglomus genera with a rare component represented by species in the Redeckera, Archaeospora and Geosiphon genera. Microscopic examination of plant roots at the end of the trial revealed the presence of diagnostic mycorrhizal structures within the root cells, confirming that colonization was successful. Plant growth response to microbial inoculation was assessed by monitoring changes in plant photosynthetic capacity over the duration of a 7 week pot trial. A significant difference in photosynthetic and biomass data was observed between drought and well-watered groups but no mycorrhizal or bacterial treatment effect was evident within the groups, despite the high levels of colonization by mycorrhizas. These results suggest that the beneficial effects of mycorrhizal colonization may be primarily attributed to improved nutrient and mineral uptake in conditions where nutrients are limiting, resulting in improved growth. The improved growth may then have secondary effects on the plant‟s ability to withstand drought. Having controlled for nutrient deficiency, it was not evident in this study that mycorrhizal fungi were able to stimulate a change in plant physiology and confer drought tolerance under the conditions imposed.
- Full Text:
- Date Issued: 2016
Investigation into the influence of an alien plant species on the soil microbiota of the sub-Antarctic, Marion Island
- Authors: Pienaar, Robert Daniel
- Date: 2020
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/124039 , vital:35531
- Description: Embargo applied. Embargo period to expire in April of 2021.
- Full Text:
- Date Issued: 2020
- Authors: Pienaar, Robert Daniel
- Date: 2020
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/124039 , vital:35531
- Description: Embargo applied. Embargo period to expire in April of 2021.
- Full Text:
- Date Issued: 2020
Investigation of the causative agents of the 1982 Gazankulu poliomyelitis outbreak, using four biochemical techniques
- Authors: Gibson, Katherine Margaret
- Date: 1989
- Subjects: Poliomyelitis -- Analysis , Poliomyelitis -- History -- South Africa , Poliomyelitis vaccine -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3878 , http://hdl.handle.net/10962/d1001612
- Description: Comparison of poliovirus strains was carried out to determine the origin of the virus in two isolates obtained during the 1982 outbreak of poliomyelitis in Gazankulu. Comparisons of the outbreak isolates with vaccine and wild-type strains of the same poliovirus type were carried out using four biochemical techniques. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional thin-layer chromatography (TLC) and reversed-phase high-performance liquid-chromatography (RP-HPLC) were used for comparing viral capsid proteins. Comparison of poliovirus strains at a genetic level was carried out using two-dimensional oligonucleotide mapping of viral RNA. Results showed the type 1 poliovirus isolate, 5061, to be a novel wild-type poliovirus. The type 2 isolate, 5068, was closely related to the poliovirus type 2 Sabin vaccine strain, P712. It was concluded that the intrinsic variability of poliovirus strains was responsible for the appearance of isolate 5068
- Full Text:
- Date Issued: 1989
- Authors: Gibson, Katherine Margaret
- Date: 1989
- Subjects: Poliomyelitis -- Analysis , Poliomyelitis -- History -- South Africa , Poliomyelitis vaccine -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3878 , http://hdl.handle.net/10962/d1001612
- Description: Comparison of poliovirus strains was carried out to determine the origin of the virus in two isolates obtained during the 1982 outbreak of poliomyelitis in Gazankulu. Comparisons of the outbreak isolates with vaccine and wild-type strains of the same poliovirus type were carried out using four biochemical techniques. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional thin-layer chromatography (TLC) and reversed-phase high-performance liquid-chromatography (RP-HPLC) were used for comparing viral capsid proteins. Comparison of poliovirus strains at a genetic level was carried out using two-dimensional oligonucleotide mapping of viral RNA. Results showed the type 1 poliovirus isolate, 5061, to be a novel wild-type poliovirus. The type 2 isolate, 5068, was closely related to the poliovirus type 2 Sabin vaccine strain, P712. It was concluded that the intrinsic variability of poliovirus strains was responsible for the appearance of isolate 5068
- Full Text:
- Date Issued: 1989
Isolation, expression and purification of the hydantoin hydrolysing enzymes of agrobacterium tumefaciens
- Authors: Clark, Sally-Ann
- Date: 2003
- Subjects: Agrobacterium tumefaciens , Amino acids Hydantoin Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4140 , http://hdl.handle.net/10962/d1016233
- Description: The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.
- Full Text:
- Date Issued: 2003
- Authors: Clark, Sally-Ann
- Date: 2003
- Subjects: Agrobacterium tumefaciens , Amino acids Hydantoin Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4140 , http://hdl.handle.net/10962/d1016233
- Description: The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.
- Full Text:
- Date Issued: 2003
Isolation, identification and genetic characterisation of a microsporidium isolated from the carob moth, Ectomyelois ceratoniae (Lepidoptera: Pyralidae)
- Authors: Lloyd, Melissa
- Date: 2018
- Subjects: Pyralidae , Pyralidae -- Genetics , Pyralidae -- Phylogeny , Pyralidae -- Pathogens , Cladistic analysis , Transmission electron microscopy , Carob moth (Ectomyelois ceratoniae)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/61894 , vital:28075
- Description: Carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) is an economically important pest, yet its biology and pest status on citrus in South Africa was, until recently, poorly understood. A study was initiated to determine the cause of collapse of a laboratory carob moth colony that was established to investigate the biology of carob moth on citrus and to develop integrated management strategies for the pest. An organism was isolated from deceased larvae and was morphologically identified as a microsporidium, based on transmission electron microscopy. Microsporidia are obligate intracellular parasites that have been found to infect almost all eukaryotes. Several Nosema species have been isolated from economically important insect pests, yet little genetic information is available from online databases for identification. Mature spores were recovered and measured using transmission electron microscopy. Spores were ovocylindrical with a wrinkled exospore, and had a length of 2.8 ± 0.02 pm and a width of 1.6 ± 0.04 pm. The identity of the microsporidium was confirmed by PCR amplification, sequencing and analysis of the regions encoding the ribosomal RNA. BLAST analysis of the different rRNA regions amplified showed that the microsporidium shared a 96 - 99 % identity with Nosema sp. M-Pr, Nosema carpocapsae, Nosema oulemae, Nosema sp. CO1, Microsporidium 57864, and Nosema bombi. Phylogenetic analysis of the SSU and LSU rRNA genes showed that the microsporidium clustered with the Nosema / Vairimorpha clade, supported by a bootstrap value of 100. The organisation of the RNA cistron was determined by PCR amplification using the primer set 18f and L1328r to be 5’-SSU-ITS-LSU-IGS-5S-3’, which confirms the placement of the microsporidium within the Nosema / Vairimorpha clade. Because the BLAST results showed a close relationship with Nosema carpocapsae, a microsporidium infecting codling moth, the pathogenicity of the microsporidium was tested against codling moth by inoculating artificial diet with a high spore concentration of 1.1 x 107 spores/ml and a low spore concentration of 1.1 x 104 spores/ml. DNA was extracted from deceased larvae inoculated with the high concentration, and PCR of the SSU rRNA gene and bacterial 16S region was performed. Mortality in the high concentration experiment was significant (p = 0.05), but the cause of infection was determined to be a bacterium, through sequencing and BLAST analysis of the bacterial 16S rDNA. The bacterium shared a 99 % identity with Bacillus cereus. Percentage mortality (p = 0.09), larval mass (p = 0.09) and instar (p = 0.24) did not differ significantly between treatments in the low concentration experiment. DNA was extracted from the larvae and PCR amplification of the SSU rRNA gene was performed to determine whether microsporidia were present. No SSU bands were observed in any of the treatments and percentage mortality was not significant, thus it was determined that no infection occurred. This is the first study to report the genetic characterisation of a microsporidium isolated from carob moth and provides important genetic information for classification of microsporidia within the Nosema / Vairimorpha clade. It is also one of few studies in which the complete rRNA cistron of a species within the Nosema / Vairimorpha clade has been sequenced. The identification of a microsporidium from a laboratory colony of carob moth is important as it provides information about pathogens infecting the carob moth and constraints to carob moth rearing, which is useful for further studies on rearing carob moth and for establishment of a clean colony for research purposes.
- Full Text:
- Date Issued: 2018
- Authors: Lloyd, Melissa
- Date: 2018
- Subjects: Pyralidae , Pyralidae -- Genetics , Pyralidae -- Phylogeny , Pyralidae -- Pathogens , Cladistic analysis , Transmission electron microscopy , Carob moth (Ectomyelois ceratoniae)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/61894 , vital:28075
- Description: Carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) is an economically important pest, yet its biology and pest status on citrus in South Africa was, until recently, poorly understood. A study was initiated to determine the cause of collapse of a laboratory carob moth colony that was established to investigate the biology of carob moth on citrus and to develop integrated management strategies for the pest. An organism was isolated from deceased larvae and was morphologically identified as a microsporidium, based on transmission electron microscopy. Microsporidia are obligate intracellular parasites that have been found to infect almost all eukaryotes. Several Nosema species have been isolated from economically important insect pests, yet little genetic information is available from online databases for identification. Mature spores were recovered and measured using transmission electron microscopy. Spores were ovocylindrical with a wrinkled exospore, and had a length of 2.8 ± 0.02 pm and a width of 1.6 ± 0.04 pm. The identity of the microsporidium was confirmed by PCR amplification, sequencing and analysis of the regions encoding the ribosomal RNA. BLAST analysis of the different rRNA regions amplified showed that the microsporidium shared a 96 - 99 % identity with Nosema sp. M-Pr, Nosema carpocapsae, Nosema oulemae, Nosema sp. CO1, Microsporidium 57864, and Nosema bombi. Phylogenetic analysis of the SSU and LSU rRNA genes showed that the microsporidium clustered with the Nosema / Vairimorpha clade, supported by a bootstrap value of 100. The organisation of the RNA cistron was determined by PCR amplification using the primer set 18f and L1328r to be 5’-SSU-ITS-LSU-IGS-5S-3’, which confirms the placement of the microsporidium within the Nosema / Vairimorpha clade. Because the BLAST results showed a close relationship with Nosema carpocapsae, a microsporidium infecting codling moth, the pathogenicity of the microsporidium was tested against codling moth by inoculating artificial diet with a high spore concentration of 1.1 x 107 spores/ml and a low spore concentration of 1.1 x 104 spores/ml. DNA was extracted from deceased larvae inoculated with the high concentration, and PCR of the SSU rRNA gene and bacterial 16S region was performed. Mortality in the high concentration experiment was significant (p = 0.05), but the cause of infection was determined to be a bacterium, through sequencing and BLAST analysis of the bacterial 16S rDNA. The bacterium shared a 99 % identity with Bacillus cereus. Percentage mortality (p = 0.09), larval mass (p = 0.09) and instar (p = 0.24) did not differ significantly between treatments in the low concentration experiment. DNA was extracted from the larvae and PCR amplification of the SSU rRNA gene was performed to determine whether microsporidia were present. No SSU bands were observed in any of the treatments and percentage mortality was not significant, thus it was determined that no infection occurred. This is the first study to report the genetic characterisation of a microsporidium isolated from carob moth and provides important genetic information for classification of microsporidia within the Nosema / Vairimorpha clade. It is also one of few studies in which the complete rRNA cistron of a species within the Nosema / Vairimorpha clade has been sequenced. The identification of a microsporidium from a laboratory colony of carob moth is important as it provides information about pathogens infecting the carob moth and constraints to carob moth rearing, which is useful for further studies on rearing carob moth and for establishment of a clean colony for research purposes.
- Full Text:
- Date Issued: 2018
Linking Hop and LANA1 in the KSHV life cycle
- Authors: Ruck, Jamie-Lee
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365291 , vital:65724
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Ruck, Jamie-Lee
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365291 , vital:65724
- Description: Thesis embargoed. Possible release date set for early 2025. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Localizing selected endocytosis protein candidates in Plasmodium falciparum using GFP-tagged fusion constructs
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Date Issued: 2016
- Authors: Basson, Travis
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2680 , vital:20316
- Description: Malaria is a mosquito-borne infectious disease caused by several obligate intracellular protozoan parasites in the Plasmodium genus, with Plasmodium falciparum causing the most widespread cases and malaria deaths. In 2013 there were approximately 190 million cases of the disease and between 584,000 and 855,000 deaths. It is essential to identify novel drug targets and develop novel drug candidates due to the increase in resistance of P. falciparum parasites to the current arsenal of antimalarial drugs. Endocytosis is an essential process in eukaryotic cells in which the external environment is internalized by the cell in order to obtain various particles from the extracellular space. This extracellular cytoplasm is internalized in membrane-bound invaginations at the plasma membrane. During the blood stage of malaria infection, the parasite requires nutrients from the host red blood cell. To obtain these nutrients, the parasite internalizes haemoglobin in large amounts and degrades it in an acidic, lysosome-like organelle, known as the digestive vacuole. Whilst the exact molecular mechanism of malaria parasite endocytosis is not yet fully understood, a number of proteins have been suggested to be involved. The most expedient approach in identifying candidate endocytosis proteins is to investigate parasite homologues of proteins known to be involved in endocytosis in mammalian cells. The three proteins selected for investigation in this study were the P. falciparum homologues of coronin, dynamin 2, and μ4. The coding sequences for the candidate endocytosis proteins were amplified by PCR and cloned into the pARL2-GFP expression vector. P. falciparum 3D7 parasites were transfected with these vectors and the episomal expression of full-length GFP-tagged fusion protein was confirmed by Western blot analysis using commercially available anti-GFP antibodies. Microscopic analysis of live parasites using fluorescence and confocal microscopy was used to determine the localization of the candidate endocytosis proteins. Coronin appeared to display diffuse cytoplasmic GFP localization during the trophozoite stage, arguing against a role in endocytosis. However, distinct localization during the schizont stage at what appears to be the inner membrane complex was observed. Coronin is thus likely required to coordinate the formation of the actin network between the merozoite IMC and the plasma membrane on which the glideosome is dependant for generating the motile forces required for the merozoite motility and invasion of RBCs. Dynamin 2 displayed localization at three potential locii: the parasite periphery (plasma membrane), punctuate regions within the cytoplasm (potentially at membrane bound organelles) and at the parasite food vacuole. The data suggested that dynamin 2 is involved in endocytosis and membrane trafficking in a similar manner to classical dynamins, potentially as a vesicle scission molecule at the plasma membrane, mediating vesicle formation at the food vacuole to recycle membrane to the plasma membrane, and possibly mitochondria organelle division. μ4 displayed transient localization, cycling between cytosolic localization, and localization to distinct regions at the plasma membrane and the food vacuole. Localization of Pfμ4 to the plasma membrane is indicative of a role for μ4 as a part of an adaptor protein (AP) complex which may be responsible for recruitment of clathrin to initiate endocytosis in a manner similar to mammalian AP-2. As was observed with PfDYN2, Pfμ4 localizes to the FV, which suggests that Pfμ4 forms part of a coat complex that mediates the formation of vesicles that recycle membrane from the FV to the parasite plasma membrane. This study showed that expressing proteins as full-length GFP-tagged fusion constructs is an effective approach in the early stages of determining the localization and function of P. falciparum proteins in vitro, and distinguishing between candidates that have a potential role in endocytosis and those that are unlikely to do so.
- Full Text:
- Date Issued: 2016
Mechanism of action of non-synonymous single nucleotide variations associated with α-carbonic anhydrases II, IV and VIII
- Authors: Sanyanga, T. Allan
- Date: 2020
- Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/167346 , vital:41470
- Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
- Full Text:
- Date Issued: 2020
- Authors: Sanyanga, T. Allan
- Date: 2020
- Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/167346 , vital:41470
- Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
- Full Text:
- Date Issued: 2020
Mechanistic analysis of two cytotoxic thiazolidinones as novel inhibitors of Triple-Negative Breast Cancer
- Authors: Vukea, Nyeleti
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365734 , vital:65780
- Description: Thesis embargoes. Expected release date early 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Vukea, Nyeleti
- Date: 2022-10-14
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365734 , vital:65780
- Description: Thesis embargoes. Expected release date early 2025. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Molecular characterization of microbial communities in the Sundays and Swartkops estuaries impacted by anthropogenic activities
- Kgomokhumo, Tlhoafalang Evah
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Molecular cloning and expression of equine CYP1A2 in Escherichia coli
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Date Issued: 2017
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
- Full Text:
- Date Issued: 2017
Molecular simulations of potential agents and targets of Alzheimer’s disease
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
- Full Text:
- Date Issued: 2020
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
- Full Text:
- Date Issued: 2020
Multiplexed Mass Spectrometry: Single, On-Bead, Detection Analysis Using MALDI-TOF MS
- Authors: Twala, Busisiwe Victoria
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164693 , vital:41155 , doi:10.21504/10962/164693
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Twala, Busisiwe Victoria
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164693 , vital:41155 , doi:10.21504/10962/164693
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Date Issued: 2015
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Date Issued: 2015
Nutrient impacts on grasses and legumes growing in communal pasture soil in relation to mycorrhizal activity
- Authors: Mkile, Zolani
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164512 , vital:41125 , doi:10.21504/10962/164512
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Mkile, Zolani
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164512 , vital:41125 , doi:10.21504/10962/164512
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Orchid mycorrhizal and endophytic fungal diversity of three co-occurring terrestrial orchids in the large African genus Disa (Orchidaceae)
- Khambule, Nondumiso Venessia
- Authors: Khambule, Nondumiso Venessia
- Date: 2020
- Subjects: Orchids South Africa , Mycorrhizal fungi South Africa , Endomycorrhizas South Africa , Endophytes , Orchids Roots
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163341 , vital:41030
- Description: Orchids (in the family Orchidaceous) are one of the richest plant families and approximately 500 species are found in South Africa. A number of orchid species are found on disturbed areas and many of the terrestrial species grow in poor soils with low mineral nutrient availability. Most orchid species are thought to be associated with mycorrhizal fungi for germination and mycorrhiza provides nutrients for the survival of adult plants. The aim of this study was to select Orchidaceous plant species and to isolate, identify and characterize the orchid endophytes and assess these isolates for potential antimicrobial and enzymatic activities Isa is the largest genus in South Africa and three Disa species co-occurring in a small geographical area were selected. These included Disa bracteata, D. cornuta and D. polygonoides which span three sections of the genus. Roots were stained to confirm the mycorrhizal status of the Disa species. Mycorrhizal pelotons structures were microscopically observed inside root cells. The presence of pelotons is indictive of mycorrhizal fungal interactions within the orchid roots and areas associated with the site of nutrient exchange between plant and fungus. The presence of pelotons, however, does not give n indication of the fungal species involved. The endophytes were successfully isolated in pure cultures on potato dextrose agar (PDA). All slow growing isolates were selected, and further molecular identification undertaken; DNA was extracted, and PCR amplified using internal transcribed spacer (ITS1F and ITS4) fungal primers. The amplified products were then sequenced and analysed by comparison to sequences in the GenBank database. Trichoderma, Penicillium, Metapochonia, Talaromyces, Oidiodendron Neopestalotiopsis, and Chaetomium were identified from these sequences. The presence of other fungal root endophytes was suspected despite the rigorous surface sterilization procedure used. The primers used to amplify the ITS region are the universal barcoding primers which are specific to fungi. ITS1F is one of the primers designed to amplify a broad range of fungi. DNA was extracted from orchid roots and amplicons were cloned into a pGEMT plasmid vector. Individual clones were sequenced and aligned with Mega software and compared to sequences in the GenBank and UNITE database. Based on percentage sequence identity, unidentified Tulasnella species, Tullasnela colaspora, and various Ascomycota endophytes were identified as contributing to the endophytic root fungal diversity of the selected Disa species. The Disa species investigated in this study were associated with several soil endophytes. D. bracteata, D. polygonoides were collected from the same site along the road verge which is regarded as being disturbed. Based on both culture – dependent and independent techniques employed Oidiodendron was found associated with both species. Antimicrobial activity was determined using a well diffusion method using extracts from the isolated fungi against the bacterial isolates Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas puptida. Most of the isolated fungi showed at least one potential inhibition effect against one of the bacterial isolates. The extracts that showed potential antimicrobial activity could be further screened to determine the compounds produced as secondary metabolites using techniques such as LC-MS Enzymatic activities of protease, cellulose and amylase were determined using solid media amended with milk protein, carboxymethylcellulose (CMC) and starch. The majority of fungal isolates tested positive with amylase and cellulose with only a few fungal isolates testing positive for protease activity. Broth cultures containing CMC and starch were shown to enhance biomass production in approximately 40 % of the fungal isolates. Degradation of the substrates is required in order to provide carbon to the fungus under test in order to optimize fungal growth as well as to gain insight into their ecological role. Enzyme activity was evident particularly when cellulose and starch were provided as substrates. All the fungal isolates tested grew on the amended medium, with 40% of the isolates preferring to utilize CMC and/or starch, indicating the ability of these fungi to utilize various resources for carbon acquisitions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
- Authors: Khambule, Nondumiso Venessia
- Date: 2020
- Subjects: Orchids South Africa , Mycorrhizal fungi South Africa , Endomycorrhizas South Africa , Endophytes , Orchids Roots
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/163341 , vital:41030
- Description: Orchids (in the family Orchidaceous) are one of the richest plant families and approximately 500 species are found in South Africa. A number of orchid species are found on disturbed areas and many of the terrestrial species grow in poor soils with low mineral nutrient availability. Most orchid species are thought to be associated with mycorrhizal fungi for germination and mycorrhiza provides nutrients for the survival of adult plants. The aim of this study was to select Orchidaceous plant species and to isolate, identify and characterize the orchid endophytes and assess these isolates for potential antimicrobial and enzymatic activities Isa is the largest genus in South Africa and three Disa species co-occurring in a small geographical area were selected. These included Disa bracteata, D. cornuta and D. polygonoides which span three sections of the genus. Roots were stained to confirm the mycorrhizal status of the Disa species. Mycorrhizal pelotons structures were microscopically observed inside root cells. The presence of pelotons is indictive of mycorrhizal fungal interactions within the orchid roots and areas associated with the site of nutrient exchange between plant and fungus. The presence of pelotons, however, does not give n indication of the fungal species involved. The endophytes were successfully isolated in pure cultures on potato dextrose agar (PDA). All slow growing isolates were selected, and further molecular identification undertaken; DNA was extracted, and PCR amplified using internal transcribed spacer (ITS1F and ITS4) fungal primers. The amplified products were then sequenced and analysed by comparison to sequences in the GenBank database. Trichoderma, Penicillium, Metapochonia, Talaromyces, Oidiodendron Neopestalotiopsis, and Chaetomium were identified from these sequences. The presence of other fungal root endophytes was suspected despite the rigorous surface sterilization procedure used. The primers used to amplify the ITS region are the universal barcoding primers which are specific to fungi. ITS1F is one of the primers designed to amplify a broad range of fungi. DNA was extracted from orchid roots and amplicons were cloned into a pGEMT plasmid vector. Individual clones were sequenced and aligned with Mega software and compared to sequences in the GenBank and UNITE database. Based on percentage sequence identity, unidentified Tulasnella species, Tullasnela colaspora, and various Ascomycota endophytes were identified as contributing to the endophytic root fungal diversity of the selected Disa species. The Disa species investigated in this study were associated with several soil endophytes. D. bracteata, D. polygonoides were collected from the same site along the road verge which is regarded as being disturbed. Based on both culture – dependent and independent techniques employed Oidiodendron was found associated with both species. Antimicrobial activity was determined using a well diffusion method using extracts from the isolated fungi against the bacterial isolates Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas puptida. Most of the isolated fungi showed at least one potential inhibition effect against one of the bacterial isolates. The extracts that showed potential antimicrobial activity could be further screened to determine the compounds produced as secondary metabolites using techniques such as LC-MS Enzymatic activities of protease, cellulose and amylase were determined using solid media amended with milk protein, carboxymethylcellulose (CMC) and starch. The majority of fungal isolates tested positive with amylase and cellulose with only a few fungal isolates testing positive for protease activity. Broth cultures containing CMC and starch were shown to enhance biomass production in approximately 40 % of the fungal isolates. Degradation of the substrates is required in order to provide carbon to the fungus under test in order to optimize fungal growth as well as to gain insight into their ecological role. Enzyme activity was evident particularly when cellulose and starch were provided as substrates. All the fungal isolates tested grew on the amended medium, with 40% of the isolates preferring to utilize CMC and/or starch, indicating the ability of these fungi to utilize various resources for carbon acquisitions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text:
- Date Issued: 2020
Prediction of interacting motifs within the protein subunits of Picornavirus capsids
- Authors: Ross, Caroline Jane
- Date: 2015
- Subjects: Picornaviruses , Antiviral agents , Poliovirus , Coxsackieviruses , Hepatitis A virus , Foot-and-mouth disease virus , Viral proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4151 , http://hdl.handle.net/10962/d1017912
- Description: The Picornaviridae family contains a number of pathogens which are economically important including Poliovirus, Coxsakievirus, Hepatitis A Virus, and Foot-and-Mouth-Disease-Virus. Recently the emergence of novel picornaviruses associated with gastrointestinal, neurological and respiratory diseases in humans has been reported. Although effective vaccines for viruses such as FMDV, PV and HAV have been developed there are currently no antivirals available for the treatment of picornavirus infections. Picornaviruses proteins are classified as: the structural proteins VP1, VP2, VP3 and VP4 which form the subunits of the viral capsid and the replication proteins which function as proteases, RNA-polymerases, primers and membrane binding proteins. Although the host specificity and viral pathogenicity varies across members of the family, the icosahedral capsid is highly conserved. The capsid consists of 60 protomers, each containing a single copy of VP1, VP2 and VP3. A fourth capsid protein, VP4, resides on the internal side of the capsid. Capsid assembly is integral to life-cycle of picornaviruses; however the process is complex and not fully-understood. The overall aim of the study was to broaden the understanding of the evolution and function of the structural proteins across the Picornaviridae family. Firstly a comprehensive analysis of the phylogenetic relationships amongst the individual structural proteins was performed. The functions of the structural proteins were further investigated by an exhaustive motif analysis. A subsequent structural analysis of highly conserved motifs was performed with respect to representative enteroviruses, Foot-and-Mouth-Disease-Virus and Theiler’s Virus. This was supplemented by the in silico prediction of interacting residues within the crystal structures of these protomers. Findings in this study suggest that the capsid proteins may be evolving independently from the replication proteins through possible inter-typic recombination of functional protein regions. Moreover the study predicts that protomer assembly may be facilitated through a network of multiple subunit-subunit interactions. Multiple conserved motifs and principle residues predicted to facilitate capsid subunit-subunit interactions were identified. It was also concluded that motif conservation may support the theory of inter-typic recombination between closely related virus sub-types. As capsid assembly is critical to the viral life-cycle, the principle interacting motifs may serve as novel drug targets for the antiviral treatment of picornavirus infections. Thus the findings in the study may be fundamental to the development of treatments which are more economically feasible or clinically effective than current vaccinations.
- Full Text:
- Date Issued: 2015
- Authors: Ross, Caroline Jane
- Date: 2015
- Subjects: Picornaviruses , Antiviral agents , Poliovirus , Coxsackieviruses , Hepatitis A virus , Foot-and-mouth disease virus , Viral proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4151 , http://hdl.handle.net/10962/d1017912
- Description: The Picornaviridae family contains a number of pathogens which are economically important including Poliovirus, Coxsakievirus, Hepatitis A Virus, and Foot-and-Mouth-Disease-Virus. Recently the emergence of novel picornaviruses associated with gastrointestinal, neurological and respiratory diseases in humans has been reported. Although effective vaccines for viruses such as FMDV, PV and HAV have been developed there are currently no antivirals available for the treatment of picornavirus infections. Picornaviruses proteins are classified as: the structural proteins VP1, VP2, VP3 and VP4 which form the subunits of the viral capsid and the replication proteins which function as proteases, RNA-polymerases, primers and membrane binding proteins. Although the host specificity and viral pathogenicity varies across members of the family, the icosahedral capsid is highly conserved. The capsid consists of 60 protomers, each containing a single copy of VP1, VP2 and VP3. A fourth capsid protein, VP4, resides on the internal side of the capsid. Capsid assembly is integral to life-cycle of picornaviruses; however the process is complex and not fully-understood. The overall aim of the study was to broaden the understanding of the evolution and function of the structural proteins across the Picornaviridae family. Firstly a comprehensive analysis of the phylogenetic relationships amongst the individual structural proteins was performed. The functions of the structural proteins were further investigated by an exhaustive motif analysis. A subsequent structural analysis of highly conserved motifs was performed with respect to representative enteroviruses, Foot-and-Mouth-Disease-Virus and Theiler’s Virus. This was supplemented by the in silico prediction of interacting residues within the crystal structures of these protomers. Findings in this study suggest that the capsid proteins may be evolving independently from the replication proteins through possible inter-typic recombination of functional protein regions. Moreover the study predicts that protomer assembly may be facilitated through a network of multiple subunit-subunit interactions. Multiple conserved motifs and principle residues predicted to facilitate capsid subunit-subunit interactions were identified. It was also concluded that motif conservation may support the theory of inter-typic recombination between closely related virus sub-types. As capsid assembly is critical to the viral life-cycle, the principle interacting motifs may serve as novel drug targets for the antiviral treatment of picornavirus infections. Thus the findings in the study may be fundamental to the development of treatments which are more economically feasible or clinically effective than current vaccinations.
- Full Text:
- Date Issued: 2015
Prediction of mass spectra for natural products using an ab initio approach
- Authors: Novokoza, Yolanda
- Date: 2020
- Subjects: Molecular dynamics , Molecular dynamics -- Computer simulation , Mass spectroscopy , Electron impact ionization
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167166 , vital:41443
- Description: Mass spectrometry (MS) is a technique that measures the fragmentation of molecules, dependent on the molecule’s chemical composition and structure, by first introducing a charge on the molecules. The instrument records the mass to charge ratio, but the energy from the ionization process causes the molecule to fragment. The resultant mass spectrum is highly indicative of not only the molecule analyzed, but also its chemical composition. MS is used in research and industry for both routine and research purposes. One such way to ionize molecules for MS is by bombarding the molecule with electrons which is the basis of electron impact mass spectrometry (EIMS). Although EIMS is widely used, prediction of electron impact mass spectra from first principles is a challenging problem due to a need to accurately determine the probability of different fragmentation pathways of a molecule. Ab initio molecular dynamics based methods are able to explore in an automatic fashion the energetically available fragmentation paths thus give reaction mechanisms in an unbiased way. The mass spectra of five molecules have been explored in work-flows leading to the prediction of mass spectra. These molecules include three natural products alpha-hispanolol, PFB oxime derivative and boronolide (for which experimental mass spectra were not available) and two compounds from the NIST database (for which experimental mass spectra were available). For each of these systems many random conformations were generated using the RDKit library. To all conformations random velocities were applied to each atom. Ab initio molecular dynamics was performed on each conformer, using these initial random velocities using CP2K software, at DFTB+ level at a variety of highly raised temperatures (to accelerate the formation of fragments) Fragmentation was monitored by iterating through all bonds, and identifying bond breakages during dynamics. Graph theoretical packages were used then to track distinct fragments generated. For each of these fragments, charges were determined from Mulliken analysis for all atoms on the fragment from the QM calculations and sum of atomic spin densities per fragment was also plotted. The fragment with the greatest charge (corresponding to the formation of a cation fragment) was taken for plotting on the mass spectrum. Finally, from the mass of the fragment and its elemental composition, the isotopic distribution for the fragment was determined, and this distribution was included by addition in to the mass spectrum. For all trajectories, the sum of all isotopic distributions determined the final mass spectrum.
- Full Text:
- Date Issued: 2020
- Authors: Novokoza, Yolanda
- Date: 2020
- Subjects: Molecular dynamics , Molecular dynamics -- Computer simulation , Mass spectroscopy , Electron impact ionization
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167166 , vital:41443
- Description: Mass spectrometry (MS) is a technique that measures the fragmentation of molecules, dependent on the molecule’s chemical composition and structure, by first introducing a charge on the molecules. The instrument records the mass to charge ratio, but the energy from the ionization process causes the molecule to fragment. The resultant mass spectrum is highly indicative of not only the molecule analyzed, but also its chemical composition. MS is used in research and industry for both routine and research purposes. One such way to ionize molecules for MS is by bombarding the molecule with electrons which is the basis of electron impact mass spectrometry (EIMS). Although EIMS is widely used, prediction of electron impact mass spectra from first principles is a challenging problem due to a need to accurately determine the probability of different fragmentation pathways of a molecule. Ab initio molecular dynamics based methods are able to explore in an automatic fashion the energetically available fragmentation paths thus give reaction mechanisms in an unbiased way. The mass spectra of five molecules have been explored in work-flows leading to the prediction of mass spectra. These molecules include three natural products alpha-hispanolol, PFB oxime derivative and boronolide (for which experimental mass spectra were not available) and two compounds from the NIST database (for which experimental mass spectra were available). For each of these systems many random conformations were generated using the RDKit library. To all conformations random velocities were applied to each atom. Ab initio molecular dynamics was performed on each conformer, using these initial random velocities using CP2K software, at DFTB+ level at a variety of highly raised temperatures (to accelerate the formation of fragments) Fragmentation was monitored by iterating through all bonds, and identifying bond breakages during dynamics. Graph theoretical packages were used then to track distinct fragments generated. For each of these fragments, charges were determined from Mulliken analysis for all atoms on the fragment from the QM calculations and sum of atomic spin densities per fragment was also plotted. The fragment with the greatest charge (corresponding to the formation of a cation fragment) was taken for plotting on the mass spectrum. Finally, from the mass of the fragment and its elemental composition, the isotopic distribution for the fragment was determined, and this distribution was included by addition in to the mass spectrum. For all trajectories, the sum of all isotopic distributions determined the final mass spectrum.
- Full Text:
- Date Issued: 2020