Identification of SANCDB compounds against G2019S and I2020T variants of leucine-rich repeat Kinase 2 (LRRK2) for the development of drugs against Parkinson’s Disease
- Authors: Baye, Bertha Cinthia
- Date: 2020
- Subjects: Antiparkinsonian agents , Parkinson's disease -- Treatment , Protein kinases , Parkinson's disease -- Chemotherapy , Molecules -- Models
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/138764 , vital:37671
- Description: Parkinson’s disease is a type of movement disorder that occurs when nerve cells in the brain stop producing dopamine. It is the second neurodegenerative disease affecting 1-2% of people above the ages of 65 years old. There is a worldwide prevalence of 7 to 10 million affected people of all cultures and race. Studies have shown that mutation that causes Parkinson’s disease result in increased kinase activity. The c.6055 G > A in exon 41 is the most prevalent LRRK2 variation which causes a substitution of glycine to serine in G2019S in the highly activated loop of its MAP kinase domain. The LRRK2 G2019S variant is the most common genetic determinant of Parkinson’s disease identified to date. This work focused on building accurate 3D models of the LRRK2 kinase domain, that were used for large-scale in silico docking against South African natural compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/). Molecular docking was performed to identify compounds that formed interactions with the active site of the protein and had the lowest binding energy scores. Molecular dynamics simulations showed different movements of the protein-ligand complexes and behavioural difference of the wildtype and the variants, all three structures proved to be compact. Network analysis was done to study residue interactions, contact maps, dynamic cross correlations, average BC and average L were used to study the residue interactions and general residue contribution to the functioning of the protein..
- Full Text:
- Date Issued: 2020
- Authors: Baye, Bertha Cinthia
- Date: 2020
- Subjects: Antiparkinsonian agents , Parkinson's disease -- Treatment , Protein kinases , Parkinson's disease -- Chemotherapy , Molecules -- Models
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/138764 , vital:37671
- Description: Parkinson’s disease is a type of movement disorder that occurs when nerve cells in the brain stop producing dopamine. It is the second neurodegenerative disease affecting 1-2% of people above the ages of 65 years old. There is a worldwide prevalence of 7 to 10 million affected people of all cultures and race. Studies have shown that mutation that causes Parkinson’s disease result in increased kinase activity. The c.6055 G > A in exon 41 is the most prevalent LRRK2 variation which causes a substitution of glycine to serine in G2019S in the highly activated loop of its MAP kinase domain. The LRRK2 G2019S variant is the most common genetic determinant of Parkinson’s disease identified to date. This work focused on building accurate 3D models of the LRRK2 kinase domain, that were used for large-scale in silico docking against South African natural compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/). Molecular docking was performed to identify compounds that formed interactions with the active site of the protein and had the lowest binding energy scores. Molecular dynamics simulations showed different movements of the protein-ligand complexes and behavioural difference of the wildtype and the variants, all three structures proved to be compact. Network analysis was done to study residue interactions, contact maps, dynamic cross correlations, average BC and average L were used to study the residue interactions and general residue contribution to the functioning of the protein..
- Full Text:
- Date Issued: 2020
Analysis of bacterial Mur amide ligase enzymes for the identification of inhibitory compounds by in silico methods
- Chamboko, Chiratidzo Respina
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
Unravelling the replication biology of Providence virus in a cell culturebased model system
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
- Date Issued: 2020
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
- Date Issued: 2020
Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
BODIPY dyes for application in the photo-oxidation of pollutants, photodynamic antimicrobial chemotherapy, and nonlinear optics
- Authors: Kelechi, Lebechi Augustus
- Date: 2020
- Subjects: Dyes and dyeing -- Chemistry , Fluorescent probes , Fluorescence spectroscopy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140298 , vital:37859
- Description: The synthesis and structural characterization of a series of BODIPY dyes to analyze both the effects of halogenations at the 2,6-positions and the introduction of styryl groups at the 3,5-positions. The photophysical properties of these dyes were investigated to determine their suitability as singlet oxygen-generating photosensitiser dyes for application in photocatalytic degradation of azo dyes and in photodynamic antimicrobial chemotherapy (PACT). Upon halogenation, the dyes showed high to moderate singlet oxygen quantum yields. The potential utility of electrospun polystyrene (PS) nanofibres embedded with halogenated BODIPY dyes for the photocatalytic degradation of Orange G and Methyl Orange from textile industry effluents were investigated. A comparison of the singlet oxygen quantum yield of the BODIPY dyes in solution and when embedded in the PS nanofibres support demonstrates that its photosensitiser properties are maintained in the nanofibre mats. The photocatalytic degradation properties of the PS nanofibres for Orange G and Methyl Orange were determined by using a 530 nm and 660 nm light-emitting diodes. The rate of photodegradation increases with both the Orange G and Methyl Orange concentrations and follows pseudo-first-order kinetics. The PACT activities of brominated BODIPYs on Escherichia coli and Staphylococcus aureus were investigated. Log reduction values of over 9 were obtained during the photoinactivation of Staphylococcus aureus. To be able to red-shift the main spectral band of the BODIPY dyes into the therapeutic window, styryl groups were introduced at the 3,5-positions through a modified Knoevenagel condensation reaction. Because the red-shifted spectral band lies above 532 nm, the second harmonic of the Nd:YAG laser, there is very minute absorption at this wavelength. One of the novel brominated BODIPY dyes was investigated for its potential utility as optical limiting materials in nonlinear optics (NLO), and the dyes demonstrated typical nonlinear absorption behaviour characterised by reverse saturable absorption (RSA) in Z-scan measurements. Excellent optical limiting parameters were obtained for third-order susceptibility and hyperpolarisability.
- Full Text:
- Date Issued: 2020
- Authors: Kelechi, Lebechi Augustus
- Date: 2020
- Subjects: Dyes and dyeing -- Chemistry , Fluorescent probes , Fluorescence spectroscopy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140298 , vital:37859
- Description: The synthesis and structural characterization of a series of BODIPY dyes to analyze both the effects of halogenations at the 2,6-positions and the introduction of styryl groups at the 3,5-positions. The photophysical properties of these dyes were investigated to determine their suitability as singlet oxygen-generating photosensitiser dyes for application in photocatalytic degradation of azo dyes and in photodynamic antimicrobial chemotherapy (PACT). Upon halogenation, the dyes showed high to moderate singlet oxygen quantum yields. The potential utility of electrospun polystyrene (PS) nanofibres embedded with halogenated BODIPY dyes for the photocatalytic degradation of Orange G and Methyl Orange from textile industry effluents were investigated. A comparison of the singlet oxygen quantum yield of the BODIPY dyes in solution and when embedded in the PS nanofibres support demonstrates that its photosensitiser properties are maintained in the nanofibre mats. The photocatalytic degradation properties of the PS nanofibres for Orange G and Methyl Orange were determined by using a 530 nm and 660 nm light-emitting diodes. The rate of photodegradation increases with both the Orange G and Methyl Orange concentrations and follows pseudo-first-order kinetics. The PACT activities of brominated BODIPYs on Escherichia coli and Staphylococcus aureus were investigated. Log reduction values of over 9 were obtained during the photoinactivation of Staphylococcus aureus. To be able to red-shift the main spectral band of the BODIPY dyes into the therapeutic window, styryl groups were introduced at the 3,5-positions through a modified Knoevenagel condensation reaction. Because the red-shifted spectral band lies above 532 nm, the second harmonic of the Nd:YAG laser, there is very minute absorption at this wavelength. One of the novel brominated BODIPY dyes was investigated for its potential utility as optical limiting materials in nonlinear optics (NLO), and the dyes demonstrated typical nonlinear absorption behaviour characterised by reverse saturable absorption (RSA) in Z-scan measurements. Excellent optical limiting parameters were obtained for third-order susceptibility and hyperpolarisability.
- Full Text:
- Date Issued: 2020
Understanding of the underlying resistance mechanism of the Kat-G protein against isoniazid in Mycobacterium tuberculosis using bioinformatics approaches
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Date Issued: 2020
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Date Issued: 2020
Functional characterization of the nuclear localisation and export signals of the human Hsp70/Hsp90 organising protein (HOP)
- Authors: Rousseau, Robert
- Date: 2019
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/97819 , vital:31489
- Description: Expected release date-April 2021
- Full Text: false
- Date Issued: 2019
- Authors: Rousseau, Robert
- Date: 2019
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/97819 , vital:31489
- Description: Expected release date-April 2021
- Full Text: false
- Date Issued: 2019
Identification of possible natural compounds as potential inhibitors against Plasmodium M1 alanyl aminopeptidase
- Soliman, Omar Samir Abdel Ghaffar
- Authors: Soliman, Omar Samir Abdel Ghaffar
- Date: 2019
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Aminopeptidases
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72284 , vital:30026
- Description: Malaria is a major tropical health problem with a 29% mortality rate among people of all ages; it also affects 35% of the children. Despite the decrease in mortality rate in recent years, malaria still results in around 2000 deaths per day. Malaria is caused by Plasmodium parasites and is transmitted to humans via the bites from infected female Anopheles mosquitoes during blood meals. There are five different Plasmodium species that can cause human malaria, which include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Among these five species, the most pathogenic ones are Plasmodium falciparum and Plasmodium vivax. Malaria is usually hard to diagnose because the symptoms are not exclusive to malaria and very similar to flu, e.g., fever, muscle pain, and chills, which lead to the misdiagnosis of malaria cases. Malaria is lethal if not treated because it can cause severe complications in the respiratory tract, liver, metabolic acidosis, and hypoglycemia. The malaria parasite life cycle includes two types of hosts, i.e., a human host and female Anopheles mosquito host. Malaria continuously develops resistance to the available drugs, which is one of the major challenges in disease control. This situation confirms the need to develop new drugs that target virulence factors of malaria. The malarial parasite has three main life cycle stages, which include the host liver stage, host blood stage and vector stage. In the blood stage, parasites degrade hemoglobin to amino acids, which is important as these parasites cannot produce their own amino acids. Different proteases are involved in this hemoglobin degradation process. M1 alanyl aminopeptidase is one of these proteases involved at the end of hemoglobin degradation. This study focused on M1 alanyl aminopeptidase as a potential drug target. M1 alanyl aminopeptidase consists of four domains: N-terminal domain, catalytic domain, middle domain and C-terminal domain. The catalytic domain remains conserved among different Plasmodium species. Inhibition of this enzyme might prevent Plasmodium growth as it can’t produce its own amino acids. In this study, sequence analysis was carried out in both human and Plasmodium M1 alanyl aminopeptidase to identify conserved and divergent regions between them. 3D protein models of the M1 alanyl aminopeptidase from Plasmodium species were built and validated. Then the generated models were used for virtual screening against 623 compounds retrieved from the South African Natural Compounds Database (SANCDB, https://sancdb.rubi.ru.ac.za/). Virtual screening was done using blind and targeted docking methods. Docking was used to identify compounds with selective high binding affinity to the active site of the parasite protein. In this study, one SANCDB compound was selected for each protein: SANC00531 was selected against P. falciparum M1 alanyl aminopeptidase, SANC00469 against P. knowlesi, SANC00660 against P. vivax, SANC00144 against P. ovale and SANC00109 against P. malariae. It was found that Plamsodium M1 alanyl aminopeptidase can be used as a potential drug target as it showed selective binding against different inhibitor compounds. This result will be investigated in future work though molecular dynamic analysis to investigate the stability of protein-ligand complexes.
- Full Text:
- Date Issued: 2019
- Authors: Soliman, Omar Samir Abdel Ghaffar
- Date: 2019
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Aminopeptidases
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/72284 , vital:30026
- Description: Malaria is a major tropical health problem with a 29% mortality rate among people of all ages; it also affects 35% of the children. Despite the decrease in mortality rate in recent years, malaria still results in around 2000 deaths per day. Malaria is caused by Plasmodium parasites and is transmitted to humans via the bites from infected female Anopheles mosquitoes during blood meals. There are five different Plasmodium species that can cause human malaria, which include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Among these five species, the most pathogenic ones are Plasmodium falciparum and Plasmodium vivax. Malaria is usually hard to diagnose because the symptoms are not exclusive to malaria and very similar to flu, e.g., fever, muscle pain, and chills, which lead to the misdiagnosis of malaria cases. Malaria is lethal if not treated because it can cause severe complications in the respiratory tract, liver, metabolic acidosis, and hypoglycemia. The malaria parasite life cycle includes two types of hosts, i.e., a human host and female Anopheles mosquito host. Malaria continuously develops resistance to the available drugs, which is one of the major challenges in disease control. This situation confirms the need to develop new drugs that target virulence factors of malaria. The malarial parasite has three main life cycle stages, which include the host liver stage, host blood stage and vector stage. In the blood stage, parasites degrade hemoglobin to amino acids, which is important as these parasites cannot produce their own amino acids. Different proteases are involved in this hemoglobin degradation process. M1 alanyl aminopeptidase is one of these proteases involved at the end of hemoglobin degradation. This study focused on M1 alanyl aminopeptidase as a potential drug target. M1 alanyl aminopeptidase consists of four domains: N-terminal domain, catalytic domain, middle domain and C-terminal domain. The catalytic domain remains conserved among different Plasmodium species. Inhibition of this enzyme might prevent Plasmodium growth as it can’t produce its own amino acids. In this study, sequence analysis was carried out in both human and Plasmodium M1 alanyl aminopeptidase to identify conserved and divergent regions between them. 3D protein models of the M1 alanyl aminopeptidase from Plasmodium species were built and validated. Then the generated models were used for virtual screening against 623 compounds retrieved from the South African Natural Compounds Database (SANCDB, https://sancdb.rubi.ru.ac.za/). Virtual screening was done using blind and targeted docking methods. Docking was used to identify compounds with selective high binding affinity to the active site of the parasite protein. In this study, one SANCDB compound was selected for each protein: SANC00531 was selected against P. falciparum M1 alanyl aminopeptidase, SANC00469 against P. knowlesi, SANC00660 against P. vivax, SANC00144 against P. ovale and SANC00109 against P. malariae. It was found that Plamsodium M1 alanyl aminopeptidase can be used as a potential drug target as it showed selective binding against different inhibitor compounds. This result will be investigated in future work though molecular dynamic analysis to investigate the stability of protein-ligand complexes.
- Full Text:
- Date Issued: 2019
Genetic characterisation of a range of geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) isolates and evaluation of biological activity against South African populations of the African bollworm, Helicoverpa armigera (Hu bner) (Lepidoptera: Noctuidae)
- Mtambanengwe, Kudzai Tapiwanashe Esau
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
- Full Text:
- Date Issued: 2019
- Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
- Date: 2019
- Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/97334 , vital:31426
- Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
- Full Text:
- Date Issued: 2019
An investigation into the interaction partners of the scaffold protein human CNK1 in the NF-κB pathway
- Authors: Moodley, Holisha
- Date: 2019
- Subjects: CNK1 , Scaffold proteins
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96031 , vital:31228
- Description: The protein connector enhancer of KSR1 (CNK1) plays a role in a number of signalling pathways including those involved in cell proliferation, cell growth and differentiation. De-regulation of these pathways has been linked to the promotion of oncogenic signalling. The involvement of CNK1 in all of these diverse pathways indicates a need to better understand the role of this protein within the cell and within key signalling networks. The research provides a platform to understand the intricate relationships that occur between these key signalling networks with the potential to identify new drug targets. CNK1 is multifunctional scaffolding protein that has binding domains that mediate and co-ordinate signalling within the MAPK, Hippo, PI3K/AKT, JNK and NF-κB pathways as well as downstream of the AT2 receptor. The activity of CNK1 is regulated through its interactions with a range of different binding partners within these pathways. Of particular interest to this research is the role of CNK1 in NF-κB signalling. The deregulation of the NF-κB pathway is implicated in chronic inflammation, tissue damage and induction of cervical and breast cancer. CNK1 has been reported to regulate the non-canonical branch of the NF-κB pathway, upstream of the IKK complex however new findings lead to uncertainty about these conclusions. In addition, the interacting partner of CNK1 in the NF-κB pathway has not been elucidated. In this thesis, we aim to identify the binding partners of CNK1 in the NF-κB pathway. First, we validate an epitope-tagged CNK1-expression construct to express elevated levels of CNK1 in cervical cancer cells. We report that the expression of myc-CNK1 is comparable to endogenous CNK1. Cells expressing elevated CNK1 levels were used in traditional co-immunoprecipitation reactions to identify potential CNK1-interacting proteins. We present data that indicates a potential role for NIK in the CNK1 signalling complex. We discuss the weaknesses of the traditional co-immunoprecipitation reactions and design an alternative co-immunoprecipitation technique with which to study CNK1-interacting partners. In this system, a promiscuous biotin ligase fused to the protein sequence for CNK1 (BirA-CNK1) is used to label proteins proximal to CNK1 with biotin. Using this BirA- CNK1-expressing construct in cervical cancer cells, we demonstrate that CNK1 interacts with IKKα-IKKβ in the NF-κB pathway.
- Full Text:
- Date Issued: 2019
- Authors: Moodley, Holisha
- Date: 2019
- Subjects: CNK1 , Scaffold proteins
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96031 , vital:31228
- Description: The protein connector enhancer of KSR1 (CNK1) plays a role in a number of signalling pathways including those involved in cell proliferation, cell growth and differentiation. De-regulation of these pathways has been linked to the promotion of oncogenic signalling. The involvement of CNK1 in all of these diverse pathways indicates a need to better understand the role of this protein within the cell and within key signalling networks. The research provides a platform to understand the intricate relationships that occur between these key signalling networks with the potential to identify new drug targets. CNK1 is multifunctional scaffolding protein that has binding domains that mediate and co-ordinate signalling within the MAPK, Hippo, PI3K/AKT, JNK and NF-κB pathways as well as downstream of the AT2 receptor. The activity of CNK1 is regulated through its interactions with a range of different binding partners within these pathways. Of particular interest to this research is the role of CNK1 in NF-κB signalling. The deregulation of the NF-κB pathway is implicated in chronic inflammation, tissue damage and induction of cervical and breast cancer. CNK1 has been reported to regulate the non-canonical branch of the NF-κB pathway, upstream of the IKK complex however new findings lead to uncertainty about these conclusions. In addition, the interacting partner of CNK1 in the NF-κB pathway has not been elucidated. In this thesis, we aim to identify the binding partners of CNK1 in the NF-κB pathway. First, we validate an epitope-tagged CNK1-expression construct to express elevated levels of CNK1 in cervical cancer cells. We report that the expression of myc-CNK1 is comparable to endogenous CNK1. Cells expressing elevated CNK1 levels were used in traditional co-immunoprecipitation reactions to identify potential CNK1-interacting proteins. We present data that indicates a potential role for NIK in the CNK1 signalling complex. We discuss the weaknesses of the traditional co-immunoprecipitation reactions and design an alternative co-immunoprecipitation technique with which to study CNK1-interacting partners. In this system, a promiscuous biotin ligase fused to the protein sequence for CNK1 (BirA-CNK1) is used to label proteins proximal to CNK1 with biotin. Using this BirA- CNK1-expressing construct in cervical cancer cells, we demonstrate that CNK1 interacts with IKKα-IKKβ in the NF-κB pathway.
- Full Text:
- Date Issued: 2019
Investigating the role of Hsp90 and LRP1 in FN matrix dynamics
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
Recombinant expression, purification and in vitro interaction analysis of HOP and RhoC
- Vaaltyn, Michaelone Chantelle
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
Synthesis of silver nanoparticles and their role against a thiazolekinase enzyme from Plasmodium falciparum
- Yao, Jia
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
Analysis of the interaction of Hsp90 with the extracellular matrix protein fibronectin (FN)
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014
Screening of entomopathogenic fungi against citrus mealybug (Planococcus citri (Risso)) and citrus thrips (Scirtothrips aurantii (Faure))
- FitzGerald, Véronique Chartier
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Date Issued: 2014
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Date Issued: 2014
The large scale bioinformatics analysis of auxiliary activity family 9 enzymes
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
Analysis of transcription factor binding specificity using ChIP-seq data.
- Authors: Kibet, Caleb Kipkurui
- Date: 2014
- Subjects: Transcription factors , Chronic myeloid leukemia , Antioncogenes , Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4115 , http://hdl.handle.net/10962/d1013131
- Description: Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.
- Full Text:
- Date Issued: 2014
- Authors: Kibet, Caleb Kipkurui
- Date: 2014
- Subjects: Transcription factors , Chronic myeloid leukemia , Antioncogenes , Cancer cells -- Growth -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4115 , http://hdl.handle.net/10962/d1013131
- Description: Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.
- Full Text:
- Date Issued: 2014
Understanding the replication biology of Providence virus: elucidating the function of non-structural proteins
- Authors: Nakayinga, Ritah
- Date: 2014
- Subjects: Insects Viruses , Viruses Reproduction , Tombusviridae , RNA viruses , RNA polymerases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/193930 , vital:45408
- Description: Tetraviruses are non-enveloped, small insect RNA viruses with a single stranded positive RNA genome that is either monopartite or bipartite. Providence virus (PrV) is the only member of the three tetravirus families with a viral replicase similar to the replicases of tombusviruses and umbraviruses. The principle aim of this thesis was to study PrV replication, focusing on subcellular localization and potential interactions between PrV replication proteins. The first objective of this study was to generate an anti-p104 antibody that does not cross-react with p40. Expression of the C-terminal portion of p104 in E. coli resulted in no detectable protein. Further expression in an insect cell based expression system resulted in the production of an insoluble protein. Attempts to improve protein solubility with a range of solubilization treatments were unsuccessful. Bioinformatic analysis was used to detect an antigenic region at the C-terminus of p104 and the peptide was used to raise anti-p104 antibodies. These antibodies did not detect native protein by western blot detection however they were used for immunoprecipitation. The establishment of the subcellular localization of PrV required two approaches; immunofluorescence in persistently infected Helicoverpa zea MG8 cells using antip40 and anti-dsRNA antibodies and the expression of EGFP-replicase fusion protein in Spodoptera frugiperda Sf9 cells. Replication of PrV was found to take place in cytosolic punctate structures. Co-immunoprecipitation experiments revealed that p40 self-interacts and interacts with p104. Bioinformatic analysis of PrV p104 suggests that the RdRp is similar to viral RdRps of the carmo-like supergroup II. Potential RNA binding regions are present within p104. A potential p40 interaction domain that shares hydrophilic and surface exposed properties with the TBSV p33 interaction domain is present. A putative arginine-rich region and disordered C-terminal region is present in p130. In conclusion, PrV p104 is the viral replicase. The resemblance of the expression strategy and putative functional domains with tombusviruses and umbraviruses suggest that PrV replication is related to the replication system of the tombusviruses and umbraviruses. This has led to propose that tetravirus replication strategies are diverse and raises questions on the origin and evolution of PrV. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
- Authors: Nakayinga, Ritah
- Date: 2014
- Subjects: Insects Viruses , Viruses Reproduction , Tombusviridae , RNA viruses , RNA polymerases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/193930 , vital:45408
- Description: Tetraviruses are non-enveloped, small insect RNA viruses with a single stranded positive RNA genome that is either monopartite or bipartite. Providence virus (PrV) is the only member of the three tetravirus families with a viral replicase similar to the replicases of tombusviruses and umbraviruses. The principle aim of this thesis was to study PrV replication, focusing on subcellular localization and potential interactions between PrV replication proteins. The first objective of this study was to generate an anti-p104 antibody that does not cross-react with p40. Expression of the C-terminal portion of p104 in E. coli resulted in no detectable protein. Further expression in an insect cell based expression system resulted in the production of an insoluble protein. Attempts to improve protein solubility with a range of solubilization treatments were unsuccessful. Bioinformatic analysis was used to detect an antigenic region at the C-terminus of p104 and the peptide was used to raise anti-p104 antibodies. These antibodies did not detect native protein by western blot detection however they were used for immunoprecipitation. The establishment of the subcellular localization of PrV required two approaches; immunofluorescence in persistently infected Helicoverpa zea MG8 cells using antip40 and anti-dsRNA antibodies and the expression of EGFP-replicase fusion protein in Spodoptera frugiperda Sf9 cells. Replication of PrV was found to take place in cytosolic punctate structures. Co-immunoprecipitation experiments revealed that p40 self-interacts and interacts with p104. Bioinformatic analysis of PrV p104 suggests that the RdRp is similar to viral RdRps of the carmo-like supergroup II. Potential RNA binding regions are present within p104. A potential p40 interaction domain that shares hydrophilic and surface exposed properties with the TBSV p33 interaction domain is present. A putative arginine-rich region and disordered C-terminal region is present in p130. In conclusion, PrV p104 is the viral replicase. The resemblance of the expression strategy and putative functional domains with tombusviruses and umbraviruses suggest that PrV replication is related to the replication system of the tombusviruses and umbraviruses. This has led to propose that tetravirus replication strategies are diverse and raises questions on the origin and evolution of PrV. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
Influence of non-synonymous sequence mutations on the architecture of HIV-1 clade C protease receptor site : docking and molecular dynamics studies
- Authors: Onywera, David Harris
- Date: 2014
- Subjects: HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , HIV infections -- Chemotherapy , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research , Antiretroviral agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4116 , http://hdl.handle.net/10962/d1013133
- Description: Despite the current interventions to avert contagions and AIDS-related deaths, sub-Saharan Africa is still the region most severely affected by the HIV/AIDS pandemic, where clade C is the dominant circulating HIV-1 strain. The pol-encoded HIV-1 protease enzyme has been extensively exploited as a drug target. Protease inhibitors have been engineered within the framework of clade B, the commonest in America, Europe and Australia. Recent studies have attested the existence of sequence and catalytic disparities between clades B and C proteases that could upset drug susceptibilities. Emergence of drug-resistant associated mutations and combinatorial explosions due to recombination thwarts the attempt to stabilize the current highly active antiretroviral therapy (HAART) baseline. The project aimed at identifying the structural and molecular mechanisms hired by mutants to affect the efficacies of both FDA approved and Rhodes University (RU)-synthesized inhibitors, in order to define how current and or future drugs ought to be modified or synthesized with the intent of combating drug resistance. The rationale involved the generation of homology models of the HIV-1 sequences from the South African infants failing treatment with two protease inhibitors: lopinavir and ritonavir (as monitored by alterations in surrogate markers: CD4 cell count decline and viral load upsurge). Consistent with previous studies, we established nine polymorphisms: 12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, and 93L, linked to subtype C wild-type; some of which are associated with protease treatment in clade B. Even though we predicted two occurrence patterns of M46I, I54V and V82A mutations as V82A→I54V→M46I and I54V→V82A→M46V, other possibilities might exist. Mutations either caused a protracted or contracted active site cleft, which enforced differential drug responses. The in silico docking indicated susceptibility discordances between clades B and C in certain polymorphisms and non-polymorphisms. The RU-synthesized ligands displayed varied efficacies that were below those of the FDA approved protease inhibitors. The flaps underwent a wide range of structural motions to accommodate and stabilize the ligands. Computational analyses unravelled the need for these potential drugs to be restructured by (de novo) drug engineers to improve their binding fits, affinities, energies and interactions with multiple key protease residues in order to target resilient HIV-1 assemblages. Accumulating evidences on contrasting drug-choice interpretations from the Stanford HIVdb should act as an impetus for the customization of a HIVdb for the sub-Saharan subcontinent.
- Full Text:
- Date Issued: 2014
- Authors: Onywera, David Harris
- Date: 2014
- Subjects: HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , HIV infections -- Chemotherapy , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research , Antiretroviral agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4116 , http://hdl.handle.net/10962/d1013133
- Description: Despite the current interventions to avert contagions and AIDS-related deaths, sub-Saharan Africa is still the region most severely affected by the HIV/AIDS pandemic, where clade C is the dominant circulating HIV-1 strain. The pol-encoded HIV-1 protease enzyme has been extensively exploited as a drug target. Protease inhibitors have been engineered within the framework of clade B, the commonest in America, Europe and Australia. Recent studies have attested the existence of sequence and catalytic disparities between clades B and C proteases that could upset drug susceptibilities. Emergence of drug-resistant associated mutations and combinatorial explosions due to recombination thwarts the attempt to stabilize the current highly active antiretroviral therapy (HAART) baseline. The project aimed at identifying the structural and molecular mechanisms hired by mutants to affect the efficacies of both FDA approved and Rhodes University (RU)-synthesized inhibitors, in order to define how current and or future drugs ought to be modified or synthesized with the intent of combating drug resistance. The rationale involved the generation of homology models of the HIV-1 sequences from the South African infants failing treatment with two protease inhibitors: lopinavir and ritonavir (as monitored by alterations in surrogate markers: CD4 cell count decline and viral load upsurge). Consistent with previous studies, we established nine polymorphisms: 12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, and 93L, linked to subtype C wild-type; some of which are associated with protease treatment in clade B. Even though we predicted two occurrence patterns of M46I, I54V and V82A mutations as V82A→I54V→M46I and I54V→V82A→M46V, other possibilities might exist. Mutations either caused a protracted or contracted active site cleft, which enforced differential drug responses. The in silico docking indicated susceptibility discordances between clades B and C in certain polymorphisms and non-polymorphisms. The RU-synthesized ligands displayed varied efficacies that were below those of the FDA approved protease inhibitors. The flaps underwent a wide range of structural motions to accommodate and stabilize the ligands. Computational analyses unravelled the need for these potential drugs to be restructured by (de novo) drug engineers to improve their binding fits, affinities, energies and interactions with multiple key protease residues in order to target resilient HIV-1 assemblages. Accumulating evidences on contrasting drug-choice interpretations from the Stanford HIVdb should act as an impetus for the customization of a HIVdb for the sub-Saharan subcontinent.
- Full Text:
- Date Issued: 2014