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
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
The effects of extracellular and intracellular Hop on cell migration processes
- Authors: Contu, Lara
- Date: 2014
- Subjects: Heat shock proteins , Metastasis , Cancer Chemotherapy , Molecular chaperones , Cell migration
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193961 , vital:45410
- Description: The Hsp70/Hsp90-organising protein (Hop) is a 60 kDa co-chaperone that acts as an adaptor molecule, facilitating the transfer of client proteins between the Hsp70 and Hsp90 chaperone systems. Hop functions both intracellularly and extracellularly and has been implicated in many processes involved in cancer progression, including cell migration and invasion. Little is known about the mechanisms or domains by which extracellular Hop functions. In addition, little is known about the effects of Hop on signalling molecules involved in cell migration and invasion through regulation of actin dynamics. It was hypothesised that both extracellular and intracellular pools of Hop would regulate distinct cell migration processes by activation of cell signalling pathways or direct interactions with signalling intermediates. HS578T cells were treated with recombinant full length and truncated murine Hop proteins (overexpressed and purified in this study) to determine the effects of extracellular Hop and the independent domains on cell migration processes. Additionally, RNA interference (RNAi) techniques were used to determine the effect of Hop knockdown on cell migration related signalling intermediates and cell morphologies. A short hairpin RNA (shRNA) system for the stable knockdown of Hop was developed and used for a number of these studies. Treatment of HS578T cells with the TPR2A2B and TPR1 domains of Hop resulted in a significant decrease in cell migration and caused changes in the actin cytoskeleton and extracellular matrix proteins, gelatin and fibronectin. RhoC immunoprecipitated in a common complex with Hop and Hsp90. Hop knockdown reduced levels of actin and total RhoC, as well as active RhoC. In addition, knockdown of Hop resulted in a reduced migratory phenotype. We interpreted these data to indicate that intracellular Hop played a role in cell migration through regulation of RhoC activity, either through a direct interaction between Hop and RhoC, or an indirect interaction of RhoC with the Hsp90 multichaperone heterocomplex. Taken together, the data suggested that extracellular and intracellular Hop played distinct roles in extracellular and intracellular processes that lead to actin dynamics and cell migration. Understanding the mechanistic role of Hop in these processes is essential as it would aid in assessing the viability of Hop as a potential drug target for the treatment of metastatic cancers. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2014
- Full Text:
- Date Issued: 2014
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
The involvement of TRAP1 in the mitochondrial localization of STAT3 in mammalian cells
- Authors: Kadye, Rose
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55760 , vital:26731
- Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
- Full Text:
- Date Issued: 2014
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
An investigation of the role of mitochondrial STAT3 and modulation of Reactive Oxygen Species in adipocyte differentiation
- Authors: Kramer, Adam Hildyard
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54632 , vital:26595
- Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
- Full Text:
- Date Issued: 2014
Bacterial degradation of waste coal
- Authors: Madikiza, Lwazikazi
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54576 , vital:26590
- Description: As an energy source coal has one of the largest agglomerations in the world. Consequently mining of coal creates large volumes of waste in the form of low ranks coals. The complex structure of coal makes it difficult for the microorganisms to degrade and relatively few bacteria and fungi have been shown to break down coal. This study aimed to investigate bacteria not previously known to degrade coal. In this study bacteria were isolated from hydrocarbon contaminated sites and inoculated in coal medium where coal served as the only carbon source. Three strains produced a yellow – brown supernatant after 14 d of incubation at 30 °C. Bacteria generating a yellow – brown coloured supernatant were presumed to possess coal degrading capabilities and the best performing of these bacterial species was identified using 16s rDNA as Bacillus flexus. Scanning electron microscopy showed that there was a close association between the bacterium and substrate coal. The close association of bacteria to substrate suggested that these organisms were able to maximize solubilisation. FT-IR spectroscopic analysis demonstrated the addition of single bonded compounds COOH, OH, CN and CH that were absent prior to bacterial interaction. The increase in oxygen rich regions indicated degradation of the coal substrate. Elemental analysis showed that there was a decrease in carbon content from 47 % to 24 % during the 14 day incubation period. Reduction in coal carbon content was assumed to be due to bacterial utilization for metabolism and growth particularly as untreated coal substrate showed minimal loss of carbon. Analysis of the residual culture medium revealed that there was a linear increase in humic-like substance concentration for 8 d, coincident with increased coal biosolubilisation and colour change. Laccase activity was insignificant, and at 13 d enzyme activity was only 5×10-3 U/L suggesting that B. flexus may use a different mechanism to degrade coal. Residual culture medium remaining after bacterial action on the coal substrate appeared to possess plant growth promoting activity. This soluble biodegradation product with characteristics similar to humic acid-like substances was shown to impact growth of radish cotyledons. Expansion of isolated radish cotyledons was enhanced by 140% when incubated in coal biodegradation product. In conclusion, this study has yielded B. flexus and two other unidentified bacteria, isolated from polyaromatic hydrocarbon contaminated soils, and demonstrated the ability of these microorganisms to degrade waste coal. Further studies to elucidate the mechanism of coal breakdown by B. flexus, synergies with other coal degrading microorganisms, and incorporation of bacterium into Fungcoal bioprocess technology is imminent.
- Full Text:
- Date Issued: 2014
An investigation into the biological treatment of platinum refinery effluent using the plant Azolla Filiculoides
- Authors: Marran, Vernon Edward
- Date: 2003
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193453 , vital:45333
- Description: In order to understand the effects of metals contained in effluent and to define effluent quality suitable for safe discharge to natural water streams, it is essential to understand the effects of the interaction of metal ions with plants. The availability of metal ions and their ability to bind to plants are dependent on the chemical speciation of metals and on the biological factors governing the availability of metals within the plant cells. This thesis will address both aspects and thereby propose a combination of an appropriate chemical and biological approach to the investigation of bioaccumulation of the plant Azolla Filiculoides. Laboratory studies have shown that varying concentrations of free metal ions in solution determine efficiency of metal uptake and that metal toxicity can also be detrimental to plant life and efficiency of metal recovery from solution. Many questions however, remain unanswered with regard to the application of a biological treatment for effluent discharge. This thesis includes the determination of metal speciation combined with the study of bioaccumulation of metals in plants and their effects from test- work utilising effluent generated from a Precious Metals Refinery (PMR). Plant species are known to differ widely in their tolerance to metals, however despite an abundant knowledge on molecular, biochemical and physiological effects of metals to plants, only a few general principles have been proposed to guide the prediction of tolerance differences. The properties of protective cellular responses as well as of the molecular target sites are important components in determining the intrinsic tolerance of a particular species to a metal. The role of the whole assembly of cellular ligands in buffering metal ions within the cells will be evaluated. Standard preparation methods combined with use of Inductively Coupled Plasma Emission Spectrophotometer (1CP) used for analytical analysis will be included to reflect analytical data in providing evidence to support a conclusion. The outcome of the test work utilising the aquatic plant Azoila has proven that it can be used as a process step to re-mediate effluent generated from Precious Metal Refining operations. This process offers an alternative to the classical chemical methods widely used in the Precious Metals Refining industry proving economically viable and ensuring environmental sustainability in comparison to the current known methods of effluent treatment. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2003
- Full Text:
- Date Issued: 2003
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
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
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)
- 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
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
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- Date Issued: 2014
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
Reactor development and process optimisation for the bioremediation of phenolic wastewaters by trametes species
- Authors: Ryan, Daniel Reginald
- Date: 2004-04
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191487 , vital:45103
- Description: In many service industries, the source of a company’s value has shifted from capital to knowledge and ideas, the quality of which is dependent on its employees (Wooldridge, 2006). In fact, human resources can be considered part of factor conditions which can positively impact on a firm’s competitive context. This impact can ultimately translate into improved financial results (Porter and Kramer, 2002). There is therefore a growing interest in ways to attract and retain talent. According to the managers of many big companies, well communicated corporate responsibility practices can improve staff attraction as well as retention rates by improving morale (CSRwire, 2002). To explore this, a small, creative company in Johannesburg which engages in charity work was selected as a case study, with the goal being to understand whether their culture of good deeds has a positive impact on staff wellbeing. While the owner of the company actively attempts to make the company an enjoyable place to work at, he appears to have initiated the philanthropic activities in a true spirit of giving, rather than with the motive of engaging staff in order to make more money. Nevertheless, the researcher’s investigative stance is that of an enlightened egoist, and the study focuses on the business case of giving being beneficial to the giver (ultimately the company) in the long term, as well as to the recipient. While the danger of suggesting that philanthropy could be instrumentalised is acknowledged (Morton, 2004), the investigation explores the possibility because such evidence could persuade other companies to become more socially concerned. Through a qualitative approach involving interviews, observation and analysis of video footage, it becomes apparent that there is clearly value for the staff in the charity work they do. Unfortunately the multiple initiatives undertaken to keep staff morale high at the company make it impossible to establish a clear link between the philanthropy and overall wellbeing, but as the study was conducted in the phenomenological paradigm the main concern was with understanding the experience of participants. However, an unexpected finding was that the employees derive great satisfaction from using their professional skills for charity work rather than just donating money to the charity. They feel that their skills uniquely position them to make significant changes to the lives of others, which gives them a sense of pride and achievement that they don’t necessarily experience in their ordinary activities at work. On the basis of this, it is recommended that companies look to involve staff with projects that require their specific expertise when evaluating philanthropic initiatives. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2004
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- Date Issued: 2004-04
An investigation into the biological treatment of platinum refinery effluent
- Authors: Smith, Roland Paul
- Date: 200u
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193464 , vital:45334
- Description: This Review and project will discuss and demonstrate the use made of Biotechnology in the production and reduction of metals. It will look at how and why metal binding takes place, known platinum group metal speciation will be included. Examples of how to improve metal binding efficiency will be discussed by stimulating ligand activity by polarisation. Various biotechnical options available, with emphasis placed on the use of the aquatic fern and algae will be given as examples of biological treatment of heavy metals in particular the aquatic fern Azolla. The method of standard preparation and the use of Inductively Coupled Plasma Emission Spectrophotometer (ICP) used for analytical analysis will be included so that consideration can be given to the collection of analytical data in the provision of evidence to support or provide a conclusion. The outcome of the test work utilising the aquatic plant Azolla has proven that it can be used to remediate platinum refinery effluent. This process can offer an alternative to the classical chemical method normally used, which is economically viable and environmentally friendly in comparison to the common methods of refinery effluent treatment. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 200u
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- Date Issued: 200u
Identification of possible natural compounds as potential inhibitors against Plasmodium M1 alanyl aminopeptidase
- 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.
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- Date Issued: 2019
Recombinant expression, purification and in vitro interaction analysis of HOP and RhoC
- 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
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- Date Issued: 2016