Generation of polyclonal antibodies against Theiler's Murine Encephalomyelitis virus protein 2C, and their use in investigating localisation of the protein in infected cells
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
Regulation of hyu gene expression in Agrobacterium tumefaciens strains RU-AE01 and RU-OR
- Authors: Jiwaji, Meesbah
- Date: 2007
- Subjects: Agrobacterium tumefaciens Amino acids Gene expression Hydrolysis Hydantoin Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3983 , http://hdl.handle.net/10962/d1004042
- Description: Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.
- Full Text:
- Date Issued: 2007
Co-utilisation of microalgae for wastewater treatment and the production of animal feed supplements
- Authors: Johnson, Hailey E
- Date: 2011
- Subjects: Microalgae -- Biotechnology , Algae culture , Algae products , Waste products as feed , Sewage -- Purification , Organic wastes -- Recycling , Food industry and trade -- Waste disposal , Agriculture -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3940 , http://hdl.handle.net/10962/d1003999 , Microalgae -- Biotechnology , Algae culture , Algae products , Waste products as feed , Sewage -- Purification , Organic wastes -- Recycling , Food industry and trade -- Waste disposal , Agriculture -- Waste disposal
- Description: Microalgae have a variety of commercial applications, the oldest of which include utilisation as a food source and for use in wastewater treatment. These applications, however, are seldom combined due to toxicity concerns, for ethical reasons, and generally the requirement for cultivation of a single algae species for use as a feed supplement. These problems might be negated if a “safer” wastewater such as that from agricultural and/or commercial food production facilities were to be utilised and if a stable algae population can be maintained. In this investigation preliminary studies were carried out using an Integrated Algae Pond System (IAPS) for domestic wastewater treatment to determine the species composition in the associated High Rate Algae Ponds (HRAPs). The effect of different modes of operation, continuous versus batch, on nutrient removal, productivity and species composition was also investigated. Furthermore, indigenous species in the HRAP were isolated and molecularly identified as, Chlorella, Micractinium, Scenedesmus and Pediastrum. Additionally, the effect of the nor amino acid, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA) and its Cu-chelated derivative, on the growth and biochemical composition of Chlorella, Micractinium, Scenedesmus, Pediastrum and Spirulina was investigated. Species composition in the HRAP was stable under continuous operation with Micractinium dominating > 90% of the algae population. Under batch operation the population dynamic shifted; Chlorella outcompeted Micractinium possibly due to nutrient depletion and selective grazing pressures caused by proliferation of Daphnia. Higher species diversity was observed during batch mode as slower growing algae were able to establish in the HRAP. Nutrient removal efficiency and biomass productivity was higher in continuous mode, however lower nutrient levels were obtained in batch operation. HMTBA did not significantly affect growth rate, however treatment with 10 mg.L-1 resulted in slightly increased growth rate in Micractinium and increased final biomass concentrations in Chlorella, Micractinium and Spirulina (although this was not statistically significant for Micractinium and Spirulina), which are known mixotrophic species. Algae treated with Cu-HMTBA, showed reduced final biomass concentration with 10 mg.L-1, caused by Cu toxicity. Biochemical composition of the algae was species-specific and differed through the growth cycle, with high protein observed during early growth and high carbohydrate during late growth/early stationary phase. Additionally, 0.1 mg.L-1 HMTBA and Cu-HMTBA significantly reduced protein content in Chlorella, Micractinium, Scenedesmus and Pediastrum. In conclusion, operation of the HRAP in continuous culture provided suitable wastewater treatment with high productivity of an ideal species, Micractinium, for use in animal feed supplementation. This species had 40% protein content during growth (higher than the other species tested) and dominated the HRAP at > 90% of the algae population during continuous mode. Addition of HMTBA (> 1 mg.L-1) to algae cultivation systems and those treating wastewater, has the potential to improve productivity and the value of the biomass by enhancing protein content. Overall, the co-utilisation of microalgae for wastewater treatment and the generation of a biomass rich in protein, for incorporation into formulated animal feed supplements, represents a closed ecosystem which conserves nutrients and regenerates a most valuable resource, water.
- Full Text:
- Date Issued: 2011
Isolation of xylanolytic multi-enzyme complexes from Bacillus subtilis SJ01
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
Isolation and characterization of a novel thermostable and catalytically efficient laccase from Peniophora sp. strain UD4
- Authors: Jordaan, Justin
- Date: 2005
- Subjects: Enzymes Enzymes -- Industrial applications Peniophora Laccase
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3970 , http://hdl.handle.net/10962/d1004029
- Description: Enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as chirally selective biocatalysis. The ligninolytic enzymes have recently received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. This group of enzymes was therefore identified as the target group for this study. Improved enzyme properties are constantly being sought to enhance the range of applications for enzymes. Biodiversity provides a wide variety of enzymes. Several researchers have concentrated on extremophiles as their primary source of superior enzymes, consequently neglecting temperate environments in their search for these enzymes. The relatively neglected fungal biodiversity of South Africa provided an opportunity to test the hypothesis that potentially important industrial enzymes with unusual properties could be isolated from mesophilic basidiomycetous fungi. Subsequent screening of Eastern Cape biodiversity for thermostable ligninolytic enzymes from basidiomycetes resulted in the isolation of a novel laccase enzyme from a basidiomycetous species. This fungus was identified as Peniophora sp. UD4 by phylogenetic analysis of rDNA ITS sequences. Initial studies indicated a superior optimum temperature of 70°C and thermostability, indicated by no loss in activity at 60°C over nine hours. Further characterization of the laccase revealed a broader than usual substrate range through its unusual ability to oxidatively couple DMAB and MBTH. The laccase also exhibited a broad pH oxidation range for ABTS (pH 2 – 6.8), and a relatively high affinity (K_m_ = 0.0123 mM) and catalytic efficiency (63 252 mM^(-1)^s^(-1)^) for ABTS as a substrate. The laccase activity from Peniophora sp. UD4 was shown to be comprised of three isozymes with a molecular weight of 62 kDa and pI’s of 6.33, 6.45 and 6.50. Investigation of the nutrient and physical factors affecting ligninolytic enzyme production and growth of Peniophora sp. UD4 indicated that the wild-type organism was unsuitable for large scale production of the thermostable laccase due to the low levels of laccase production. The thermostable laccase was applied to defouling of ultrafiltration membranes, bioremediation of industrial waste streams, biocatalysis, and biosensor technology as potential applications. Application of the Peniophora sp. UD4 laccase to defouling of membranes used for ultrafiltration of brown water showed large flux recoveries of 31, 21 and 21% after the first three defouling recycles respectively, compared to 3% for the control without immobilized enzyme. The novel laccase showed potential for the bioremediation of industrial waste streams, the most successful being that of bleach plant effluent, where a reduction of 66% of the phenolic load was achieved. Application of the novel laccase to biocatalytic oxidation of ferulic acid and (±)-α-pinene showed higher product yield as compared to oxidation of these compounds by Trametes versicolor laccase in mediated and non-mediated systems. The major products of (±)-α-pinene oxidation were identified as verbenol and trans-sorberol. The Peniophora sp. UD4 laccase was successfully applied to biosensor technology, which benchmarked significantly better than Trametes versicolor laccase for the detection of 4-chlorophenol. The biosensor developed with laccase from UD4 by covalent binding to a glassy carbon electrode exhibited the best combination of sensitivity and stability. This thesis shows that a laccase with superior properties was obtained from a mesophilic South African basidiomycete. The catalytic properties displayed by the novel laccase from Peniophora sp. UD4 all contribute to the increased industrial applicability of laccases, and may be the most industrially feasible enzyme of its class isolated to date.
- Full Text:
- Date Issued: 2005
A study of carbonate-rich brines from Sua Pan to characterize organic contaminants in the soda ash process
- Authors: Joseph, Manjusha
- Date: 2001
- Subjects: Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4031 , http://hdl.handle.net/10962/d1004091 , Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Description: Botswana Ash (Pty) Ltd which is situated in Sua Pan, north east Bostwana, is one of Africa's largest suppliers of salt and soda ash. For a number of years, the company has been experiencing problems which have resulted in the final soda ash product being contaminated and discoloured. The problems experienced at Sua Pan have been reported also to occur in other salt works all over the world. It has been suggested that contamination in many salt works could be possibly be due to the microbial activity by halophilic algae and bacteria that grow in the solar ponds. This study was undertaken to investigate the nature of the contaminating organic compounds present in the brine, to identify the compounds, and to establish how these components vary during the various stages of the soda ash processing. For this study, two sets of brine samples were used; the first set was collected before the summer rains and the second set was collected after the summer rains. Solid bicarbonate and soda ash samples were also used. Extractions, desalting, UV and HPLC analysis and oxidative biotransformations using four enzymes, were used for developing profiles and characterizing the brine components. From these studies, we were able to confirm that the components of the brine are organic in nature. A thorough study of one of the compounds isolated,from solid bicarbonate and soda ash was conducted using UV, HPLC, IR, NMR, HPLC-MS, GC-MS and TLC. The results of these analyses, show that the. isolated compound was benzyl butyl phthalate which is generally regarded to be humic in nature. This compound was found to be present in all the brine samples collected after the summer rains including the well brine, suggesting this compound occurs naturally and is not formed during the processing.
- Full Text:
- Date Issued: 2001
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
Falcipains as malarial drug targets
- Authors: Kanzi, Aquillah Mumo
- Date: 2013
- Subjects: Malaria Malaria -- Chemotherapy Plasmodium falciparum Antimalarials -- Development Cysteine proteinases Cysteine proteinases -- Inhibitors Papain Drug development Bioinformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3897 , http://hdl.handle.net/10962/d1003842
- Description: Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.
- Full Text:
- Date Issued: 2013
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
Expression of heat shock proteins on the plasma membrane of cancer cells : a potential multi-chaperone complex that mediates migration
- Authors: Kenyon, Amy
- Date: 2011 , 2011-03-29
- Subjects: Heat shock proteins , Protein folding , Molecular chaperones , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4122 , http://hdl.handle.net/10962/d1013362
- Description: Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.
- Full Text:
- Date Issued: 2011
The dissociation of ammonium salts and their effect on the physiology and biochemistry of L-lysine synthesis by Corynebacterium glutamicum FP6
- Authors: Kenyon, Colin Peter
- Date: 1994
- Subjects: Ammonium salts Lysine -- Synthesis Corynebacterium Dissociation -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4034 , http://hdl.handle.net/10962/d1004094
- Description: The availability and assimilation of NH₄⁺ plays an integral role in the growth of microorganisms and the production of amino acids by these organisms. This study investigated the dissociation of NH₄⁺in aqueous solution, its availability and effect on the enzymes of NH₄⁺ assimilation and its influence on lysine production by Corynebacterium glutamicum.In aqueous solution the extent of dissociation of NH₄C1, {NH₄)₂S0₄ and (NH₄)₂HP0₄ increases with decreasing concentration. A model is proposed for the dissociation of these molecules. It is believed that at very low concentrations, dissociation to NH₃ plus the respective counter-ions occurs. At these low concentrations the NH₃ acts as the substrate for glutamine synthetase. At the higher concentrations dissociation is to NH₄⁺ which is the substrate for glutamate dehydrogenase. At these higher concentrations the enzyme activities obtained for glutamate dehydrogenase, at equivalent concentrations of the above ammonium salts, were different when based on the total concentration of NH₄⁺, and similar when based on the concentration of free NH₄⁺. L-Iysine occurs in the +1 ionic form, at pH 7,2. The lysine which is produced during fermentation associates with the anionic counter-ion of the ammonium salt used. The concentration of the free NH₄⁺ in the media appears to affect both the rate of lysine synthesis as well as the yield. The lysine fermentation occurs in two stages; a growth (or replicative) phase, during which very little lysine is produced, and a lysine synthesis (or maturation) phase. During the lysine synthesis phase there is no cell replication, however an increase in the mass of the biomass produced is apparent. Evidence is provided for the possible concomitant synthesis of the the cell wall polymer, glycerol teichoic acid, and lysine. On the basis of this evidence, a nucleotide balance is proposed for lysine and teichoic acid synthesis. The replicative phase and the maturation phase have to be effectively separated to obtain optimal lysine yields and titres. It is believed that teichoic acid synthesis during the replicative phase must be kept to a minimum for optimal yields and titres to be obtained, and on completion of the cell wall and therefore teichoic acid synthesis, lysine synthesis ceases. As the production of lysine appears to be affected by the NH₄⁺ concentration in the culture media, it is proposed that a futile cycle may exist around the transport and assimilation of the NH₄⁺. If the fermentations are run at low free NH₄⁺ concentrations, it was shown that lysine yields of 0,66, on the glucose utilised, are attainable during the fermentation.
- Full Text:
- Date Issued: 1994
Molecular and biochemical analysis of the diet of the black rhinoceros
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
- Date Issued: 2009
The development of the emerging technologies sustainability assessment (ETSA) and its application in the design of a bioprocess for the treatment of wine distillery effluent
- Authors: Khan, Nuraan
- Date: 2005
- Subjects: Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3953 , http://hdl.handle.net/10962/d1004012 , Technology assessment , Wine and wine making -- Waste disposal , Distilleries -- Waste disposal
- Description: Emerging Technologies Sustainability Assessment (ETSA) is a new technology assessment tool that was developed in order to compare emerging processes or technologies to existing alternatives. It utilizes infoIDlation modules, with the minimum use of resources such as time and money, in order to deteIDline if the process under development is comparatively favourable and should be developed beyond the early conceptual phase. The preliminary ETSA is vital in order to identify the gaps in the existing information and the specific methodologies to be used for data capture and analysis. The use of experimental design tools, such as Design-Expert, can facilitate rapid and efficient collection of necessary data and fits in well with the rationale for the ETSA. Wine distillery effluent (vinasse) is the residue left after alcohol has been distilled from fennented grape juice. It is an acidic, darkly coloured effluent, with a high COD and polyphenol content. The most popular method of disposal of this effluent, land application, is no longer viable due to stricter legislation and pressure on the industry to better manage its wastes. Although the ability of whiterot fungi to degrade a number of pollutants is well-known, fungal treatment of wine distillery effluent is still in the conceptual phase. The perfoIDlance of the fungal remediation system was assessed experimentally in terms of COD removal and laccase production using Design-Expert. Although Pycnoporus sanguine us was found to be most efficient at COD removal (85%) from 30% vinasse, laccase production was low (0.021 U/I). The optimum design for economically viable fungal treatment used Trametespubescens. This fungus was able to remove over 50% of the COD from undiluted vinasse while producing almost 800U/l of the valuable laccase enzyme within three days. Since the effluent from the fungal system did not meet the legal limits for wastewater disposal, a two-stage aerobicanaerobic system is suggested to improve the quality of the effluent prior to disposal. The ETSA was used to assess the fungal technology in relation to the two current methods of vinasse treatment and disposal, namely land application and anaerobic digestion. Based on the ETSA, which considered environmental, social and economic impacts, the fungal system proved to be potentially competitive and further development of the technology is suggested.
- Full Text:
- Date Issued: 2005
Development of a novel, quantitative assay for determining the rate of activity of antimalarial drugs
- Authors: Khan, Tasmiyah
- Date: 2013
- Subjects: Assay , ATP , Antimalarials -- Therapeutic use Malaria Malaria -- Drug therapy Adenosine triphosphate Luciferases Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3884 , http://hdl.handle.net/10962/d1001618
- Description: Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery
- Full Text:
- Date Issued: 2013
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
An investigation of the isolation, characterisation and application of hydantoinases for the industrial production of amino acids
- Authors: Kirchmann, Shaun
- Date: 2003
- Subjects: Hydantoin Amino acids Hydrolysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3969 , http://hdl.handle.net/10962/d1004028
- Description: This thesis describes a series of investigations into the hydantoin-hydrolysing activity of bacterial strains RU-KM1 and RU-OR, which were previously isolated for their ability to hydrolyse hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids using a bioreactor based system utilising the hydantoin hydrolysing enzymes of the two isolated microorganisms. Different substituted hydantoins may be used as substrates by these enzymes for the production of a variety of amino acids. These are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as semisynthetic penicillin/ampicillin, L-aspartame (sweetener), Fluvalinate (insecticide), Enalapril (ACE inhibitor). Thus, the ability of the above-mentioned strains to hydrolyse these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an N-carbamylamino acid intermediate, and subsequently, an amino acid. The hydantoin-hydrolysing enzymes of a Pseudomonas sp. RU-KM1, and an Agrobacterium sp. RU-OR were characterised as whole cells and in a crude extract preparation, and reaction conditions for its biocatalytic application were optimised. The optimum conditions for conversion of hydantoin to glycine were found to be 1 hour at 40 °C, with conversion yields greater than 30 % achieved. The enzymes of RU-KM1 demonstrated considerable stability, retaining 80 % of their activity after storage for 2 weeks at 4 °C. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamoylase of RU-KM1 suggested that these enzymes required metal ions for activity, with metal ions such as Cu[superscript (2+)], Fe[superscript (2+)], and Co[superscript (2+)] resulting in no significant change in enzyme activity, however there was an activation of the enzymes when Mn[superscript (2+)] was added to the enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was D-selective, whereas the N-carbamoylase was shown to be L-selective by other researchers. The hydantoin substrate selectivity of RU-KM1 and RU-OR was investigated, and the organisms were shown to be able to hydrolyse all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing slightly lower activity than whole cells in RU-KM1, and the whole cells or RU-OR showing the lower activity than its crude extract. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolysed p-hydroxyphenylhydantoin, achieving 57 % and 52 % conversions respectively. RU-OR whole cells preferred methylhydantoin where as the crude extract preferred isopropylhydantoin, and showed 49 % and 51 % conversions respectively. The enzymes were characterised in terms of their temperature and pH optima, inducer requirements, and product inhibition studies. The hydantoinase of RU-KM1 was shown to be inducible with low levels of hydantoin, and thermostable upto 75 °C with its optima between 60 and 70 °C. The N-carbamoylase was shown to have its optima at 50 °C. The addition of ATP (0.5 mM), DTT (1 mM) and a protease inhibitor (2 mg.mL[superscript (-1)]) all increased the hydantoinase activity of RU-KM1 crude extract, however they had very little effect on the N-carbamoylase activity. The hydantoinase enzyme from extracts of RU-KM1 was partially purified by development of cell disruption methods using mechanical and lysing enzymes, followed by precipitation and chromatographic resolution. The results obtained showed a hydantoinase enzyme of between 48 and 66 kDa. RU-KM1 was grown under fermentation conditions using different minimal media. The activity and yields under these conditions were low. Previous attempt to grow the organism in a rich medium had resulted in an increase in biomass but no hydantoinase activity. A rich medium was developed by carbon and nitrogen optimisation and yielded biomass up to 30 g.L[superscript (-1)] dry cell weight. The hydantoinase activity was restored by nitrogen starvation in stationary phase. This resulted in high biomass with increased activity. This data is currently in press. Crude extract and whole cells were immobilised on flat sheet membranes, hollow fibre membranes and in alginate beads. Low hydantoinase activity was measured in bioreactors using membranes in different configurations. A significant increase in hydantoinase activity was measured when the crude extract was immobilised in sodium alginate, as a result of stabilisation of the N-carbamoylase. Temperature and pH optima were unaffected by the immobilisation procedure, however the durability of the enzymes increased 2-fold. Different configurations of the bioreactor were investigated, as well as a hydroxyphenylhydantoin as an alternative substrate in this study. The bioreactors showed a near 95 % conversion of the hydantoin to glycine, and a 99 % conversion using HPG. In conclusion, the hydantoin-hydrolysing enzymes of RU-KM1 have been shown to be possibly membrane associated, which is a novel finding. This study has shown that the hydantoinase of RU-KM1 is D-stereoselective, with high temperature stability. A growth medium was developed for the rapid production of active biomass. A bioreactor was developed using a single and a dual biocatalyst configuration, which was capable of hydrolysing hydantoin and monosubstituted hydantoins to produce amino acids. To our knowledge this system is the first such dual biocatalyst system reported for the production of amino acids.
- Full Text:
- Date Issued: 2003
Isolation and identification of Beta-Lactam Producing Microorganisms using PCR based methodologies
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
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
High-throughput modelling and structural investigation of cysteine protease complexes with protein inhibitors
- Authors: Kroon, Matthys Christoffel
- Date: 2013
- Subjects: Cysteine proteinases Cysteine proteinases -- Inhibitors Papain Cystatins Malaria -- Chemotherapy Homology (Biology) Protein-protein interactions
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3885 , http://hdl.handle.net/10962/d1001619
- Description: The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb
- Full Text:
- Date Issued: 2013
Voltammetric investigation of microbiological growth media and carbon nanotube modified electrodes : a case study of oxytetracycline
- Authors: Kruid, Jan
- Date: 2013
- Subjects: Voltammetry , Electrodes , Oxytetracycline , Carbon nanotubes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4156 , http://hdl.handle.net/10962/d1018238
- Description: Oxytetracycline (OTC) is a broad spectrum antibiotic used extensively in the agricultural and human-health sector, and is effective against various gram positive and –negative bacteria as well as large viruses and certain pathogenic Rickettsiae. This study addresses the lack of voltammetric knowledge regarding the electroanalytical characterisation of OTC and its analysis in complex matrices. Cyclic voltammetry (CV) revealed several irreversible anodic peaks for OTC at a bare glassy carbon electrode (GCE). These current responses were improved through the selection of a diluent for OTC stock preparation, electrolyte solution and electrolyte pH, stir time and applied preconditioning potential. Under enhanced adsorptive conditions and using square wave voltammetry (SWV), a detection limit of 24.3 nM was achieved. The electrode surface could be renewed in vitro for 10 successive scans. OTC oxidation was characterised as a one electron:one proton ECiE mechanisms. Next, investigating the viability of voltammetry in various complex microbiological growth media revealed that selected growth media contained interfering redox active components, which, while simultaneously coating the electrode surface, effectively reduced GCE performance and lowered the active electrode surface area, as ascertained through CV and electrochemical impedance spectroscopy (EIS) studies. This interference lowered OTC current response in the presence of growth media which was partially recovered by appropriate growth media selection and sample dilution. In testing the use of acid functionalised multi-walled carbon nanotubes (MWCNTs) to improve anodic OTC response, charge-based attraction was observed between the MWCNT dispersal agent Nafion® and OTC, while increased surface area associated with prolonged acid functionalisation time aided in improving OTC current response.
- Full Text:
- Date Issued: 2013