The synthesis of fructooligosaccharides by the fructofuranosidase FopAp from Aspergillus niger
- Pindura, Mitchell Kingsley Chido
- Authors: Pindura, Mitchell Kingsley Chido
- Date: 2012
- Subjects: Aspergillus niger , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4158 , http://hdl.handle.net/10962/d1018267
- Description: Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. β-fructofuranosidase (FFase) and fructosyltransferase (FTase) enzymes are capable of synthesing FOS from carbohydrate raw materials such as chicory and sugar beet. The aim of this study was to investigate the synthesis of FOS of a pre-defined chain length, from sucrose, by the enzyme FopAp; a β-fructofuranosidase from Aspergillus niger. ATCC 20611. The crude enzyme FopAp was successfully purified, with a yield of 78.20 %, by ammonium sulphate precipitation and anion exchange chromatography. Two protein fractions, named FA and FB were shown to exhibit FFase activity. SDS PAGE analysis revealed two proteins with molecular weights of 112 kDa and 78 kDa, which were identified as a FFase and a hydrolase. Temperature and pH optima of 20 ºC and 9, respectively, were observed for the transfructosylation activity in the FFase. The purified FFase exhibited a half life of 1.5 hrs under optimal conditions. Substrate kinetic studies indicated a high hydrolytic activity at low sucrose concentrations, with Vmax and Km of 1.25 μmol/ml/min and 3.28 mM, respectively. Analysis by response surface methodology identified temperature and pH to be significant factors for the production of kestose and nystose, at a 95 % level of confidence. These findings were confirmed by neural networks constructed to identify optimal conditions of FOS synthesis.FOS synthesis was found to be optimal between pH 6 and pH 9 at 25 ºC. The factor of reaction time was found to be insignificant within the selected experimental constraints, for both FOS species. The findings of this investigation are very important as the foundations of a commercially viable synthetic process for the production of FOS.
- Full Text:
- Date Issued: 2012
- Authors: Pindura, Mitchell Kingsley Chido
- Date: 2012
- Subjects: Aspergillus niger , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4158 , http://hdl.handle.net/10962/d1018267
- Description: Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. β-fructofuranosidase (FFase) and fructosyltransferase (FTase) enzymes are capable of synthesing FOS from carbohydrate raw materials such as chicory and sugar beet. The aim of this study was to investigate the synthesis of FOS of a pre-defined chain length, from sucrose, by the enzyme FopAp; a β-fructofuranosidase from Aspergillus niger. ATCC 20611. The crude enzyme FopAp was successfully purified, with a yield of 78.20 %, by ammonium sulphate precipitation and anion exchange chromatography. Two protein fractions, named FA and FB were shown to exhibit FFase activity. SDS PAGE analysis revealed two proteins with molecular weights of 112 kDa and 78 kDa, which were identified as a FFase and a hydrolase. Temperature and pH optima of 20 ºC and 9, respectively, were observed for the transfructosylation activity in the FFase. The purified FFase exhibited a half life of 1.5 hrs under optimal conditions. Substrate kinetic studies indicated a high hydrolytic activity at low sucrose concentrations, with Vmax and Km of 1.25 μmol/ml/min and 3.28 mM, respectively. Analysis by response surface methodology identified temperature and pH to be significant factors for the production of kestose and nystose, at a 95 % level of confidence. These findings were confirmed by neural networks constructed to identify optimal conditions of FOS synthesis.FOS synthesis was found to be optimal between pH 6 and pH 9 at 25 ºC. The factor of reaction time was found to be insignificant within the selected experimental constraints, for both FOS species. The findings of this investigation are very important as the foundations of a commercially viable synthetic process for the production of FOS.
- Full Text:
- Date Issued: 2012
Understanding the biomolecular interactions involved in dimerisation of the Saccharomyces cerevisiae eukaryotic translation initiation factor 5A
- Authors: Charlton, Jane Laura
- Date: 2012
- Subjects: Proteins -- Synthesis -- Research , Saccharomyces cerevisiae -- Research , Dimers , Dimers -- Research , Eukaryotic cells -- Research , Yeast -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4057 , http://hdl.handle.net/10962/d1004118 , Proteins -- Synthesis -- Research , Saccharomyces cerevisiae -- Research , Dimers , Dimers -- Research , Eukaryotic cells -- Research , Yeast -- Research
- Description: Translation initiation factor 5A (IF5A) is an essential, highly conserved protein found within all eukaryotic (eIF5A) and archaeal (aIF5A) cells. The IF5A protein is unique in that it contains the amino acid hypusine; a two-step post translational modification of a single, conserved lysine residue. Although hypusination of eIF5A is vital for eukaryotic cell viability, the primary role of the protein and its hypusine side chain remain a mystery. eIF5A, initially identified as a translation initiation factor, is not required for global protein synthesis leading to the prevailing proposal that eIF5A is purely involved in the translation of a select subset of mRNAs. Recently a number of mutational studies have focused on the conserved, hypusine-containing loop region of eIF5A where specific residues have been found to be essential for activity without affecting hypusination. It has been postulated that eIF5A exists as a dimer (40 kDa) under native conditions and that these residues may be at the interface of dimerisation. The aim of this research was therefore to conduct a mutational analysis of the loop region in support of this hypothesis. A functional analysis of the Saccharomyces cerevisiae eIF5A mutant proteins K48D, G50A, H52A and K56A revealed that these substitutions impaired growth to varying degrees in vivo with G50A and K48D mutant proteins displaying the most convincing defects. Gel filtration profiles gave unexpected results determining eIF5A mutant and wild type proteins to have a native molecular weight of 30 to 31 kDa, suggesting that the eIF5A oligomeric state may be transitory and subject to certain conditions. The inconclusive results obtained from using gel filtration studies led to an investigation into the feasibility of producing native, hypusinated peptides for future structural studies using nuclear magnetic resonance. Hypusinated and unhypusinated eIF5A were successfully separated into their domains making this a possibility. Finally, this study proposes a role for eIF5A in eukaryotic IRES-driven translation initiation.
- Full Text:
- Date Issued: 2012
- Authors: Charlton, Jane Laura
- Date: 2012
- Subjects: Proteins -- Synthesis -- Research , Saccharomyces cerevisiae -- Research , Dimers , Dimers -- Research , Eukaryotic cells -- Research , Yeast -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4057 , http://hdl.handle.net/10962/d1004118 , Proteins -- Synthesis -- Research , Saccharomyces cerevisiae -- Research , Dimers , Dimers -- Research , Eukaryotic cells -- Research , Yeast -- Research
- Description: Translation initiation factor 5A (IF5A) is an essential, highly conserved protein found within all eukaryotic (eIF5A) and archaeal (aIF5A) cells. The IF5A protein is unique in that it contains the amino acid hypusine; a two-step post translational modification of a single, conserved lysine residue. Although hypusination of eIF5A is vital for eukaryotic cell viability, the primary role of the protein and its hypusine side chain remain a mystery. eIF5A, initially identified as a translation initiation factor, is not required for global protein synthesis leading to the prevailing proposal that eIF5A is purely involved in the translation of a select subset of mRNAs. Recently a number of mutational studies have focused on the conserved, hypusine-containing loop region of eIF5A where specific residues have been found to be essential for activity without affecting hypusination. It has been postulated that eIF5A exists as a dimer (40 kDa) under native conditions and that these residues may be at the interface of dimerisation. The aim of this research was therefore to conduct a mutational analysis of the loop region in support of this hypothesis. A functional analysis of the Saccharomyces cerevisiae eIF5A mutant proteins K48D, G50A, H52A and K56A revealed that these substitutions impaired growth to varying degrees in vivo with G50A and K48D mutant proteins displaying the most convincing defects. Gel filtration profiles gave unexpected results determining eIF5A mutant and wild type proteins to have a native molecular weight of 30 to 31 kDa, suggesting that the eIF5A oligomeric state may be transitory and subject to certain conditions. The inconclusive results obtained from using gel filtration studies led to an investigation into the feasibility of producing native, hypusinated peptides for future structural studies using nuclear magnetic resonance. Hypusinated and unhypusinated eIF5A were successfully separated into their domains making this a possibility. Finally, this study proposes a role for eIF5A in eukaryotic IRES-driven translation initiation.
- Full Text:
- Date Issued: 2012
A central enrichment-based comparison of two alternative methods of generating transcription factor binding motifs from protein binding microarray data
- Authors: Mahaye, Ntombikayise
- Date: 2013 , 2013-03-13
- Subjects: Transcription factors , Bioinformatics , Protein binding , Protein microarrays , Cell lines
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3890 , http://hdl.handle.net/10962/d1003049 , Transcription factors , Bioinformatics , Protein binding , Protein microarrays , Cell lines
- Description: Characterising transcription factor binding sites (TFBS) is an important problem in bioinformatics, since predicting binding sites has many applications such as predicting gene regulation. ChIP-seq is a powerful in vivo method for generating genome-wide putative binding regions for transcription factors (TFs). CentriMo is an algorithm that measures central enrichment of a motif and has previously been used as motif enrichment analysis (MEA) tool. CentriMo uses the fact that ChIP-seq peak calling methods are likely to be biased towards the centre of the putative binding region, at least in cases where there is direct binding. CentriMo calculates a binomial p-value representing central enrichment, based on the central bias of the binding site with the highest likelihood ratio. In cases where binding is indirect or involves cofactors, a more complex distribution of preferred binding sites may occur but, in many cases, a low CentriMo p-value and low width of maximum enrichment (about 100bp) are strong evidence that the motif in question is the true binding motif. Several other MEA tools have been developed, but they do not consider motif central enrichment. The study investigates the claim made by Zhao and Stormo (2011) that they have identified a simpler method than that used to derive the UniPROBE motif database for creating motifs from protein binding microarray (PBM) data, which they call BEEML-PBM (Binding Energy Estimation by Maximum Likelihood-PBM). To accomplish this, CentriMo is employed on 13 motifs from both motif databases. The results indicate that there is no conclusive difference in the quality of motifs from the original PBM and BEEML-PBM approaches. CentriMo provides an understanding of the mechanisms by which TFs bind to DNA. Out of 13 TFs for which ChIP-seq data is used, BEEML-PBM reports five better motifs and twice it has not had any central enrichment when the best PBM motif does. PBM approach finds seven motifs with better central enrichment. On the other hand, across all variations, the number of examples where PBM is better is not high enough to conclude that it is overall the better approach. Some TFs bind directly to DNA, some indirect or in combination with other TFs. Some of the predicted mechanisms are supported by literature evidence. This study further revealed that the binding specificity of a TF is different in different cell types and development stages. A TF is up-regulated in a cell line where it performs its biological function. The discovery of cell line differences, which has not been done before in any CentriMo study, is interesting and provides reasons to study this further.
- Full Text:
- Date Issued: 2013
- Authors: Mahaye, Ntombikayise
- Date: 2013 , 2013-03-13
- Subjects: Transcription factors , Bioinformatics , Protein binding , Protein microarrays , Cell lines
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3890 , http://hdl.handle.net/10962/d1003049 , Transcription factors , Bioinformatics , Protein binding , Protein microarrays , Cell lines
- Description: Characterising transcription factor binding sites (TFBS) is an important problem in bioinformatics, since predicting binding sites has many applications such as predicting gene regulation. ChIP-seq is a powerful in vivo method for generating genome-wide putative binding regions for transcription factors (TFs). CentriMo is an algorithm that measures central enrichment of a motif and has previously been used as motif enrichment analysis (MEA) tool. CentriMo uses the fact that ChIP-seq peak calling methods are likely to be biased towards the centre of the putative binding region, at least in cases where there is direct binding. CentriMo calculates a binomial p-value representing central enrichment, based on the central bias of the binding site with the highest likelihood ratio. In cases where binding is indirect or involves cofactors, a more complex distribution of preferred binding sites may occur but, in many cases, a low CentriMo p-value and low width of maximum enrichment (about 100bp) are strong evidence that the motif in question is the true binding motif. Several other MEA tools have been developed, but they do not consider motif central enrichment. The study investigates the claim made by Zhao and Stormo (2011) that they have identified a simpler method than that used to derive the UniPROBE motif database for creating motifs from protein binding microarray (PBM) data, which they call BEEML-PBM (Binding Energy Estimation by Maximum Likelihood-PBM). To accomplish this, CentriMo is employed on 13 motifs from both motif databases. The results indicate that there is no conclusive difference in the quality of motifs from the original PBM and BEEML-PBM approaches. CentriMo provides an understanding of the mechanisms by which TFs bind to DNA. Out of 13 TFs for which ChIP-seq data is used, BEEML-PBM reports five better motifs and twice it has not had any central enrichment when the best PBM motif does. PBM approach finds seven motifs with better central enrichment. On the other hand, across all variations, the number of examples where PBM is better is not high enough to conclude that it is overall the better approach. Some TFs bind directly to DNA, some indirect or in combination with other TFs. Some of the predicted mechanisms are supported by literature evidence. This study further revealed that the binding specificity of a TF is different in different cell types and development stages. A TF is up-regulated in a cell line where it performs its biological function. The discovery of cell line differences, which has not been done before in any CentriMo study, is interesting and provides reasons to study this further.
- Full Text:
- Date Issued: 2013
A role for heat shock protein 90 (Hsp90) in fibronectin matrix dynamics
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
- Full Text:
- Date Issued: 2013
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
- Full Text:
- Date Issued: 2013
Biochemical characterisation of Pfj2, a Plasmodium falciparum heat shock protein 40 chaperone potentially involved in protein quality control in the endoplasmic reticulum
- Authors: Afolayan, Omolola Folasade
- Date: 2013
- Subjects: Plasmodium falciparum Endoplasmic reticulum Heat shock proteins Malaria , Mosquito-borne infectious disease
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3883 , http://hdl.handle.net/10962/d1001617
- Description: Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease
- Full Text:
- Date Issued: 2013
- Authors: Afolayan, Omolola Folasade
- Date: 2013
- Subjects: Plasmodium falciparum Endoplasmic reticulum Heat shock proteins Malaria , Mosquito-borne infectious disease
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3883 , http://hdl.handle.net/10962/d1001617
- Description: Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease
- Full Text:
- Date Issued: 2013
Characterisation of a plasmodium falciparum type II Hsp40 chaperone exported to the cytosol of infected erythrocytes
- Maphumulo, Philile Nompumelelo
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- Full Text:
- Date Issued: 2013
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- Full Text:
- Date Issued: 2013
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
- 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
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
- 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
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
- 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
Immobilisation of electric eel acetylcholinesterase on nanofibres electrospun from a nylon and chitosan blend
- Authors: Mafuma, Tendai Simbarashe
- Date: 2013
- Subjects: Acetylcholinesterase Acetylcholinesterase -- Inhibitors Electric eel Biosensors Immobilized enzymes Pesticides -- Environmental aspects Pesticides -- Toxicology Nylon Chitosan Nanofibers Electrospinning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3886 , http://hdl.handle.net/10962/d1001620
- Description: Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity
- Full Text:
- Date Issued: 2013
- Authors: Mafuma, Tendai Simbarashe
- Date: 2013
- Subjects: Acetylcholinesterase Acetylcholinesterase -- Inhibitors Electric eel Biosensors Immobilized enzymes Pesticides -- Environmental aspects Pesticides -- Toxicology Nylon Chitosan Nanofibers Electrospinning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3886 , http://hdl.handle.net/10962/d1001620
- Description: Organophosphates and carbamates are potent inhibitors of the neurotransmitter acetylcholinesterase. This inhibition results in the blocking of nerve signal transference into the post synaptic neuron leading to loss of muscle action and death. Because of the universal mechanisms of signal transduction in animals, these inhibitors have been widely used as agricultural pesticides as well as chemical warfare agents (nerve agents). Health issues associated with pesticide usage result from the fact that both the pesticides and their breakdown products often end up in water and food sources as well as in the soil. As a result, there has been an increase in the number of studies aimed at the detection of these pesticides in the environment. One popular research area is enzyme based biosensor construction. Some important criteria for consideration during the construction of biosensors are the importance of a suitable solid support as well as the enzyme immobilisation method. Recently, there has been increased interest in using nano-scale material e.g. using nanoparticles as enzyme support material. This is largely due to their advantages such as large surface area to volume ratio as well as reduced mass transfer resistance. Electrospinning is a straight forward and cost effective method for producing nanofibres from any soluble polymer(s). The applications of electrospun nanofibres have been reported in clinical studies, biofuel production as well as bioremediation. In this study two polymers were selected: nylon for its mechanical stability and chitosan for its biocompatibility and hydrophilicity, for the fabrication of electrospun nanofibres which would function as immobilisation support material for acetylcholinesterase. The first objective of this study was to electrospin nanofibres from a nylon-6 and chitosan blend solution. A binary solvent system consisting of formic acid and acetic acid (50:50) successfully dissolved and blended the polymers which were subsequently electrospun. Scanning electron microscopy characterisation of the nanofibres showed that (i) a nylon-6: chitosan ratio of 16%: 3% resulted in the formation of bead free nanofibres and (ii) the fibres were collected in non-woven mats characterised by different size nanofibres with average diameters of 250 nm for the main fibres and 40 nm for the smaller nanofibres. Fourier transform infra-red (FT-IR) analysis of the nanofibres indicated that a new product had been formed during the blending of the two polymers. The second aim of the study was to carry out a facile immobilisation of electric eel acetylcholinesterase via glutaraldehyde (GA) cross-linking. Glutaraldehyde solution 5% (v/v) resulted in the immobilisation of 0.334 mg/cm² of acetylcholinesterase onto the nanofibres. The immobilisation procedure was optimised with reference to acetylcholinestease and crosslinker concentrations, incubation time and the cross-linking method. A comparative investigation into the optimum pH and temperature conditions, pH and thermal stabilities, substrate and inhibition kinetics was then carried out on free and immobilised acetylcholinesterase. The final objective of this study was to determine the storage stabilities of the immobilised and free enzymes as well as the reusability characteristics of the immobilised acetylcholinesterase. Several conclusions were drawn from this study. Acetylcholinesterase was successfully immobilised onto the surface of nylon-6:chitosan nanofibres with retention of its activity. There was a shift in the pH optimum of the immobilised acetylcholineseterase by 0.5 units towards a neutral pH. Although both free and immobilised acetylcholinesterase exhibited the same optimum temperature, immobilised acetylcholinesterase showed enhanced thermal stability. In terms of pH stability, immobilised acetylcholinesterase showed greater stability at acidic pH whilst free acetylcholinesterase was more stable under alkaline pH conditions. Relative to free acetylcholinesterase, the immobilised enzyme showed considerable storage stability retaining ~50% of its activity when stored for 49 days at 4°C. Immobilised acetylcholinesterase also retained > 20% of its initial activity after 9 consecutive reuse cycles. When exposed to fixed concentrations of carbofuran or demeton-S-methyl sulfone, immobilised acetylcholinesterase showed similar inhibition characteristics to that of the free enzyme. The decrease in enzyme activity observed after immobilisation to the nanofibres may have been due to several reasons which include some enzyme molecules being immobilised in structural conformations which reduced substrate access to the catalytic site, participation of the catalytic residues in immobilisation and enzyme denaturation due to the reaction conditions used for acetylcholinesterase immobilisation. Similar observations have been widely reported in literature and this is one of the major drawbacks of enzyme immobilisation. In conclusion, nylon-6:chitosan electrospun nanofibres were shown to be suitable supports for facile acetylcholinesterase immobilisation and the immobilised enzyme has potential for use in pesticide detection. Future recommendations for this study include a comparative study of the GA cross-linking method for AChE immobilisation which will lead to more intensely bound enzyme molecules to prevent non-specific binding. An investigation into the effect of inhibitors on stored immobilised AChE, as well as reactivation and reuse studies, may also be useful for determining the cost-effectiveness of reusing immobilised AChE for pesticide detection in environmental water samples. Several models have been designed for the determination of the kinetic parameters for immobilised enzymes. These take into account the mass transfer resistance as well as the overall charge of the immobilisation matrix. The use of these models to analyse experimental data will give a clear understanding of the effects of immobilisation on enzyme activity
- Full Text:
- Date Issued: 2013
In-silico analysis of Plasmodium falciparum Hop protein and its interactions with Hsp70 and Hsp90
- Authors: Clitheroe, Crystal-Leigh
- Date: 2013
- Subjects: Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3896 , http://hdl.handle.net/10962/d1003819 , Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Description: A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
- Full Text:
- Date Issued: 2013
- Authors: Clitheroe, Crystal-Leigh
- Date: 2013
- Subjects: Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3896 , http://hdl.handle.net/10962/d1003819 , Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Description: A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
- Full Text:
- Date Issued: 2013
Interaction of metallic nanoparticles with biomedical enzyme target: neuronal nitric oxide synthase
- Authors: Ngqwala, Nosiphiwe Patience
- Date: 2013
- Subjects: Nitric-oxide synthase Alzheimer's disease Arginine Nanoparticles Biochemical markers Biochemical markers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3875 , http://hdl.handle.net/10962/d1001536
- Description: Alzheimer's disease (AD) is the most common type of dementia characterized by intracellular appearance of neurofibrillary tangles, synaptic and neuronal loss; and extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques. The initial causes leading to AD are unknown, and the available treatments are only effective at slowing the degeneration process. The accumulation of arginine in the brain of Alzheimer patients indicates a possible disruption of enzymes responsible for its metabolism. One such enzyme is neuronal nitric oxide synthase (nNOS) and controlling its activity by interacting with nanoparticles may lead to a delay in the onset of the disease. Neuronal nitric oxide synthase was purified using DEAE-Sephacel ion exchange resulting in 10 % yield, 0.43 fold recovery and specific activity 0.09 U/mg. The enzyme was found to be a dimer with a molecular mass of 150 kDa. Characterisation of the nNOS showed an optimum temperature and pH of 50°C and 7.5 respectively, and it was relatively stable at the optimum conditions (t½ = 100 min). The purity was analysed by SDS-PAGE followed by Western blot. Purified nNOS was challenged with 3-7 nm silver and 4-15 nm gold nanoparticles of between synthesized chemical using AgNO3 and either sodium borohydride or sodium citrate. Results showed that gold nanoparticles are more effective at low concentration (5 μM) than silver nanoparticles due to their size difference. Incubation of different concentration of nanoparticles (5, 15, 25, 50 μM) with the purified nNOS showed an initial decrease of 5% in enzyme activity which over time was restored to 80%. This suggests that different nanoparticles are produced in different sizes and interaction over a given time may result in enzyme association–dissociation mechanism. Inhibition studies showed a strong binding of both nanoparticles with Ki values of 1.4 μM and 0.2 μM for silver and gold, respectively. Both nanoparticles inhibited the activity of nNOS extensively as they bound strongly to the inhibition site on the enzyme and were more in contact with fluorophores nanoparticles. This was confirmed by fluorimetry with binding constants of 0.0084 μM and 0.01092 μM for silver and gold, respectively. Results of this study suggest that silver and gold nanoparticles competitively inhibit nNOS.
- Full Text:
- Date Issued: 2013
- Authors: Ngqwala, Nosiphiwe Patience
- Date: 2013
- Subjects: Nitric-oxide synthase Alzheimer's disease Arginine Nanoparticles Biochemical markers Biochemical markers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3875 , http://hdl.handle.net/10962/d1001536
- Description: Alzheimer's disease (AD) is the most common type of dementia characterized by intracellular appearance of neurofibrillary tangles, synaptic and neuronal loss; and extracellular accumulation of amyloid-β (Aβ) peptide in senile plaques. The initial causes leading to AD are unknown, and the available treatments are only effective at slowing the degeneration process. The accumulation of arginine in the brain of Alzheimer patients indicates a possible disruption of enzymes responsible for its metabolism. One such enzyme is neuronal nitric oxide synthase (nNOS) and controlling its activity by interacting with nanoparticles may lead to a delay in the onset of the disease. Neuronal nitric oxide synthase was purified using DEAE-Sephacel ion exchange resulting in 10 % yield, 0.43 fold recovery and specific activity 0.09 U/mg. The enzyme was found to be a dimer with a molecular mass of 150 kDa. Characterisation of the nNOS showed an optimum temperature and pH of 50°C and 7.5 respectively, and it was relatively stable at the optimum conditions (t½ = 100 min). The purity was analysed by SDS-PAGE followed by Western blot. Purified nNOS was challenged with 3-7 nm silver and 4-15 nm gold nanoparticles of between synthesized chemical using AgNO3 and either sodium borohydride or sodium citrate. Results showed that gold nanoparticles are more effective at low concentration (5 μM) than silver nanoparticles due to their size difference. Incubation of different concentration of nanoparticles (5, 15, 25, 50 μM) with the purified nNOS showed an initial decrease of 5% in enzyme activity which over time was restored to 80%. This suggests that different nanoparticles are produced in different sizes and interaction over a given time may result in enzyme association–dissociation mechanism. Inhibition studies showed a strong binding of both nanoparticles with Ki values of 1.4 μM and 0.2 μM for silver and gold, respectively. Both nanoparticles inhibited the activity of nNOS extensively as they bound strongly to the inhibition site on the enzyme and were more in contact with fluorophores nanoparticles. This was confirmed by fluorimetry with binding constants of 0.0084 μM and 0.01092 μM for silver and gold, respectively. Results of this study suggest that silver and gold nanoparticles competitively inhibit nNOS.
- Full Text:
- Date Issued: 2013
Interactions of arbuscular mycorrhizal fungi and spore-associated bacteria
- Authors: Ridsdale, Carmen Jane
- Date: 2013
- Subjects: Mycorrhizal fungi , Host plants , Bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4159 , http://hdl.handle.net/10962/d1018269
- Description: Arbuscular mycorrhizal (AM) fungi are naturally occurring in roots of terrestrial plants. AM fungi are capable of benefiting the host plant through various mechanisms such as enhanced nutrient supply, alleviation of environmental stress and inhibition of plant fungal pathogens. AM fungal spore-associated bacteria have been previously isolated and shown to have plant growthpromoting (PGP) abilities by several authors. Some bacterial isolates are able to promote AM fungal colonisation of host plants and are known to be mycorrhizal helper bacteria (MHB). This study focused on the isolation of AM fungal spore-associated bacteria, characterization of the isolates according to plant growth promoting abilities and evaluation of their potential to enhance plant growth and mycorrhizal colonisation. AM fungi were extracted from soils sampled from natural indigenous forest sources, raspberry (Rubus idaeus cv. Heritage) and strawberry (Fragaria ananassa) farms in South Africa and from a raspberry (Rubus idaeus cv. Autumn Bliss) plantation in Argentina. A total of 52 sporeassociated bacteria were isolated from the external and internal surfaces of AM fungal spore morphotypes from the two countries. The bacterial isolates were evaluated for their PGP abilities such as phosphate solubilisation, indole-3-acetic acid production, ammonia production and inhibition of the fungal pathogens Fusarium oxysporum and Phythophthora nicotianae through mechanisms such as siderophore and/ or hydrolytic enzyme production. A total of 23 bacterial isolates from both South Africa and Argentina showing the most potential to be PGP, were identified molecularly as belonging to the genera Acinetobacter, Alcaligenes, Bacillus, Microbacterium, Micrococcus, Serratia and Staphylococcus. The ability of ten selected bacterial isolates showing multiple PGP capacity were evaluated for their plant growth promotion and mycorrhizal colonisation enhancement ability on raspberry (Rubus idaeus cv. Meeker). Significant differences in increased shoot and root dry weights were shown by the treatments compared to the uninoculated control. The highest increase in shoot and root dry weights were shown by South African (Bacillus mycoides) and Argentinean (Alcaligenes faecalis) isolates. AM fungal colonisation was significantly enhanced by the South African (Bacillus mycoides) and Argentinean (Micrococcus luteus) isolates compared to the AM fungal singly inoculated control.
- Full Text:
- Date Issued: 2013
- Authors: Ridsdale, Carmen Jane
- Date: 2013
- Subjects: Mycorrhizal fungi , Host plants , Bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4159 , http://hdl.handle.net/10962/d1018269
- Description: Arbuscular mycorrhizal (AM) fungi are naturally occurring in roots of terrestrial plants. AM fungi are capable of benefiting the host plant through various mechanisms such as enhanced nutrient supply, alleviation of environmental stress and inhibition of plant fungal pathogens. AM fungal spore-associated bacteria have been previously isolated and shown to have plant growthpromoting (PGP) abilities by several authors. Some bacterial isolates are able to promote AM fungal colonisation of host plants and are known to be mycorrhizal helper bacteria (MHB). This study focused on the isolation of AM fungal spore-associated bacteria, characterization of the isolates according to plant growth promoting abilities and evaluation of their potential to enhance plant growth and mycorrhizal colonisation. AM fungi were extracted from soils sampled from natural indigenous forest sources, raspberry (Rubus idaeus cv. Heritage) and strawberry (Fragaria ananassa) farms in South Africa and from a raspberry (Rubus idaeus cv. Autumn Bliss) plantation in Argentina. A total of 52 sporeassociated bacteria were isolated from the external and internal surfaces of AM fungal spore morphotypes from the two countries. The bacterial isolates were evaluated for their PGP abilities such as phosphate solubilisation, indole-3-acetic acid production, ammonia production and inhibition of the fungal pathogens Fusarium oxysporum and Phythophthora nicotianae through mechanisms such as siderophore and/ or hydrolytic enzyme production. A total of 23 bacterial isolates from both South Africa and Argentina showing the most potential to be PGP, were identified molecularly as belonging to the genera Acinetobacter, Alcaligenes, Bacillus, Microbacterium, Micrococcus, Serratia and Staphylococcus. The ability of ten selected bacterial isolates showing multiple PGP capacity were evaluated for their plant growth promotion and mycorrhizal colonisation enhancement ability on raspberry (Rubus idaeus cv. Meeker). Significant differences in increased shoot and root dry weights were shown by the treatments compared to the uninoculated control. The highest increase in shoot and root dry weights were shown by South African (Bacillus mycoides) and Argentinean (Alcaligenes faecalis) isolates. AM fungal colonisation was significantly enhanced by the South African (Bacillus mycoides) and Argentinean (Micrococcus luteus) isolates compared to the AM fungal singly inoculated control.
- Full Text:
- Date Issued: 2013
Polyunsaturated fatty acid metabolism and effects on colon cancer cell biology in vitro.
- Authors: Bulcao, Candice
- Date: 2013
- Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
- Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
- Full Text:
- Date Issued: 2013
- Authors: Bulcao, Candice
- Date: 2013
- Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
- Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
- Full Text:
- Date Issued: 2013
Sequence and structural investigation of the nonribosomal peptide synthetases of Bacillus atrophaeus UCMB 5137(63Z)
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- 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
- 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
The effect of sewage effluent from De Beers marine diamond mining operations on the expression of cytochrome P450 (CYP1A) and vitellogenin (vtg)
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
A step forward in defining Hsp90s as potential drug targets for human parasitic diseases
- Authors: Faya, Ngonidzashe
- Date: 2014
- Subjects: Heat shock proteins -- Research , Malaria -- Chemotherapy -- Research , Antimalarials -- Development -- Research , Parasitic diseases -- Research , Plasmodium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4110 , http://hdl.handle.net/10962/d1012993
- Description: Parasitic diseases remain a health burden affecting more than 500 million people worldwide with malaria having the highest mortality rate. The parasites can be transferred to the human bodies either through the mouth by ingestion of contaminated food and water or through the skin by bug bites or direct contact to environments harbouring them. Epidemiological control seems to be impossible since there is failure to control the insect vectors as well as practice of hygiene. Therefore, this has led to the development of a number of vaccines, chemotherapy and disease control programs. However, parasites have increasingly developed resistance to traditionally used anti-parasitic drugs and due to that fact there is need for alternative medication for parasitic diseases. Heat shock protein 90 (Hsp90) facilitates the folding of proteins in all living cells and their role is more important to parasites because of their environmental changes, from vector to host. Hsp90s play a major role; therefore this justifies the need for a deeper analysis of the parasitic Hsp90s. Recent studies have revealed that, the Plasmodium sp. Hsp90 has an extended linker region which increases the protein’s affinity for ATP and its inhibitors. Therefore we hypothesize that there are also significant features in other parasitic Hsp90s which would lead to Hsp90 being defined as potential drug targets. In the present study an attempt was made to gain more insight into the differences in primary structure of human and parasitic Hsp90s. The sequences were retrieved from the NCBI database and analysis was done in three groups basing on the localization of the Hsp90. The physicochemical properties were calculated and in every group, the protozoan Hsp90s showed significant differences when compared to the human orthologs. Multiple sequence alignments (MSA) showed that endoplasmic reticulum Hsp90s have an extended region in the middle domain indicating their ability to bind to a unique subset of client proteins. Sequence identities between the human and parasites showed that the protozoan Hsp90s are less related to the human Hsp90s as compared to the other parasites. Likewise, motif analysis showed the trypanosomatids and apicomplexan groups have their own unique set of motifs and they were grouped together in the phylogenetic analysis. Phylogenetic analysis also showed that, the protozoan Hsp90s forms their own clades in each group while the helminths did not form in endoplasmic reticulum group. In this study, we concluded that, Hsp90 can be a potential drug target for the protozoan species and more specifically those from the apicomplexan and trypanosomatids groups.
- Full Text:
- Date Issued: 2014
- Authors: Faya, Ngonidzashe
- Date: 2014
- Subjects: Heat shock proteins -- Research , Malaria -- Chemotherapy -- Research , Antimalarials -- Development -- Research , Parasitic diseases -- Research , Plasmodium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4110 , http://hdl.handle.net/10962/d1012993
- Description: Parasitic diseases remain a health burden affecting more than 500 million people worldwide with malaria having the highest mortality rate. The parasites can be transferred to the human bodies either through the mouth by ingestion of contaminated food and water or through the skin by bug bites or direct contact to environments harbouring them. Epidemiological control seems to be impossible since there is failure to control the insect vectors as well as practice of hygiene. Therefore, this has led to the development of a number of vaccines, chemotherapy and disease control programs. However, parasites have increasingly developed resistance to traditionally used anti-parasitic drugs and due to that fact there is need for alternative medication for parasitic diseases. Heat shock protein 90 (Hsp90) facilitates the folding of proteins in all living cells and their role is more important to parasites because of their environmental changes, from vector to host. Hsp90s play a major role; therefore this justifies the need for a deeper analysis of the parasitic Hsp90s. Recent studies have revealed that, the Plasmodium sp. Hsp90 has an extended linker region which increases the protein’s affinity for ATP and its inhibitors. Therefore we hypothesize that there are also significant features in other parasitic Hsp90s which would lead to Hsp90 being defined as potential drug targets. In the present study an attempt was made to gain more insight into the differences in primary structure of human and parasitic Hsp90s. The sequences were retrieved from the NCBI database and analysis was done in three groups basing on the localization of the Hsp90. The physicochemical properties were calculated and in every group, the protozoan Hsp90s showed significant differences when compared to the human orthologs. Multiple sequence alignments (MSA) showed that endoplasmic reticulum Hsp90s have an extended region in the middle domain indicating their ability to bind to a unique subset of client proteins. Sequence identities between the human and parasites showed that the protozoan Hsp90s are less related to the human Hsp90s as compared to the other parasites. Likewise, motif analysis showed the trypanosomatids and apicomplexan groups have their own unique set of motifs and they were grouped together in the phylogenetic analysis. Phylogenetic analysis also showed that, the protozoan Hsp90s forms their own clades in each group while the helminths did not form in endoplasmic reticulum group. In this study, we concluded that, Hsp90 can be a potential drug target for the protozoan species and more specifically those from the apicomplexan and trypanosomatids groups.
- Full Text:
- Date Issued: 2014
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
- 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
Analysis of the interaction of Hsp90 with the extracellular matrix protein fibronectin (FN)
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014