A baculovirus-mediated expression system for the analysis of HaSV RNA packaging
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- Full Text:
- Date Issued: 2012
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- 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 comparative bioinformatic analysis of zinc binuclear cluster proteins
- Authors: Mthombeni, Jabulani S
- Date: 2005
- Subjects: Bioinformatics , Zinc proteins , GABA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4004 , http://hdl.handle.net/10962/d1004064 , Bioinformatics , Zinc proteins , GABA
- Description: Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.
- Full Text:
- Date Issued: 2005
- Authors: Mthombeni, Jabulani S
- Date: 2005
- Subjects: Bioinformatics , Zinc proteins , GABA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4004 , http://hdl.handle.net/10962/d1004064 , Bioinformatics , Zinc proteins , GABA
- Description: Members of the zinc binuclear cluster family are important fungal transcriptional regulators sharing a common DNA binding domain. Da181p is a pleotropic zinc binuclear cluster protein involved in the induction of the UGA genes required for the γ-aminobutyrate nitrogen catabolic pathway in Saccharomyces cerevisiae. The zinc binuclear cluster domain is indispensable for function in Da181p and little is known about other domains in this protein. The aim of the study was to explore the zinc binuclear cluster protein family using comparative bioinformatics as a complement to biochemical and structural approaches. A database of all zinc binuclear cluster proteins was composed. A total of 118 zinc binuclear proteins are reported in this work. Thirty nine previously unidentified zinc binuclear cluster proteins were found. Four homologues of Da181p were identified by homology searching. Important sequence motifs were identified in the aligned sequences of Da181p and its homologues. The coiled coil motif found in the Ga14p zinc binuclear cluster protein could not be identified in Da181p and its homologues. This suggested that Da181p did not dimerise through this structural motif as other zinc binuclear cluster proteins. Solvent accessible site that could be phosphorylated by protein kinase C or casein kinase II and the role of such sites in the possible regulation of Da181p function were discussed.
- Full Text:
- Date Issued: 2005
A novel adjuvant : polymerised serum albumin beads
- Authors: Dewar, John Barr
- Date: 1985
- Subjects: Antigens , Serum albumin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4104 , http://hdl.handle.net/10962/d1011146 , Antigens , Serum albumin
- Description: Lee, T. et al (1981) proposed the encapsulation of hormones such as progesterone into serum albumin beads, such that their in vivo proteolysis would allow a gradual release of hormone at low levels, for extended hormone action. It was proposed, in the Department of Microbiology, Rhodes University, to replace the hormone component of the above bead formulation, with virus as antigen, in the development of a vaccine. Beads optimally crosslinked at 1% final glutaraldehyde concentration, containing Nodamura virus, were shown to promote an adjuvant effect in vivo, analogous to the release of antigen from Freund's Complete Adjuvant (FCA), so that extended immunostimulation resulted. It was shown that soluble antigen promoted a short-lived primary immune response, peaking around day 25 following inoculation. Antigen presented in beads, on the other hand, initially elicited a low humoral response, but this response gradually increased up to a peak around day 110 post inoculation, before decreasing. No apparent adverse side-effects were noted following inoculation of antigen-containing serum albumin beads, compared to necrosis following antigen in FCA inoculation, supporting the proposal of using albumin homotypic for the test inoculee animal, so that the beads would themselves be non-immunogenic and would merely act as a vehicle in the vaccine formulation. The indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the humoral response to antigen following inoculation. Results showed that covalent crosslinking of albumin in the formation of the beads did not promote immunogenicity on the part of the chemically altered albumin. The ELISA test was used to indicate the kinetics of the IgG response to Nodamura virus when presented in formulations such as: Freely soluble virus or its subunit; soluble intact virus inactivated by treatment with glutaraldehyde; intact virus entrapped in serum albumin beads cross; linked at different percentage final glutaraldehyde concentrations and also virus subunit prepared in albumin beads. The presence of virus-neutral ising antibodies was noted in serum obtained from rabbits inoculated with virus entrapped in albumin beads. Virus infectivity, titrated in mice, showed protection against virus challenge after incubation of virus with serum obtained above.
- Full Text:
- Date Issued: 1985
- Authors: Dewar, John Barr
- Date: 1985
- Subjects: Antigens , Serum albumin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4104 , http://hdl.handle.net/10962/d1011146 , Antigens , Serum albumin
- Description: Lee, T. et al (1981) proposed the encapsulation of hormones such as progesterone into serum albumin beads, such that their in vivo proteolysis would allow a gradual release of hormone at low levels, for extended hormone action. It was proposed, in the Department of Microbiology, Rhodes University, to replace the hormone component of the above bead formulation, with virus as antigen, in the development of a vaccine. Beads optimally crosslinked at 1% final glutaraldehyde concentration, containing Nodamura virus, were shown to promote an adjuvant effect in vivo, analogous to the release of antigen from Freund's Complete Adjuvant (FCA), so that extended immunostimulation resulted. It was shown that soluble antigen promoted a short-lived primary immune response, peaking around day 25 following inoculation. Antigen presented in beads, on the other hand, initially elicited a low humoral response, but this response gradually increased up to a peak around day 110 post inoculation, before decreasing. No apparent adverse side-effects were noted following inoculation of antigen-containing serum albumin beads, compared to necrosis following antigen in FCA inoculation, supporting the proposal of using albumin homotypic for the test inoculee animal, so that the beads would themselves be non-immunogenic and would merely act as a vehicle in the vaccine formulation. The indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the humoral response to antigen following inoculation. Results showed that covalent crosslinking of albumin in the formation of the beads did not promote immunogenicity on the part of the chemically altered albumin. The ELISA test was used to indicate the kinetics of the IgG response to Nodamura virus when presented in formulations such as: Freely soluble virus or its subunit; soluble intact virus inactivated by treatment with glutaraldehyde; intact virus entrapped in serum albumin beads cross; linked at different percentage final glutaraldehyde concentrations and also virus subunit prepared in albumin beads. The presence of virus-neutral ising antibodies was noted in serum obtained from rabbits inoculated with virus entrapped in albumin beads. Virus infectivity, titrated in mice, showed protection against virus challenge after incubation of virus with serum obtained above.
- Full Text:
- Date Issued: 1985
A polarimetric method for collagenase activity measurement
- Brüning, Adrian Rudolf Nicolaus Ernst
- Authors: Brüning, Adrian Rudolf Nicolaus Ernst
- Date: 1992
- Subjects: Collagenases -- Research , Hides and skins -- Preservation -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4052 , http://hdl.handle.net/10962/d1004113 , Collagenases -- Research , Hides and skins -- Preservation -- Research
- Description: A polarimetric method for monitoring the rate of soluble collagen breakdown by collagenase enzyme action has been developed. The method represents an extension of previous physicochemical techniques based on viscometry, but is simpler and easier to carry out, particularly in the case of reaction rate studies. The method was developed arising from reports of collagenase activity measurement on inappropriate substrates such as gelatin, modified collagens and synthetic polypeptides. The optical method depends on measurement of the loss in optical rotation in solutions of soluble calfskin collagen resulting from initial enzymic cleavage of the collagen trip1e-helix, followed by spontaneous unwinding of the resultant unstable helical fragments. Specific assay conditions were chosen to ensure that the loss in optical rotation following enzymic cleavage was rapid and complete. The method is specific since in the absence of collagenase, non-specific proteinases produce only a limited decrease in solution optical activity. The method has also been compared with established physicochemical assay techniques and compares favourably with both viscometric and titrimetric collagenase assays. The availability of a rapid, sensitive and quantitative procedure for measurement of collagenase activity provides a convenient means for detecting the presence of collagenase in solution and examination of hide bacterial cultures for collagenase production. In addition, a study of biocidal compounds of potential interest in hide preservation for possible inhibitory effects on collagenase is conveniently carried out with the method. Fundamental research into synergistic action in enzymic hydrolysis of collagen is now possible, providing valuable insight into the mechanism of raw hide biodeterioration.
- Full Text:
- Date Issued: 1992
- Authors: Brüning, Adrian Rudolf Nicolaus Ernst
- Date: 1992
- Subjects: Collagenases -- Research , Hides and skins -- Preservation -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4052 , http://hdl.handle.net/10962/d1004113 , Collagenases -- Research , Hides and skins -- Preservation -- Research
- Description: A polarimetric method for monitoring the rate of soluble collagen breakdown by collagenase enzyme action has been developed. The method represents an extension of previous physicochemical techniques based on viscometry, but is simpler and easier to carry out, particularly in the case of reaction rate studies. The method was developed arising from reports of collagenase activity measurement on inappropriate substrates such as gelatin, modified collagens and synthetic polypeptides. The optical method depends on measurement of the loss in optical rotation in solutions of soluble calfskin collagen resulting from initial enzymic cleavage of the collagen trip1e-helix, followed by spontaneous unwinding of the resultant unstable helical fragments. Specific assay conditions were chosen to ensure that the loss in optical rotation following enzymic cleavage was rapid and complete. The method is specific since in the absence of collagenase, non-specific proteinases produce only a limited decrease in solution optical activity. The method has also been compared with established physicochemical assay techniques and compares favourably with both viscometric and titrimetric collagenase assays. The availability of a rapid, sensitive and quantitative procedure for measurement of collagenase activity provides a convenient means for detecting the presence of collagenase in solution and examination of hide bacterial cultures for collagenase production. In addition, a study of biocidal compounds of potential interest in hide preservation for possible inhibitory effects on collagenase is conveniently carried out with the method. Fundamental research into synergistic action in enzymic hydrolysis of collagen is now possible, providing valuable insight into the mechanism of raw hide biodeterioration.
- Full Text:
- Date Issued: 1992
A process for the detanning of chrome leather wastes utilising tannery effluents
- Authors: Glaum, Deanne Melanie
- Date: 1994
- Subjects: Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4029 , http://hdl.handle.net/10962/d1004089 , Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Description: The considerable volume of chromium-bearing wastes generated during the process of leather tanning, exacerbated by the potential for trivalent chromium in the wastes to be oxidised to the toxic hexavalent state, has created a major waste disposal dilemma for the tanning industry. While methods are available for the safe and effective treatment of residual chrome-tanning liquors, little has been done to address the issue of the chrome-bearing solid wastes. Given the increasingly stringent environmental compliance standards facing tanneries, unless an appropriate treatment process is developed in the immediate future, the continued use of chromium as a tanning agent could be compromised. Recent investigations have demonstrated the potential of heated alkaline conditions for dechroming these solid wastes. This study expanded upon these considerations and examined the feasibility of utilising the highly alkaline tannery waste effluents as cost-effective, substitute alkaline media. The three effluents considered in this study, classed as lime sulphide liquors, were shown to be capable of dechroming wet blue shavings, with resultant separation of the solid wastes into a protein and a concentrated chromium product. The solubilised protein product contained low chromium concentrations which comply with legal discharge limits. The precipitated chromium product offers opportunity for reutilisation in the tannery. A novel industrial-scale treatment process, based on these investigations, indicated the process to be capable of treating the quantity of shavings produced on a daily basis by a medium to large scale tannery. Application of this method for the dechroming of other chrome-tanned solid wastes was also shown to be feasible.
- Full Text:
- Date Issued: 1994
- Authors: Glaum, Deanne Melanie
- Date: 1994
- Subjects: Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4029 , http://hdl.handle.net/10962/d1004089 , Tanneries -- Waste disposal , Recycling (Waste, etc.) , Leather
- Description: The considerable volume of chromium-bearing wastes generated during the process of leather tanning, exacerbated by the potential for trivalent chromium in the wastes to be oxidised to the toxic hexavalent state, has created a major waste disposal dilemma for the tanning industry. While methods are available for the safe and effective treatment of residual chrome-tanning liquors, little has been done to address the issue of the chrome-bearing solid wastes. Given the increasingly stringent environmental compliance standards facing tanneries, unless an appropriate treatment process is developed in the immediate future, the continued use of chromium as a tanning agent could be compromised. Recent investigations have demonstrated the potential of heated alkaline conditions for dechroming these solid wastes. This study expanded upon these considerations and examined the feasibility of utilising the highly alkaline tannery waste effluents as cost-effective, substitute alkaline media. The three effluents considered in this study, classed as lime sulphide liquors, were shown to be capable of dechroming wet blue shavings, with resultant separation of the solid wastes into a protein and a concentrated chromium product. The solubilised protein product contained low chromium concentrations which comply with legal discharge limits. The precipitated chromium product offers opportunity for reutilisation in the tannery. A novel industrial-scale treatment process, based on these investigations, indicated the process to be capable of treating the quantity of shavings produced on a daily basis by a medium to large scale tannery. Application of this method for the dechroming of other chrome-tanned solid wastes was also shown to be feasible.
- Full Text:
- Date Issued: 1994
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
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
A study of carbonate-rich brines from Sua Pan to characterize organic contaminants in the soda ash process
- Authors: Joseph, Manjusha
- Date: 2001
- Subjects: Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4031 , http://hdl.handle.net/10962/d1004091 , Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Description: Botswana Ash (Pty) Ltd which is situated in Sua Pan, north east Bostwana, is one of Africa's largest suppliers of salt and soda ash. For a number of years, the company has been experiencing problems which have resulted in the final soda ash product being contaminated and discoloured. The problems experienced at Sua Pan have been reported also to occur in other salt works all over the world. It has been suggested that contamination in many salt works could be possibly be due to the microbial activity by halophilic algae and bacteria that grow in the solar ponds. This study was undertaken to investigate the nature of the contaminating organic compounds present in the brine, to identify the compounds, and to establish how these components vary during the various stages of the soda ash processing. For this study, two sets of brine samples were used; the first set was collected before the summer rains and the second set was collected after the summer rains. Solid bicarbonate and soda ash samples were also used. Extractions, desalting, UV and HPLC analysis and oxidative biotransformations using four enzymes, were used for developing profiles and characterizing the brine components. From these studies, we were able to confirm that the components of the brine are organic in nature. A thorough study of one of the compounds isolated,from solid bicarbonate and soda ash was conducted using UV, HPLC, IR, NMR, HPLC-MS, GC-MS and TLC. The results of these analyses, show that the. isolated compound was benzyl butyl phthalate which is generally regarded to be humic in nature. This compound was found to be present in all the brine samples collected after the summer rains including the well brine, suggesting this compound occurs naturally and is not formed during the processing.
- Full Text:
- Date Issued: 2001
- Authors: Joseph, Manjusha
- Date: 2001
- Subjects: Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4031 , http://hdl.handle.net/10962/d1004091 , Sua Pan Soda Ash Project -- Botswana , Sodium carbonate -- Research , Biotic communities , Organic compounds
- Description: Botswana Ash (Pty) Ltd which is situated in Sua Pan, north east Bostwana, is one of Africa's largest suppliers of salt and soda ash. For a number of years, the company has been experiencing problems which have resulted in the final soda ash product being contaminated and discoloured. The problems experienced at Sua Pan have been reported also to occur in other salt works all over the world. It has been suggested that contamination in many salt works could be possibly be due to the microbial activity by halophilic algae and bacteria that grow in the solar ponds. This study was undertaken to investigate the nature of the contaminating organic compounds present in the brine, to identify the compounds, and to establish how these components vary during the various stages of the soda ash processing. For this study, two sets of brine samples were used; the first set was collected before the summer rains and the second set was collected after the summer rains. Solid bicarbonate and soda ash samples were also used. Extractions, desalting, UV and HPLC analysis and oxidative biotransformations using four enzymes, were used for developing profiles and characterizing the brine components. From these studies, we were able to confirm that the components of the brine are organic in nature. A thorough study of one of the compounds isolated,from solid bicarbonate and soda ash was conducted using UV, HPLC, IR, NMR, HPLC-MS, GC-MS and TLC. The results of these analyses, show that the. isolated compound was benzyl butyl phthalate which is generally regarded to be humic in nature. This compound was found to be present in all the brine samples collected after the summer rains including the well brine, suggesting this compound occurs naturally and is not formed during the processing.
- Full Text:
- Date Issued: 2001
A study of petrol and diesel fuel blends with special reference to their thermodynamic propeties and phase equilibria
- Authors: Hayward, Caroline
- Date: 1986
- Subjects: Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4065 , http://hdl.handle.net/10962/d1004902 , Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Description: The ternary phase behaviour of the n-heptane-l-propanol-water system was studied and compared with the theoretical prediction based on the UNIQUAC model for non-electrolyte solutions. The results showed that this model adequately approximated experimental studies. The excess enthalpies and excess volumes for several binary mixtures were determined. The excess enthalpies were measured using a LKB flow microcalorimeter and the excess -volumes determined using a PAAR densitometer. The study showed that no significant enthalpy or volume changes occurred when petrol/n-heptane were mixed with alcohols . Ternary phase diagrams, including tie lines have been determined for a number of petrol-alcohol-water systems (including the Sasol blend of alcohols). The tie line results show that the concentration of water in the water-rich layer is strongly dependent on the type of alcohol used. The Sasol alcohol blended with petrol resulted in a high water concentration in the water-rich layer which forms on phase separation. This is believed to contribute significantly to the corrosion problems experienced by motorists using the Sasol blended fuel on the Witwatersrand. The effect of temperature on several of these blends was included in the study. Diesel-alcohol blends and the co-solvent properties of ethyl acetate investigated. Ethyl acetate ensures miscibility at low concentrations for diesel-ethanol blends. Octyl nitrate and two cetane improvers from AECI were assessed in terms of their ability to restore cetane rating of blended diesel fuel to that of pure diesel fuel. The results indicated that all three samples were successful in this application. , KMBT_363
- Full Text:
- Date Issued: 1986
- Authors: Hayward, Caroline
- Date: 1986
- Subjects: Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4065 , http://hdl.handle.net/10962/d1004902 , Gasoline , Diesel fuels , Thermodynamics , Liquid-liquid equilibrium , Alcohol as fuel
- Description: The ternary phase behaviour of the n-heptane-l-propanol-water system was studied and compared with the theoretical prediction based on the UNIQUAC model for non-electrolyte solutions. The results showed that this model adequately approximated experimental studies. The excess enthalpies and excess volumes for several binary mixtures were determined. The excess enthalpies were measured using a LKB flow microcalorimeter and the excess -volumes determined using a PAAR densitometer. The study showed that no significant enthalpy or volume changes occurred when petrol/n-heptane were mixed with alcohols . Ternary phase diagrams, including tie lines have been determined for a number of petrol-alcohol-water systems (including the Sasol blend of alcohols). The tie line results show that the concentration of water in the water-rich layer is strongly dependent on the type of alcohol used. The Sasol alcohol blended with petrol resulted in a high water concentration in the water-rich layer which forms on phase separation. This is believed to contribute significantly to the corrosion problems experienced by motorists using the Sasol blended fuel on the Witwatersrand. The effect of temperature on several of these blends was included in the study. Diesel-alcohol blends and the co-solvent properties of ethyl acetate investigated. Ethyl acetate ensures miscibility at low concentrations for diesel-ethanol blends. Octyl nitrate and two cetane improvers from AECI were assessed in terms of their ability to restore cetane rating of blended diesel fuel to that of pure diesel fuel. The results indicated that all three samples were successful in this application. , KMBT_363
- Full Text:
- Date Issued: 1986
A study of the molecular variation between orbivirus proteins
- Authors: Whistler, Toni
- Date: 1985 , 2013-03-13
- Subjects: Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3892 , http://hdl.handle.net/10962/d1003290 , Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Description: The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985
- Authors: Whistler, Toni
- Date: 1985 , 2013-03-13
- Subjects: Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3892 , http://hdl.handle.net/10962/d1003290 , Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Description: The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985
Accelerated carbon dioxide deliming of cattle hides and sheepskins
- Authors: Flowers, Karl Bernard
- Date: 2002
- Subjects: Tanning , Hides and skins , Carbon dioxide
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3918 , http://hdl.handle.net/10962/d1003977 , Tanning , Hides and skins , Carbon dioxide
- Description: To avoid environmental pressure from water authorities, specifically regarding nitrogen and sulfate limits in tannery wastewater, modifications to existing deliming processes have been made. Conventional ammonium salt deliming methods contribute to Total Kjeldahl Nitrogen values in the region of 0.5 – 1.0g/L (33-67% of total TKN). Sulfate levels are increased with the use of organic deliming and ammonium sulfate deliming to the extent of 0.9g/L (27% of total sulfate). To understand the dynamics and kinetics of carbon dioxide equilibrium, the movement of carbon dioxide into deliming water, through carbonic acid, bicarbonate and ultimately into carbonates at liming or early deliming pH was studied. It was shown in this study that effective lime removal, at optimum conditions, resulted in fully delimed pelts at highly comparable quality and times compared to conventional ammonium salt deliming
- Full Text:
- Date Issued: 2002
- Authors: Flowers, Karl Bernard
- Date: 2002
- Subjects: Tanning , Hides and skins , Carbon dioxide
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3918 , http://hdl.handle.net/10962/d1003977 , Tanning , Hides and skins , Carbon dioxide
- Description: To avoid environmental pressure from water authorities, specifically regarding nitrogen and sulfate limits in tannery wastewater, modifications to existing deliming processes have been made. Conventional ammonium salt deliming methods contribute to Total Kjeldahl Nitrogen values in the region of 0.5 – 1.0g/L (33-67% of total TKN). Sulfate levels are increased with the use of organic deliming and ammonium sulfate deliming to the extent of 0.9g/L (27% of total sulfate). To understand the dynamics and kinetics of carbon dioxide equilibrium, the movement of carbon dioxide into deliming water, through carbonic acid, bicarbonate and ultimately into carbonates at liming or early deliming pH was studied. It was shown in this study that effective lime removal, at optimum conditions, resulted in fully delimed pelts at highly comparable quality and times compared to conventional ammonium salt deliming
- Full Text:
- Date Issued: 2002
An investigation into cholinergic interactions in the rat pineal gland
- Authors: Eason, Jason Shane
- Date: 1993
- Subjects: Pineal gland -- Research , Acetylcholine -- Receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4048 , http://hdl.handle.net/10962/d1004109 , Pineal gland -- Research , Acetylcholine -- Receptors
- Description: The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.
- Full Text:
- Date Issued: 1993
- Authors: Eason, Jason Shane
- Date: 1993
- Subjects: Pineal gland -- Research , Acetylcholine -- Receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4048 , http://hdl.handle.net/10962/d1004109 , Pineal gland -- Research , Acetylcholine -- Receptors
- Description: The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.
- Full Text:
- Date Issued: 1993
An investigation into the antioxidative potential and regulatory aspects of liver tryptophan 2,3-dioxygenase by tryptophan and related analogues
- Authors: Antunes, Ana Paula Martins
- Date: 1998
- Subjects: Tryptophan -- Physiological effect , Antioxidants , Liver
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4010 , http://hdl.handle.net/10962/d1004070 , Tryptophan -- Physiological effect , Antioxidants , Liver
- Description: The amino acid, tryptophan, obtained through dietary means, is metabolised by the enzymes tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase. All the enzymes have an effect on circulating tryptophan levels, especially TDO, since it is the major site of tryptophan catabolism in the liver and results in the production of kynurenine metabolites, viz. kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and quinolinic acid. Extrahepatically, IDO is responsible for the synthesis of the kynurenine metabolites. Tryptophan 2,3-dioxygenase and IDO activity is increased by hormones or substrates such as tryptophan, and inflammation, in the case of IDO. Tryptophan availability for serotonin (5-HT) synthesis by the enzyme tryptophan hydroxylase is primarily dependent on TDO activity. A study was attempted in order to ascertain whether any of the endogenous metabolites of the kynurenine and serotonergic pathways would be able to inhibit TDO activity. Results showed that although the kynurenines had no effect, the indoleamines, except for the indoleacetic acids, were able to reduce TDO activity. 6-Methoxy-2-benzoxazolinone (6-MBOA), a structural analogue to melatonin, was the most potent inhibitor with a reduction in activity of 55 % compared with the control. The pineal gland in the rat brain has been shown to have the highest IDO activity. With induction, the kynurenine metabolite concentrations of kynurenic acid and quinolinic acid are increased. The effects of both compounds were determined on the serotonergic pathway. Although kynurenic acid produced no significant effect, quinolinic acid significantly reduced N-acetylserotonin and melatonin synthesis at concentrations of lOJLM and 100 JLM respectively. Many authors have implicated oxygen derived species as causative agents in the important neurodegenerative disorders such as Parkinson's and Huntington's disease. Increased radical generation and lipid peroxidation have been suggested to be responsible for the toxic destruction of neurons, especially in the brain because of its high lipid content and oxygen demand. The brain is therefore vulnerable to oxidative attack. During inflammatory diseases, IDO is induced with a resultant increase in kynurenines. This study was also an attempt at determining the effect of kynurenines on lipid peroxidation. All metabolites of the kynurenine pathway were able to induce lipid peroxidation significantly. The antioxidative potential of various tryptophan analogues, viz. serotonin, melatonin and 6-methoxy-2-benzoxazolinone, was determined using quinolinic acid-induced lipid peroxidation. Serotonin, melatonin and 6-MBOA were able to significantly reduce quinolinic acid-induced lipid peroxidation.
- Full Text:
- Date Issued: 1998
- Authors: Antunes, Ana Paula Martins
- Date: 1998
- Subjects: Tryptophan -- Physiological effect , Antioxidants , Liver
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4010 , http://hdl.handle.net/10962/d1004070 , Tryptophan -- Physiological effect , Antioxidants , Liver
- Description: The amino acid, tryptophan, obtained through dietary means, is metabolised by the enzymes tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase. All the enzymes have an effect on circulating tryptophan levels, especially TDO, since it is the major site of tryptophan catabolism in the liver and results in the production of kynurenine metabolites, viz. kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and quinolinic acid. Extrahepatically, IDO is responsible for the synthesis of the kynurenine metabolites. Tryptophan 2,3-dioxygenase and IDO activity is increased by hormones or substrates such as tryptophan, and inflammation, in the case of IDO. Tryptophan availability for serotonin (5-HT) synthesis by the enzyme tryptophan hydroxylase is primarily dependent on TDO activity. A study was attempted in order to ascertain whether any of the endogenous metabolites of the kynurenine and serotonergic pathways would be able to inhibit TDO activity. Results showed that although the kynurenines had no effect, the indoleamines, except for the indoleacetic acids, were able to reduce TDO activity. 6-Methoxy-2-benzoxazolinone (6-MBOA), a structural analogue to melatonin, was the most potent inhibitor with a reduction in activity of 55 % compared with the control. The pineal gland in the rat brain has been shown to have the highest IDO activity. With induction, the kynurenine metabolite concentrations of kynurenic acid and quinolinic acid are increased. The effects of both compounds were determined on the serotonergic pathway. Although kynurenic acid produced no significant effect, quinolinic acid significantly reduced N-acetylserotonin and melatonin synthesis at concentrations of lOJLM and 100 JLM respectively. Many authors have implicated oxygen derived species as causative agents in the important neurodegenerative disorders such as Parkinson's and Huntington's disease. Increased radical generation and lipid peroxidation have been suggested to be responsible for the toxic destruction of neurons, especially in the brain because of its high lipid content and oxygen demand. The brain is therefore vulnerable to oxidative attack. During inflammatory diseases, IDO is induced with a resultant increase in kynurenines. This study was also an attempt at determining the effect of kynurenines on lipid peroxidation. All metabolites of the kynurenine pathway were able to induce lipid peroxidation significantly. The antioxidative potential of various tryptophan analogues, viz. serotonin, melatonin and 6-methoxy-2-benzoxazolinone, was determined using quinolinic acid-induced lipid peroxidation. Serotonin, melatonin and 6-MBOA were able to significantly reduce quinolinic acid-induced lipid peroxidation.
- Full Text:
- Date Issued: 1998
An investigation into the effects of inorganic toxins and tryptophan metabolites on the forebrain cholinergic system and the pineal gland of the rat
- Authors: Mahabeer, Rajeshree
- Date: 1997
- Subjects: Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4018 , http://hdl.handle.net/10962/d1004078 , Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Description: As soon as the building of the body is completed, the ageing process begins. In the natural course of events, the functioning of some organ systems finally ebbs below the threshold necessary to maintain the body, resulting in death. This occurrence is relatively rare, because diseases superimpose themselves upon the ageing process, bringing premature death resulting from pathological causes. This study focused on the cholinergic system of the rat forebrain. The cholinergic neurons in the brain are said to be involved in memory and learning, and a decrease in the activity of its enzymes has been reported in certain diseases, such as Alzheimer's disease. In the present study, the in vitro effects on the cholinergic system, of aluminium and mercury and tryptophan metabolites, kynurenic acid and quinolinic acid, are determined. Aluminium has been considered as a possible factor in Alzheimer's disease. Mercury in high concentrations is toxic, and its use in amalgam for dental treatment is under consideration with regard to its possible role in promoting neurological disease. The tryptophan metabolites increase in the brain with age and may have a role in pathological diseases. Quinolinic acid, when administered in toxic concentrations produces a possible model for Huntington's disease. This study investigated the effects of the above mentioned toxins on: (1) The synthesis of acetylcholine by choline acetyltransferase; (2) The specific binding of acetylcholine muscarinic receptors; (3) The degradation of acetylcholine by acetyl cholinesterase, Choline acetyltransferase activity did not change in the presence of aluminium chloride, kynurenic acid and quinolinic acid from 1 nM to 1 mM. Mercuric chloride had no significant effect on the enzymes activity from a concentration of 1 nM- 1 pM. At 10 pM there was a significant decrease in cholineacetyltransferase activity (P < 0.001). Enzyme activity continued to decrease at 100 pM (P < 0.0002). At 1 mM, enzyme activity was virtually non existent (P < 0.0001). Acetyl cholinesterase activity was not affected by aluminium chloride, kynurenic acid and quinolinic acid. Mercuric chloride from 1 pM - 1 mM significantly reduced the enzyme activity (P < 0.05). The binding of the antagonist, [³H] quinuclidinyl benzilate (QNB), to acetylcholine muscarinic receptors, revealed that aluminium chloride did not affect the binding of the antagonist, in the concentration range of 1 nM - 100 pM, to the receptors. At 1 mM, aluminium chloride appears to increase the sensitivity of the receptors for the ligand (P < 0.01). Mercuric chloride also does not appear to have any significant effect on receptor binding in this range. However, at 1 mM there appears to be a very significant decrease in receptor binding (P < 0.01). This decrease may be attributed to the interaction of mercury with the sulfhydryl groups in muscarinic receptors. Kynurenic acid had no effect on the receptor binding. Quinolinic acid, in the concentration range from 10 nM - 1 mM increased the binding ofthe receptor to [3Hi QNB significantly (P < 0.001). The study also investigated the effect of the tryptophan metabolites of the kynurenine pathway on pineal indole metabolism. The kynurenine pathway is a major route of tryptophan metabolism in the pineal gland, along with indole metabolism. Investigations showed that kynurenic acid produced a decrease in N-acetylserotonin concentrations ( P < 0.001) and melatonin concentrations (P < 0.003). Further experiments using quinolinic acid produced a similar decrease in N-acetylserotonin (P < 0.001) and melatonin (P < 0.015). A decrease was also noted in the level of 5-methoxytryptophol (P < 0.0005). These findings suggest that aluminium chloride, kynurenic acid and quinolinic acid have no possible role in the decrease of activity of cholinergic enzymes which is observered in diseases such as Alzheimer's disease. The results regarding the effect of mercury chloride on the cholinergic system suggest that low exposure to the toxin will not adversely effect the enzymes. The decrease in N-acetylserotonin and melatonin concentrations reported here, may be a result of kynurenic acid and quinolinic acid having an inhibitory effect on the enzyme, serotonin Nacetyltransferase, which is responsible for the conversion of serotonin to N-acety/serotonin.
- Full Text:
- Date Issued: 1997
- Authors: Mahabeer, Rajeshree
- Date: 1997
- Subjects: Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4018 , http://hdl.handle.net/10962/d1004078 , Toxins -- Physiological effect , Metabolites -- Physiological effect , Pineal gland , Brain -- Physiological aspects
- Description: As soon as the building of the body is completed, the ageing process begins. In the natural course of events, the functioning of some organ systems finally ebbs below the threshold necessary to maintain the body, resulting in death. This occurrence is relatively rare, because diseases superimpose themselves upon the ageing process, bringing premature death resulting from pathological causes. This study focused on the cholinergic system of the rat forebrain. The cholinergic neurons in the brain are said to be involved in memory and learning, and a decrease in the activity of its enzymes has been reported in certain diseases, such as Alzheimer's disease. In the present study, the in vitro effects on the cholinergic system, of aluminium and mercury and tryptophan metabolites, kynurenic acid and quinolinic acid, are determined. Aluminium has been considered as a possible factor in Alzheimer's disease. Mercury in high concentrations is toxic, and its use in amalgam for dental treatment is under consideration with regard to its possible role in promoting neurological disease. The tryptophan metabolites increase in the brain with age and may have a role in pathological diseases. Quinolinic acid, when administered in toxic concentrations produces a possible model for Huntington's disease. This study investigated the effects of the above mentioned toxins on: (1) The synthesis of acetylcholine by choline acetyltransferase; (2) The specific binding of acetylcholine muscarinic receptors; (3) The degradation of acetylcholine by acetyl cholinesterase, Choline acetyltransferase activity did not change in the presence of aluminium chloride, kynurenic acid and quinolinic acid from 1 nM to 1 mM. Mercuric chloride had no significant effect on the enzymes activity from a concentration of 1 nM- 1 pM. At 10 pM there was a significant decrease in cholineacetyltransferase activity (P < 0.001). Enzyme activity continued to decrease at 100 pM (P < 0.0002). At 1 mM, enzyme activity was virtually non existent (P < 0.0001). Acetyl cholinesterase activity was not affected by aluminium chloride, kynurenic acid and quinolinic acid. Mercuric chloride from 1 pM - 1 mM significantly reduced the enzyme activity (P < 0.05). The binding of the antagonist, [³H] quinuclidinyl benzilate (QNB), to acetylcholine muscarinic receptors, revealed that aluminium chloride did not affect the binding of the antagonist, in the concentration range of 1 nM - 100 pM, to the receptors. At 1 mM, aluminium chloride appears to increase the sensitivity of the receptors for the ligand (P < 0.01). Mercuric chloride also does not appear to have any significant effect on receptor binding in this range. However, at 1 mM there appears to be a very significant decrease in receptor binding (P < 0.01). This decrease may be attributed to the interaction of mercury with the sulfhydryl groups in muscarinic receptors. Kynurenic acid had no effect on the receptor binding. Quinolinic acid, in the concentration range from 10 nM - 1 mM increased the binding ofthe receptor to [3Hi QNB significantly (P < 0.001). The study also investigated the effect of the tryptophan metabolites of the kynurenine pathway on pineal indole metabolism. The kynurenine pathway is a major route of tryptophan metabolism in the pineal gland, along with indole metabolism. Investigations showed that kynurenic acid produced a decrease in N-acetylserotonin concentrations ( P < 0.001) and melatonin concentrations (P < 0.003). Further experiments using quinolinic acid produced a similar decrease in N-acetylserotonin (P < 0.001) and melatonin (P < 0.015). A decrease was also noted in the level of 5-methoxytryptophol (P < 0.0005). These findings suggest that aluminium chloride, kynurenic acid and quinolinic acid have no possible role in the decrease of activity of cholinergic enzymes which is observered in diseases such as Alzheimer's disease. The results regarding the effect of mercury chloride on the cholinergic system suggest that low exposure to the toxin will not adversely effect the enzymes. The decrease in N-acetylserotonin and melatonin concentrations reported here, may be a result of kynurenic acid and quinolinic acid having an inhibitory effect on the enzyme, serotonin Nacetyltransferase, which is responsible for the conversion of serotonin to N-acety/serotonin.
- Full Text:
- Date Issued: 1997
An investigation into the interaction partners of the scaffold protein human CNK1 in the NF-κB pathway
- Authors: Moodley, Holisha
- Date: 2019
- Subjects: CNK1 , Scaffold proteins
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96031 , vital:31228
- Description: The protein connector enhancer of KSR1 (CNK1) plays a role in a number of signalling pathways including those involved in cell proliferation, cell growth and differentiation. De-regulation of these pathways has been linked to the promotion of oncogenic signalling. The involvement of CNK1 in all of these diverse pathways indicates a need to better understand the role of this protein within the cell and within key signalling networks. The research provides a platform to understand the intricate relationships that occur between these key signalling networks with the potential to identify new drug targets. CNK1 is multifunctional scaffolding protein that has binding domains that mediate and co-ordinate signalling within the MAPK, Hippo, PI3K/AKT, JNK and NF-κB pathways as well as downstream of the AT2 receptor. The activity of CNK1 is regulated through its interactions with a range of different binding partners within these pathways. Of particular interest to this research is the role of CNK1 in NF-κB signalling. The deregulation of the NF-κB pathway is implicated in chronic inflammation, tissue damage and induction of cervical and breast cancer. CNK1 has been reported to regulate the non-canonical branch of the NF-κB pathway, upstream of the IKK complex however new findings lead to uncertainty about these conclusions. In addition, the interacting partner of CNK1 in the NF-κB pathway has not been elucidated. In this thesis, we aim to identify the binding partners of CNK1 in the NF-κB pathway. First, we validate an epitope-tagged CNK1-expression construct to express elevated levels of CNK1 in cervical cancer cells. We report that the expression of myc-CNK1 is comparable to endogenous CNK1. Cells expressing elevated CNK1 levels were used in traditional co-immunoprecipitation reactions to identify potential CNK1-interacting proteins. We present data that indicates a potential role for NIK in the CNK1 signalling complex. We discuss the weaknesses of the traditional co-immunoprecipitation reactions and design an alternative co-immunoprecipitation technique with which to study CNK1-interacting partners. In this system, a promiscuous biotin ligase fused to the protein sequence for CNK1 (BirA-CNK1) is used to label proteins proximal to CNK1 with biotin. Using this BirA- CNK1-expressing construct in cervical cancer cells, we demonstrate that CNK1 interacts with IKKα-IKKβ in the NF-κB pathway.
- Full Text:
- Date Issued: 2019
- Authors: Moodley, Holisha
- Date: 2019
- Subjects: CNK1 , Scaffold proteins
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96031 , vital:31228
- Description: The protein connector enhancer of KSR1 (CNK1) plays a role in a number of signalling pathways including those involved in cell proliferation, cell growth and differentiation. De-regulation of these pathways has been linked to the promotion of oncogenic signalling. The involvement of CNK1 in all of these diverse pathways indicates a need to better understand the role of this protein within the cell and within key signalling networks. The research provides a platform to understand the intricate relationships that occur between these key signalling networks with the potential to identify new drug targets. CNK1 is multifunctional scaffolding protein that has binding domains that mediate and co-ordinate signalling within the MAPK, Hippo, PI3K/AKT, JNK and NF-κB pathways as well as downstream of the AT2 receptor. The activity of CNK1 is regulated through its interactions with a range of different binding partners within these pathways. Of particular interest to this research is the role of CNK1 in NF-κB signalling. The deregulation of the NF-κB pathway is implicated in chronic inflammation, tissue damage and induction of cervical and breast cancer. CNK1 has been reported to regulate the non-canonical branch of the NF-κB pathway, upstream of the IKK complex however new findings lead to uncertainty about these conclusions. In addition, the interacting partner of CNK1 in the NF-κB pathway has not been elucidated. In this thesis, we aim to identify the binding partners of CNK1 in the NF-κB pathway. First, we validate an epitope-tagged CNK1-expression construct to express elevated levels of CNK1 in cervical cancer cells. We report that the expression of myc-CNK1 is comparable to endogenous CNK1. Cells expressing elevated CNK1 levels were used in traditional co-immunoprecipitation reactions to identify potential CNK1-interacting proteins. We present data that indicates a potential role for NIK in the CNK1 signalling complex. We discuss the weaknesses of the traditional co-immunoprecipitation reactions and design an alternative co-immunoprecipitation technique with which to study CNK1-interacting partners. In this system, a promiscuous biotin ligase fused to the protein sequence for CNK1 (BirA-CNK1) is used to label proteins proximal to CNK1 with biotin. Using this BirA- CNK1-expressing construct in cervical cancer cells, we demonstrate that CNK1 interacts with IKKα-IKKβ in the NF-κB pathway.
- Full Text:
- Date Issued: 2019
An investigation into the synergistic association between the major Clostridium cellulovorans cellulosomal endoglucanase and two hemicellulases on plant cell wall degradation
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
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 bacterial Mur amide ligase enzymes for the identification of inhibitory compounds by in silico methods
- Chamboko, Chiratidzo Respina
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
Analysis of the anti-cancer activity of novel indigenous algal compounds in breast cancer: towards the development of a model for screening anti-cancer stem cell activity
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010